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  1. Silencing Nicotiana attenuata LHY and ZTL alters circadian rhythms in flowers.

    PubMed

    Yon, Felipe; Joo, Youngsung; Cortés Llorca, Lucas; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

    2016-02-01

    The rhythmic opening/closing and volatile emissions of flowers are known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach has led to the identification of core circadian clock components in Arabidopsis thaliana, the involvement of these clock components in floral rhythms has remained untested, probably because of the weak diurnal rhythms in A. thaliana flowers. Here, we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents and move vertically through a 140° arc. We first measured N. attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission and pedicel movement, but not flower closing. We generated transgenic N. attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known to be core clock components. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N. attenuata coordinate these floral rhythms.

  2. Silencing Nicotiana attenuata LHY and ZTL alters circadian rhythms in flowers

    PubMed Central

    Yon, Felipe; Joo, Youngsung; Cortés Llorca, Lucas; Rothe, Eva; Baldwin, Ian T.; Kim, Sang-Gyu

    2016-01-01

    Summary The rhythmic opening/closing and volatile emissions of flowers is known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach led to the identification of core circadian clock components in Arabidopsis thaliana, involvement of these clock components for floral rhythms remained untested likely due to weak diurnal rhythms in A. thaliana flowers.Here we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents, and move vertically through a 140° arc.We first measured N. attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission, and pedicel movement, but not flower closing.We generated transgenic N. attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known as a core clock component. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N. attenuata coordinate these floral rhythms. PMID:26439540

  3. Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata.

    PubMed

    Brütting, Christoph; Schäfer, Martin; Vanková, Radomíra; Gase, Klaus; Baldwin, Ian T; Meldau, Stefan

    2017-01-01

    Plant defense metabolites are well known to be regulated developmentally. The optimal defense (OD) theory posits that a tssue's fitness values and probability of attack should determine defense metabolite allocations. Young leaves are expected to provide a larger fitness value to the plant, and therefore their defense allocations should be higher when compared with older leaves. The mechanisms that coordinate development with defense remain unknown and frequently confound tests of the OD theory predictions. Here we demonstrate that cytokinins (CKs) modulate ontogeny-dependent defenses in Nicotiana attenuata. We found that leaf CK levels highly correlate with inducible defense expressions with high levels in young and low levels in older leaves. We genetically manipulated the developmental patterns of two different CK classes by using senescence- and chemically inducible expression of CK biosynthesis genes. Genetically modifying the levels of different CKs in leaves was sufficient to alter ontogenic patterns of defense metabolites. We conclude that the developmental regulation of growth hormones that include CKs plays central roles in connecting development with defense and therefore in establishing optimal patterns of defense allocation in plants.

  4. Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism

    PubMed Central

    Onkokesung, Nawaporn; Galis, Ivan; Baldwin, Ian T.

    2013-01-01

    In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N′,N′′-dicoumaroylspermidine, N′,N′′-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense. PMID:23704878

  5. Ectopic Terpene Synthase Expression Enhances Sesquiterpene Emission in Nicotiana attenuata without Altering Defense or Development of Transgenic Plants or Neighbors1[W

    PubMed Central

    Schuman, Meredith C.; Palmer-Young, Evan C.; Schmidt, Axel; Gershenzon, Jonathan; Baldwin, Ian T.

    2014-01-01

    Sesquiterpenoids, with approximately 5,000 structures, are the most diverse class of plant volatiles with manifold hypothesized functions in defense, stress tolerance, and signaling between and within plants. These hypotheses have often been tested by transforming plants with sesquiterpene synthases expressed behind the constitutively active 35S promoter, which may have physiological costs measured as inhibited growth and reduced reproduction or may require augmentation of substrate pools to achieve enhanced emission, complicating the interpretation of data from affected transgenic lines. Here, we expressed maize (Zea mays) terpene synthase10 (ZmTPS10), which produces (E)-α-bergamotene and (E)-β-farnesene, or a point mutant ZmTPS10M, which produces primarily (E)-β-farnesene, under control of the 35S promoter in the ecological model plant Nicotiana attenuata. Transgenic N. attenuata plants had specifically enhanced emission of target sesquiterpene(s) with no changes detected in their emission of any other volatiles. Treatment with herbivore or jasmonate elicitors induces emission of (E)-α-bergamotene in wild-type plants and also tended to increase emission of (E)-α-bergamotene and (E)-β-farnesene in transgenics. However, transgenics did not differ from the wild type in defense signaling or chemistry and did not alter defense chemistry in neighboring wild-type plants. These data are inconsistent with within-plant and between-plant signaling functions of (E)-β-farnesene and (E)-α-bergamotene in N. attenuata. Ectopic sesquiterpene emission was apparently not costly for transgenics, which were similar to wild-type plants in their growth and reproduction, even when forced to compete for common resources. These transgenics would be well suited for field experiments to investigate indirect ecological effects of sesquiterpenes for a wild plant in its native habitat. PMID:25187528

  6. A Jasmonate ZIM-Domain Protein NaJAZd Regulates Floral Jasmonic Acid Levels and Counteracts Flower Abscission in Nicotiana attenuata Plants

    PubMed Central

    Oh, Youngjoo; Baldwin, Ian T.; Galis, Ivan

    2013-01-01

    Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ) proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd) by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions played by jasmonates

  7. Herbivore induction of jasmonic acid and chemical defences reduce photosynthesis in Nicotiana attenuata.

    PubMed

    Nabity, Paul D; Zavala, Jorge A; DeLucia, Evan H

    2013-01-01

    Herbivory initiates a shift in plant metabolism from growth to defence that may reduce fitness in the absence of further herbivory. However, the defence-induced changes in carbon assimilation that precede this reallocation in resources remain largely undetermined. This study characterized the response of photosynthesis to herbivore induction of jasmonic acid (JA)-related defences in Nicotiana attenuata to increase understanding of these mechanisms. It was hypothesized that JA-induced defences would immediately reduce the component processes of photosynthesis upon attack and was predicted that wild-type plants would suffer greater reductions in photosynthesis than plants lacking JA-induced defences. Gas exchange, chlorophyll fluorescence, and thermal spatial patterns were measured together with the production of defence-related metabolites after attack and through recovery. Herbivore damage immediately reduced electron transport and gas exchange in wild-type plants, and gas exchange remained suppressed for several days after attack. The sustained reductions in gas exchange occurred concurrently with increased defence metabolites in wild-type plants, whereas plants lacking JA-induced defences suffered minimal suppression in photosynthesis and no increase in defence metabolite production. This suppression in photosynthesis occurred only after sustained defence signalling and defence chemical mobilization, whereas a short bout of feeding damage only transiently altered components of photosynthesis. It was identified that lipoxygenase signalling interacted with photosynthetic electron transport and that the resulting JA-related metabolites reduced photosynthesis. These data represent a metabolic cost to mounting a chemical defence against herbivory and link defence-signalling networks to the differential effects of herbivory on photosynthesis in remaining leaf tissues in a time-dependent manner.

  8. Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata.

    PubMed

    Kessler, Danny; Bhattacharya, Samik; Diezel, Celia; Rothe, Eva; Gase, Klaus; Schöttner, Matthias; Baldwin, Ian T

    2012-08-01

    Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.

  9. Constraints to herbivore-induced systemic responses: bidirectional signaling along orthostichies in Nicotiana attenuata.

    PubMed

    Schittko, Ursula; Baldwin, Ian T

    2003-03-01

    We investigated the impact of leaf vascular connections on systemically transmitted herbivore-induced gene expression in Nicotiana attenuata. Although systemic signaling is clearly associated with the plant vascular system, few studies consider vascular architecture when measuring systemically induced defenses. N. attenuata is a plant with dispersed phyllotaxis approximating 3/8 in the rosette stage of growth. We mimicked Manduca sexta herbivory by introducing larval regurgitant to wounds produced with a standardized continuous mechanical wounding and investigated mRNA accumulation of genes. Herbivory in N. attenuata induces the expression of genes coding for a proteinase inhibitor protein (PI), threonine deaminase (TD, EC 4.3.1.19), a luminal-binding protein (BiP), and an alpha-dioxygenase (alpha-DOX). We measured the systemic response of sink leaves when orthostichous (growing at an angular distance of 0 degrees) source leaves were treated, and vice versa, and compared it to the systemic response of leaves growing at the maximum angular distance of 180 degrees. Vascular architecture clearly controlled the intensity of systemic mRNA accumulation within the 4-hr time frame of the experiment. In addition, we found signal translocation to be bidirectional, travelling from source to sink as well as from sink to source leaves, which argues against a phloem-based assimilate-linked signal identity.

  10. Priming of anti-herbivore defence in Nicotiana attenuata by insect oviposition: herbivore-specific effects.

    PubMed

    Bandoly, Michele; Grichnik, Roland; Hilker, Monika; Steppuhn, Anke

    2016-04-01

    Oviposition by Spodoptera exigua on Nicotiana attenuata primes plant defence against its larvae that consequently suffer reduced performance. To reveal whether this is a general response of tobacco to insect oviposition or species-specific, we investigated whether also Manduca sexta oviposition primes N. attenuata's anti-herbivore defence. The plant response to M. sexta and S. exigua oviposition overlapped in the egg-primed feeding-induced production of the phenylpropanoid caffeoylputrescine. While M. sexta larvae were unaffected in their performance, they showed a novel response to the oviposition-mediated plant changes: a reduced antimicrobial activity in their haemolymph. In a cross-resistance experiment, S. exigua larvae suffered reduced performance on M. sexta-oviposited plants like they did on S. exigua-oviposited plants. The M. sexta oviposition-mediated plant effects on the S. exigua larval performance and on M. sexta larval immunity required expression of the NaMyb8 transcription factor that is governing biosynthesis of phenylpropanoids such as caffeoylputrescine. Thus, NaMyb8-dependent defence traits mediate the effects that oviposition by both lepidopteran species exerts on the plant's anti-herbivore defence. These results suggest that oviposition by lepidopteran species on N. attenuata leaves may generally prime the feeding-induced production of certain plant defence compounds but that different herbivore species show different susceptibility to egg-primed plant effects.

  11. Silencing of a germin-like gene in Nicotiana attenuata improves performance of native herbivores.

    PubMed

    Lou, Yonggen; Baldwin, Ian T

    2006-03-01

    Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval oral secretions (OS) to wounds up-regulates transcripts of a GLP. To understand the function of this gene, which occurs as a single copy, we cloned the full-length NaGLP and silenced its expression in N. attenuata by expressing a 250-bp fragment in an antisense orientation with an Agrobacterium-based transformation system and by virus-induced gene silencing (VIGS). Homozygous lines harboring a single insert and VIGS plants had significantly reduced constitutive (measured in roots) and elicited NaGLP transcript levels (in leaves). Silencing NaGLP improved M. sexta larval performance and Tupiocoris notatus preference, two native herbivores of N. attenuata. Silencing NaGLP also attenuated the OS-induced hydrogen peroxide (H(2)O(2)), diterpene glycosides, and trypsin proteinase inhibitor responses, which may explain the observed susceptibility of antisense or VIGS plants to herbivore attack and increased nicotine contents, but did not influence the OS-elicited jasmonate and salicylate bursts, or the release of the volatile organic compounds (limonene, cis-alpha-bergamotene, and germacrene-A) that function as an indirect defense. This suggests that NaGLP is involved in H(2)O(2) production and might also be related to ethylene production and/or perception, which in turn influences the defense responses of N. attenuata via H(2)O(2) and ethylene-signaling pathways.

  12. HSPRO controls early Nicotiana attenuata seedling growth during interaction with the fungus Piriformospora indica.

    PubMed

    Schuck, Stefan; Camehl, Iris; Gilardoni, Paola A; Oelmueller, Ralf; Baldwin, Ian T; Bonaventure, Gustavo

    2012-10-01

    In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1(pro)(-)(1)) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica.

  13. Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata.

    PubMed

    Ling, Zhihao; Zhou, Wenwu; Baldwin, Ian T; Xu, Shuqing

    2015-10-01

    Changes in gene expression and alternative splicing (AS) are involved in many responses to abiotic and biotic stresses in eukaryotic organisms. In response to attack and oviposition by insect herbivores, plants elicit rapid changes in gene expression which are essential for the activation of plant defenses; however, the herbivory-induced changes in AS remain unstudied. Using mRNA sequencing, we performed a genome-wide analysis on tobacco hornworm (Manduca sexta) feeding-induced AS in both leaves and roots of Nicotiana attenuata. Feeding by M. sexta for 5 h reduced total AS events by 7.3% in leaves but increased them in roots by 8.0% and significantly changed AS patterns in leaves and roots of existing AS genes. Feeding by M. sexta also resulted in increased (in roots) and decreased (in leaves) transcript levels of the serine/arginine-rich (SR) proteins that are involved in the AS machinery of plants and induced changes in SR gene expression that were jasmonic acid (JA)-independent in leaves but JA-dependent in roots. Changes in AS and gene expression elicited by M. sexta feeding were regulated independently in both tissues. This study provides genome-wide evidence that insect herbivory induces changes not only in the levels of gene expression but also in their splicing, which might contribute to defense against and/or tolerance of herbivory.

  14. Oviposition by Spodoptera exigua on Nicotiana attenuata primes induced plant defence against larval herbivory.

    PubMed

    Bandoly, Michele; Hilker, Monika; Steppuhn, Anke

    2015-08-01

    Plants exhibit multifarious defence traits against herbivory that are constitutively expressed or induced upon attack. Insect egg deposition often precedes impending larval attack, and several plants can increase their resistance against larvae after experiencing the oviposition by an herbivore. The nature of such oviposition-mediated resistance remains unknown, and here we aim to determine plant traits that explain it. We test whether oviposition on a host plant can induce plant defence responses or enhance (prime) the induction of defence traits in response to larval herbivory. We exposed Nicotiana attenuata plants to oviposition by moths of a generalist herbivore, Spodoptera exigua. Its larvae suffered higher mortality, retarded development and inflicted less feeding damage on oviposition-experienced than on oviposition-unexperienced plants. While oviposition alone did not induce any of the examined defence traits, oviposited plants exhibited a stronger inducibility of known defence traits, i.e. caffeoylputrescine (CP) and trypsin protease inhibitors (TPIs). We found no effects of oviposition on phytohormone levels, but on the feeding-inducible accumulation of the transcription factor NaMyb8 that is governing biosynthesis of phenylpropanoid-polyamine conjugates, including CP. Comparison of larval performance on wild-type plants, CP-deficient plants (silenced NaMyb8 gene), and TPI-deficient plants (silenced NaPI gene) revealed that priming of plant resistance to larvae by prior oviposition required NaMyb8-mediated defence traits. Our results show that plants can use insect egg deposition as a warning signal to prime their feeding-induced defence.

  15. PR-13/Thionin but not PR-1 mediates bacterial resistance in Nicotiana attenuata in nature, and neither influences herbivore resistance.

    PubMed

    Rayapuram, Cbgowda; Wu, Jianqiang; Haas, Christiane; Baldwin, Ian T

    2008-07-01

    Increases in pathogenesis-related (PR) transcripts are commonly interpreted as evidence of plants' resistance responses to pathogens; however, few studies have examined whether increases in PR proteins protect plants growing under natural conditions. Pseudomonas syringae pv. tomato DC3,000, which is virulent and causes disease in Arabidopsis, is also pathogenic to the native tobacco Nicotiana attenuata. N. attenuata responds to P. syringae pv. tomato DC3,000's challenges with increases in salicylic acid and transcripts of at least two PR genes, PR-1 and PR13/Thionin. To determine if either of these PR proteins functions in bacterial resistance, we independently silenced both genes by RNAi and found that only PR-13/Thionin mediates resistance to P. syringae pv. tomato DC3,000 in glasshouse experiments. When NaPR-1- and NaThionin-silenced plants were transplanted into the plant's native habitat in the Great Basin Desert of Utah, opportunistic Pseudomonas spp. performed better on NaThionin-silenced plants compared with NaPR-1-silenced and wild-type (WT) plants, and accounted for increased plant mortality. The native herbivore community of N. attenuata attacked both NaPR-1- and PR-13/NaThionin-silenced plants to the same degree as it did in WT plants, indicating that neither PR protein provides resistance to herbivores. Although PR-1 is generally considered a marker gene of disease resistance, we found no evidence that it has an antimicrobial function. In contrast, PR-13/NaThionin is clearly an ecologically relevant defense protein involved in resisting pathogens in N. attenuata.

  16. Pithy protection: Nicotiana attenuata's jasmonic acid-mediated defenses are required to resist stem-boring weevil larvae.

    PubMed

    Diezel, Celia; Kessler, Danny; Baldwin, Ian T

    2011-04-01

    Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C(6) aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant's apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata's fitness.

  17. The HERBIVORE ELICITOR-REGULATED1 gene enhances abscisic acid levels and defenses against herbivores in Nicotiana attenuata plants.

    PubMed

    Dinh, Son Truong; Baldwin, Ian T; Galis, Ivan

    2013-08-01

    Nicotiana attenuata plants can distinguish the damage caused by herbivore feeding from other types of damage by perceiving herbivore-associated elicitors, such as the fatty acid-amino acid conjugates (FACs) in oral secretions (OS) of Manduca sexta larvae, which are introduced into wounds during feeding. However, the transduction of FAC signals into downstream plant defense responses is still not well established. We identified a novel FAC-regulated protein in N. attenuata (NaHER1; for herbivore elicitor regulated) and show that it is an indispensable part of the OS signal transduction pathway. N. attenuata plants silenced in the expression of NaHER1 by RNA interference (irHER1) were unable to amplify their defenses beyond basal, wound-induced levels in response to OS elicitation. M. sexta larvae performed 2-fold better when reared on irHER1 plants, which released less volatile organic compounds (indirect defense) and had strongly reduced levels of several direct defense metabolites, including trypsin proteinase inhibitors, 17-hydroxygeranyllinallool diterpene glycosides, and caffeoylputrescine, after real and/or simulated herbivore attack. In parallel to impaired jasmonate signaling and metabolism, irHER1 plants were more drought sensitive and showed reduced levels of abscisic acid (ABA) in the leaves, suggesting that silencing of NaHER1 interfered with ABA metabolism. Because treatment of irHER1 plants with ABA results in both the accumulation of significantly more ABA catabolites and the complete restoration of normal wild-type levels of OS-induced defense metabolites, we conclude that NaHER1 acts as a natural suppressor of ABA catabolism after herbivore attack, which, in turn, activates the full defense profile and resistance against herbivores.

  18. Polymorphism in jasmonate signaling partially accounts for the variety of volatiles produced by Nicotiana attenuata plants in a native population.

    PubMed

    Schuman, Meredith C; Heinzel, Nicolas; Gaquerel, Emmanuel; Svatos, Ales; Baldwin, Ian T

    2009-01-01

    Herbivore- and jasmonate-induced volatile organic compounds (VOCs), which mediate indirect defense, must provide reliable information for predators that frequently learn to associate their release with feeding herbivores. Yet little is known about variation of these cues within populations of native plants, on a scale encountered by predators. We examined variation in herbivore-elicited VOC emissions and patterns of herbivore-induced jasmonate signaling from accessions of Nicotiana attenuata co-occurring in a native population. VOC emissions elicited by herbivore oral secretions (OS) and by methyl jasmonate (MJ) were characterized using gas chromatography-mass spectrometry (GC-MS), high-resolution two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-ToF-MS) and micro-hydrolysis and micro-hydrogenation reactions. Accessions varied in emissions of abundant (trans-alpha-bergamotene, alpha-duprezianene, trans-beta-ocimene, and cis-3-hexenol) and total detectable VOCs, as well as the accumulation of jasmonates, the jasmonate antagonist salicylic acid (SA), abscisic acid (ABA) and jasmonate signaling-related transcripts after OS elicitation. Yet MJ treatment exacerbated differences in VOC emission, suggesting that much variation in VOC emission is caused by processes downstream of jasmonate signaling. Co-occurring N. attenuata plants emit different VOCs following simulated herbivore elicitation as a result in part of differences in jasmonate production and responsiveness, which could reduce the effectiveness of induced indirect defense.

  19. Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance.

    PubMed

    Fragoso, Variluska; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

    2014-06-01

    While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied. To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature. Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In N. attenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus. We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses.

  20. Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata.

    PubMed

    Barazani, Oz; Benderoth, Markus; Groten, Karin; Kuhlemeier, Cris; Baldwin, Ian T

    2005-12-01

    A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.

  1. Jasmonoyl-L-isoleucine coordinates metabolic networks required for anthesis and floral attractant emission in wild tobacco (Nicotiana attenuata).

    PubMed

    Stitz, Michael; Hartl, Markus; Baldwin, Ian T; Gaquerel, Emmanuel

    2014-10-01

    Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study.

  2. New insights into mechanisms regulating differential accumulation of phenylpropanoid-polyamine conjugates (PPCs) in herbivore-attacked Nicotiana attenuata plants.

    PubMed

    Gális, Ivan; Onkokesung, Nawaporn; Baldwin, Ian T

    2010-05-01

    Ubiquitously occurring phenylpropanoid-polyamine conjugates (PPCs) in plants show both developmental and stress inducible patterns of accumulation. The presence of high PPC levels in reproductive organs of plants resulted in a long lasting controversy whether PPCs could play important roles in the development of reproductive tissues; their accumulation in response to stresses, suggested a potential defensive function. The discovery and use of a key regulator, MYB8 in jasmonic acid signal transduction pathway, that controls the accumulation of major PPCs in native tobacco (Nicotiana attenuata) plants, has now provided the first clear evidence of defense-related functions for PPCs in higher plants, and contributed to our understanding of how the perception of herbivore-associated elicitors can modulate anti-herbivore defenses in plants.

  3. Diverting the flux of the JA pathway in Nicotiana attenuata compromises the plant's defense metabolism and fitness in nature and glasshouse.

    PubMed

    Stitz, Michael; Baldwin, Ian T; Gaquerel, Emmanuel

    2011-01-01

    A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature.

  4. Jasmonate and ppHsystemin regulate key Malonylation steps in the biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides, an abundant and effective direct defense against herbivores in Nicotiana attenuata.

    PubMed

    Heiling, Sven; Schuman, Meredith C; Schoettner, Matthias; Mukerjee, Purba; Berger, Beatrice; Schneider, Bernd; Jassbi, Amir R; Baldwin, Ian T

    2010-01-01

    We identified 11 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) that occur in concentrations equivalent to starch (mg/g fresh mass) in aboveground tissues of coyote tobacco (Nicotiana attenuata) and differ in their sugar moieties and malonyl sugar esters (0-2). Concentrations of HGL-DTGs, particularly malonylated compounds, are highest in young and reproductive tissues. Within a tissue, herbivore elicitation changes concentrations and biosynthetic kinetics of individual compounds. Using stably transformed N. attenuata plants silenced in jasmonate production and perception, or production of N. attenuata Hyp-rich glycopeptide systemin precursor by RNA interference, we identified malonylation as the key biosynthetic step regulated by herbivory and jasmonate signaling. We stably silenced N. attenuata geranylgeranyl diphosphate synthase (ggpps) to reduce precursors for the HGL-DTG skeleton, resulting in reduced total HGL-DTGs and greater vulnerability to native herbivores in the field. Larvae of the specialist tobacco hornworm (Manduca sexta) grew up to 10 times as large on ggpps silenced plants, and silenced plants suffered significantly more damage from herbivores in N. attenuata's native habitat than did wild-type plants. We propose that high concentrations of HGL-DTGs effectively defend valuable tissues against herbivores and that malonylation may play an important role in regulating the distribution and storage of HGL-DTGs in plants.

  5. Differences in Nicotine Metabolism of Two Nicotiana attenuata Herbivores Render Them Differentially Susceptible to a Common Native Predator

    PubMed Central

    Kumar, Pavan; Rathi, Preeti; Schöttner, Matthias; Baldwin, Ian T.; Pandit, Sagar

    2014-01-01

    Background Nicotiana attenuata is attacked by larvae of both specialist (Manduca sexta) and generalist (Spodoptera exigua) lepidopteran herbivores in its native habitat. Nicotine is one of N. attenuata's important defenses. M. sexta is highly nicotine tolerant; whether cytochrome P450 (CYP)-mediated oxidative detoxification and/or rapid excretion is responsible for its exceptional tolerance remains unknown despite five decades of study. Recently, we demonstrated that M. sexta uses its nicotine-induced CYP6B46 to efflux midgut-nicotine into the hemolymph, facilitating nicotine exhalation that deters predatory wolf spiders (Camptocosa parallela). S. exigua's nicotine metabolism is uninvestigated. Methodology/Principal Findings We compared the ability of these two herbivores to metabolize, tolerate and co-opt ingested nicotine for defense against the wolf spider. In addition, we analyzed the spider's excretion to gain insights into its nicotine metabolism. Contrary to previous reports, we found that M. sexta larvae neither accumulate the common nicotine oxides (cotinine, cotinine N-oxide and nicotine N-oxide) nor excrete them faster than nicotine. In M. sexta larvae, ingestion of nicotine as well as its oxides increases the accumulation of CYP6B46 transcripts. In contrast, S. exigua accumulates nicotine oxides and exhales less (66%) nicotine than does M. sexta. Spiders prefer nicotine-fed S. exigua over M. sexta, a preference reversed by topical or headspace nicotine supplementation, but not ingested or topically-coated nicotine oxides, suggesting that externalized nicotine but not the nicotine detoxification products deter spider predation. The spiders also do not accumulate nicotine oxides. Conclusions Nicotine oxidation reduces S. exigua's headspace-nicotine and renders it more susceptible to predation by spiders than M. sexta, which exhales unmetabolized nicotine. These results are consistent with the hypothesis that generalist herbivores incur costs of

  6. Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata.

    PubMed

    Halitschke, Rayko; Baldwin, Ian T

    2003-12-01

    Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.

  7. Dimethyl disulfide produced by the naturally associated bacterium bacillus sp B55 promotes Nicotiana attenuata growth by enhancing sulfur nutrition.

    PubMed

    Meldau, Dorothea G; Meldau, Stefan; Hoang, Long H; Underberg, Stefanie; Wünsche, Hendrik; Baldwin, Ian T

    2013-07-01

    Bacillus sp B55, a bacterium naturally associated with Nicotiana attenuata roots, promotes growth and survival of wild-type and, particularly, ethylene (ET)-insensitive (35)S-ethylene response1 (etr1) N. attenuata plants, which heterologously express the mutant Arabidopsis thaliana receptor ETR1-1. We found that the volatile organic compound (VOC) blend emitted by B55 promotes seedling growth, which is dominated by the S-containing compound dimethyl disulfide (DMDS). DMDS was depleted from the headspace during cocultivation with seedlings in bipartite Petri dishes, and (35)S was assimilated from the bacterial VOC bouquet and incorporated into plant proteins. In wild-type and (35)S-etr1 seedlings grown under different sulfate (SO(4)(-2)) supply conditions, exposure to synthetic DMDS led to genotype-dependent plant growth promotion effects. For the wild type, only S-starved seedlings benefited from DMDS exposure. By contrast, growth of (35)S-etr1 seedlings, which we demonstrate to have an unregulated S metabolism, increased at all SO(4)(-2) supply rates. Exposure to B55 VOCs and DMDS rescued many of the growth phenotypes exhibited by ET-insensitive plants, including the lack of root hairs, poor lateral root growth, and low chlorophyll content. DMDS supplementation significantly reduced the expression of S assimilation genes, as well as Met biosynthesis and recycling. We conclude that DMDS by B55 production is a plant growth promotion mechanism that likely enhances the availability of reduced S, which is particularly beneficial for wild-type plants growing in S-deficient soils and for (35)S-etr1 plants due to their impaired S uptake/assimilation/metabolism.

  8. NaJAZh regulates a subset of defense responses against herbivores and spontaneous leaf necrosis in Nicotiana attenuata plants.

    PubMed

    Oh, Youngjoo; Baldwin, Ian T; Gális, Ivan

    2012-06-01

    The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development.

  9. Jasmonate signaling in the field, part I: elicited changes in jasmonate pools of transgenic Nicotiana attenuata populations.

    PubMed

    Gaquerel, Emmanuel; Stitz, Michael; Kallenbach, Mario; Baldwin, Ian T

    2013-01-01

    Nicotiana attenuata, a wild tobacco species native of the southwestern USA that grows in the immediate postfire environment, is one of the important host plants for herbivore populations recolonizing recently burned habitats in the Great Basin Desert. Based on more than 20 years of field research on this eco-genomics model system established in our group, we have developed a genetic and analytical toolbox that allows us to assess the importance of particular genes and metabolites for the survival of this plant in its native habitat. This toolbox has been extensively applied to study the activation of jasmonate signaling after the attack of different herbivore species. Here, we provide detailed guidelines for the analysis, under field conditions, of induced changes in jasmonate pools during insect herbivory. The procedures range from selection and field release of well-characterized transgenic lines for testing the physiological consequences of manipulating jasmonate biogenesis, metabolism, or perception to the metabolic elicitation of chewing herbivore attack and the quantification of the resulting changes in jasmonate fluxes.

  10. Making sense of nectar scents: the effects of nectar secondary metabolites on floral visitors of Nicotiana attenuata.

    PubMed

    Kessler, Danny; Baldwin, Ian T

    2007-03-01

    Flowers produce a plethora of secondary metabolites but only nectar sugars, floral pigments and headspace volatiles have been examined in the context of pollinator behavior. We identify secondary metabolites in the headspace and nectar of glasshouse- and field-grown Nicotiana attenuata plants, infer within-flower origins by analyzing six flower parts, and compare the attractiveness of 16 constituents in standardized choice tests with two guilds of natural pollinators (Manducasexta moths and Archilochus alexandri and Selasphorus rufus hummingbirds) and one nectar thief (Solenopsis xyloni ants) to determine whether nectar metabolites can 'filter' flower visitors: only two could. Moths responded more strongly than did hummingbirds to headspace presentation of nicotine and benzylacetone, the most abundant repellent and attractant compounds, respectively. For both pollinators, nectar repellents decreased nectaring time and nectar volume removed, but increased visitation number, particularly for hummingbirds. Fewer ants visited if the nectar contained repellents. To determine whether nicotine reduced nectar removal rates in nature, we planted transformed, nicotine-silenced plants into native populations in Utah over 2 years. Plants completely lacking nicotine in their nectar had 68-70% more nectar removed per night by the native community of floral visitors than did wild-type plants. We hypothesize that nectar repellents optimize the number of flower visitors per volume of nectar produced, allowing plants to keep their nectar volumes small.

  11. Mechanisms of optimal defense patterns in Nicotiana attenuata: flowering attenuates herbivory-elicited ethylene and jasmonate signaling.

    PubMed

    Diezel, Celia; Allmann, Silke; Baldwin, Ian T

    2011-12-01

    To defend themselves against herbivore attack, plants produce secondary metabolites, which are variously inducible and constitutively deployed, presumably to optimize their fitness benefits in light of their fitness costs. Three phytohormones, jasmonates (JA) and their active forms, the JA-isoleucine (JA-Ile) and ethylene (ET), are known to play central roles in the elicitation of induced defenses, but little is known about how this mediation changes over ontogeny. The Optimal Defense Theory (ODT) predicts changes in the costs and benefits of the different types of defenses and has been usefully extrapolated to their modes of deployment. Here we studied whether the herbivore-induced accumulation of JA, JA-Ile and ET changed over ontogeny in Nicotiana attenuata, a native tobacco in which inducible defenses are particularly well studied. Herbivore-elicited ET production changed dramatically during six developmental stages, from rosette through flowering, decreasing with the elongation of the first corollas during flower development. This decrease was largely recovered within a day after flower removal by decapitation. A similar pattern was found for the herbivore-induced accumulation of JA and JA-Ile. These results are consistent with ODT predictions and suggest that the last steps in floral development control the inducibility of at least three plant hormones, optimizing defense-growth tradeoffs.

  12. Induced plant defenses in the natural environment: Nicotiana attenuata WRKY3 and WRKY6 coordinate responses to herbivory.

    PubMed

    Skibbe, Melanie; Qu, Nan; Galis, Ivan; Baldwin, Ian T

    2008-07-01

    A plant-specific family of WRKY transcription factors regulates plant responses to pathogens and abiotic stresses. Here, we identify two insect-responsive WRKY genes in the native tobacco Nicotiana attenuata: WRKY3, whose transcripts accumulate in response to wounding, and WRKY6, whose wound responses are significantly amplified when fatty acid-amino acid conjugates (FACs) in larval oral secretions are introduced into wounds during feeding. WRKY3 is required for WRKY6 elicitation, yet neither is elicited by treatment with the phytohormone wound signal jasmonic acid. Silencing either WRKY3 or WRKY6, or both, by stable transformation makes plants highly vulnerable to herbivores under glasshouse conditions and in their native habitat in the Great Basin Desert, Utah, as shown in three field seasons. This susceptibility is associated with impaired jasmonate (JA) accumulation and impairment of the direct (trypsin proteinase inhibitors) and indirect (volatiles) defenses that JA signaling mediates. The response to wounding and herbivore-specific signals (FACs) demonstrates that these WRKYs help plants to differentiate mechanical wounding from herbivore attack, mediating a plant's herbivore-specific defenses. Differences in responses to single and multiple elicitations indicate an important role of WRKY3 and WRKY6 in potentiating and/or sustaining active JA levels during continuous insect attack.

  13. The hydroxyproline-rich glycopeptide systemin precursor NapreproHypSys does not play a central role in Nicotiana attenuata's anti-herbivore defense responses.

    PubMed

    Berger, Beatrice; Baldwin, Ian T

    2007-11-01

    To determine whether the Nicotiana tabacum preproHypSys homolog in Nicotiana attenuata (NapreproHypSys) mediates anti-herbivore responses, we silenced (IRsys) and ectopically over-expressed (OVsys) NapreproHypSys in N. attenuata. Neither herbivore simulation nor methyl jasmonate (MeJA) application increased transcripts in wild-type (WT) or transformed lines. Compared to WT plants, OVsys plants had marginally higher constitutive levels but normally induced levels of trypsin proteinase inhibitors (TPIs) and nicotine; IRsys plants did not differ from WT plants. Herbivory-associated signalling [salicylic acid-induced protein kinase (SIPK) activity, jasmonic acid (JA), jasmonic acid-isoleucine/leucine (JA-Ile/Leu) and ethylene production or perception] did not differ strongly among the lines, but JA, JA-Ile/Leu and ethylene were marginally higher in OVsys plants. Manduca sexta larval performance did not differ among the lines, but feeding induced levels of TPI and nicotine in OVsys plants and decreased them in IRsys plants relative to WT. The secondary metabolite profiles of plants transplanted into N. attenuata's native habitat in the Great Basin Desert (UT, USA) mirrored those of glasshouse-grown plants, and compared to WT plants, OVsys plants suffered marginally less damage from grasshoppers, mirids and flea beetles but did not differ in their ability to attract Geocoris predators. We conclude that NapreproHypSys does not play a central role in anti-herbivore defense signalling in this native tobacco.

  14. Solar Ultraviolet-B Radiation and Insect Herbivory Trigger Partially Overlapping Phenolic Responses in Nicotiana attenuata and Nicotiana longiflora

    PubMed Central

    Izaguirre, Miriam M.; Mazza, Carlos A.; SvatoŠ, AleŠ; Baldwin, Ian T.; BallarÉ, Carlos L.

    2007-01-01

    Background and Aims Plants exposed to solar ultraviolet-B radiation (UV-B, 280–315 nm) frequently suffer less insect herbivory than do plants that receive attenuated levels of UV-B. This anti-herbivore effect of solar UV-B exposure, which has been documented in several ecosystems, is in part mediated by changes in plant tissue quality. Exposure to UV-B can modify the abundance of a number of secondary metabolites, including phenolic compounds with potential impacts on insect herbivores. The aim of this study is to assess the potential anti-herbivore role of UV-B-induced phenolic compounds by comparing the phenolic profiles induced by UV-B and simulated insect herbivory in two wild species of the genus Nicotiana. Methods Plants grown under field and glasshouse conditions were exposed to contrasting levels of UV-B. Half of the plants of the attenuated UV-B treatment were given a simulated herbivory treatment, where leaves were mechanically damaged and immediately treated with oral secretions of Manduca sexta caterpillars. This treatment is known to mimic the impact of real herbivory on the expression of plant defences in Nicotiana. Phenolic profiles induced by UV-B and simulated herbivory were characterized using high-performance liquid chromatography–mass spectrometry (HPLC–MS). Key Results UV-B induced the accumulation of several UV-absorbing phenolic compounds that are known to play a significant role in UV-B screening. Interestingly, there was a significant convergence in the phenolic profiles induced by UV-B and simulated herbivory: chlorogenic acid and dicaffeoylspermidine isomers, in particular, displayed a similar pattern of response to these stimuli. In contrast, rutin, the only flavonoid that accumulated in significant quantities in the experiments, was only induced by UV-B. Conclusions The results suggest that the anti-herbivory effect induced by UV-B may be mediated at least in part by the accumulation of phenylpropanoid derivatives that are

  15. MAPK-dependent JA and SA signalling in Nicotiana attenuata affects plant growth and fitness during competition with conspecifics

    PubMed Central

    2012-01-01

    Background Induced defense responses to herbivores are generally believed to have evolved as cost-saving strategies that defer the fitness costs of defense metabolism until these defenses are needed. The fitness costs of jasmonate (JA)-mediated defenses have been well documented. Those of the early signaling units mediating induced resistance to herbivores have yet to be examined. Early signaling components that mediate herbivore-induced defense responses in Nicotiana attenuata, have been well characterized and here we examine their growth and fitness costs during competition with conspecifics. Two mitogen-activated protein kinases (MAPKs), salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) are rapidly activated after perception of herbivory and both kinases regulate herbivory-induced JA levels and JA-mediated defense metabolite accumulations. Since JA-induced defenses result in resource-based trade-offs that compromise plant productivity, we evaluated if silencing SIPK (irSIPK) and WIPK (irWIPK) benefits the growth and fitness of plants competiting with wild type (WT) plants, as has been shown for plants silenced in JA-signaling by the reduction of Lipoxygenase 3 (LOX3) levels. Results As expected, irWIPK and LOX3-silenced plants out-performed their competing WT plants. Surprisingly, irSIPK plants, which have the largest reductions in JA signaling, did not. Phytohormone profiling of leaves revealed that irSIPK plants accumulated higher levels of SA compared to WT. To test the hypothesis that these high levels of SA, and their presumed associated fitness costs of pathogen associated defenses in irSIPK plants had nullified the JA-deficiency-mediated growth benefits in these plants, we genetically reduced SA levels in irSIPK plants. Reducing SA levels partially recovered the biomass and fitness deficits of irSIPK plants. We also evaluated whether the increased fitness of plants with reduced SA or JA levels resulted from

  16. Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuata.

    PubMed

    Halitschke, Rayko; Ziegler, Jörg; Keinänen, Markku; Baldwin, Ian T

    2004-10-01

    The fatty acid hydroperoxide (HP) substrates required for the biosynthesis of jasmonic acid (JA) and green leaf volatiles (GLVs) are supplied by separate lipoxygenases (LOX). We silenced the expression of two genes downstream of the LOX: allene oxide synthase (AOS) and HP lyase (HPL) by antisense expression of endogenous genes (NaAOS, NaHPL) in Nicotiana attenuata, in which the biosynthesis of JA is amplified by herbivore-specific elicitors. We report that these elicitors also amplify wound-induced GLV releases, but suppress the wound-induced increase of NaHPL transcripts, suggesting that substrate flux controls GLV biosynthesis. As expected, silencing of NaHPL and NaAOS reduced GLV release and JA accumulation, respectively. Surprisingly, HPL- and AOS-silenced plants had enhanced JA and GLV responses, suggesting substrate 'crosstalk' between these two oxylipin cascades. Plants with depleted GLVs (as-hpl) were less attractive than wild type (WT) or empty vector control plants in choice-tests with native lepidopteran herbivores. In feeding trials, Manduca sexta larvae developed slower on as-hpl plants. The reduced larval consumption and performance, which was not caused by increases in defense responses in as-hpl plants, could be restored to WT levels by the addition of synthetic GLVs, demonstrating that GLVs function as feeding stimulants. Gene expression profiling by cDNA microarray analysis and characterization of several induced defenses in herbivore-elicited as-hpl and as-aos plants revealed differential involvement of JA and GLVs in defense signaling. Elicitation of volatile terpenoids (an indirect defense) requires JA signaling, where as trypsin protease inhibitor elicitation (a direct defense) requires both functional JA and GLV cascades.

  17. Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. VII. Changes in the Plant's Proteome1[W

    PubMed Central

    Giri, Ashok P.; Wünsche, Hendrik; Mitra, Sirsha; Zavala, Jorge A.; Muck, Alexander; Svatoš, Aleš; Baldwin, Ian T.

    2006-01-01

    When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvae's oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plant's proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully

  18. Co(i)-ordinating defenses: NaCOI1 mediates herbivore- induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses.

    PubMed

    Paschold, Anja; Halitschke, Rayko; Baldwin, Ian T

    2007-07-01

    Arabidopsis and tomato plants mutated in the F-box protein COI1 mediating jasmonate (JA) responses are more susceptible to herbivores in laboratory trials, but the exact mechanisms of COI1-mediated resistance are not known. We silenced COI1 by transformation with an inverted repeat construct (ir-coi1) in Nicotiana attenuata, a plant the direct and indirect defenses of which against various herbivores have been well studied. ir-coi1 plants are male sterile and impaired in JA-elicited direct [nicotine, caffeoylputrescine and trypsin proteinase inhibitor (TPI) activity] and indirect (cis-alpha-bergamotene emission) defense responses; responses not elicited by JA treatment (ethylene production and flower TPI activity) were unaffected. Larvae of Manduca sexta, a common herbivore of N. attenuata, gained three times more mass feeding on ir-coi1 than on wild-type (WT) plants in glasshouse experiments. By regularly moving caterpillars to unattacked leaves of the same plant, we demonstrate that larvae on WT plants can grow and consume leaves as fast as those on ir-coi1 plants, a result that underscores the role of COI1 in mediating locally induced resistance in attacked leaves, and the importance of herbivore movement in avoiding the induced defenses of a plant. When transplanted into native habitats in the Great Basin Desert, ir-coi1 plants suffer greatly from damage by the local herbivore community, which includes herbivores not commonly found on N. attenuata WT plants. Choice assays with field-grown plants confirmed the increased attractiveness of ir-coi1 plants for both common and unusual herbivores. We conclude that NaCOI1 is essential for induced resistance in N. attenuata, and that ir-coi1 plants highlight the benefits of herbivore movement for avoiding induced defenses.

  19. Development and validation of a liquid chromatography-electrospray ionization-time-of-flight mass spectrometry method for induced changes in Nicotiana attenuata leaves during simulated herbivory.

    PubMed

    Gaquerel, Emmanuel; Heiling, Sven; Schoettner, Matthias; Zurek, Gabriela; Baldwin, Ian T

    2010-09-08

    A liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC/ESI-TOF-MS) procedure was developed to characterize changes induced in Nicotiana attenuata leaves 1 h and 5 days after wounding and application of Manduca sexta elicitors. The constancy of the measurement conditions was first confirmed for 22 selected analytes spanning the entire chromatogram. Using the Profile Analysis software, we extracted 367 buckets, which were analyzed by principal component analysis and two-factorial ANOVA. One hundred seventy-three buckets were found to be statistically regulated, 128 due to time effects, and 85 due to treatment effects. In vivo 15N-isotope labeling was used to facilitate the annotation and the interpretation of the fragmentation pattern of nitrogen-containing metabolites, and a correlation analysis was performed to test mathematical relationships existing among potential in-source fragments. Additionally, tandem MS measurements of the most regulated ions are presented. Altogether, this study defines a framework for the mining and annotation of major herbivory-elicited changes in Nicotiana attenuata.

  20. Alternative oxidase in resistance to biotic stresses: Nicotiana attenuata AOX contributes to resistance to a pathogen and a piercing-sucking insect but not Manduca sexta larvae.

    PubMed

    Zhang, Lu; Oh, Youngjoo; Li, Hongyu; Baldwin, Ian T; Galis, Ivan

    2012-11-01

    The role of the alternative respiratory pathway in the protection of plants against biotic stress was examined in transgenic tobacco (Nicotiana attenuata) plants (irAOX) silenced in the expression of ALTERNATIVE OXIDASE (AOX) gene. Wild-type and irAOX plants were independently challenged with (1) chewing herbivores (Manduca sexta), (2) piercing-sucking insects (Empoasca spp.), and (3) bacterial pathogens (Pseudomonas syringae pv tomato DC3000), showing that all these treatments can strongly elicit accumulation of AOX gene transcripts in wild-type plants. When N. attenuata chemical defenses and resistance were examined, irAOX plants showed wild-type levels of defense-related phytohormones, secondary metabolites, and resistance to M. sexta. In contrast, piercing-sucking leafhoppers (Empoasca spp.) caused more leaf damage and induced significantly higher salicylic acid levels in irAOX compared with wild-type plants in the field and/or glasshouse. Subsequently, irAOX plants accumulated lower levels of defense metabolites, 17-hydroxygeranyllinalool diterpene glycosides, caffeoylputrescine, and nicotine compared with wild-type plants under prolonged attack of Empoasca spp. in the glasshouse. Finally, an accelerated cell death phenotype was observed in irAOX plants infected with P. syringae, which correlated with higher levels of salicylic acid and hydrogen peroxide levels in pathogen-infected irAOX compared with wild-type leaves. Overall, the AOX-associated changes in phytohormone and/or redox levels appear to support the resistance of N. attenuata plants against cell piercing-sucking insects and modulate the progression of cell death in pathogen-infected tissues but are not effective against rapidly feeding specialist herbivore M. sexta.

  1. RNA-directed RNA polymerase 1 (RdR1) mediates the resistance of Nicotiana attenuata to herbivore attack in nature.

    PubMed

    Pandey, Shree P; Baldwin, Ian T

    2007-04-01

    Small RNAs are important regulators of plant development and resistance to viruses. To determine whether small RNAs mediate defense responses to herbivore attack, we silenced the expression of three RNA-directed RNA polymerases (RdRs) in the native tobacco Nicotiana attenuata by virus-induced gene silencing. Larvae of the leaf-chewing solanaceous specialist Manduca sexta grew faster on the RdR1-silenced plants than on empty vector (EV) controls; silencing RdR3 and 2 had little to no effect on larval performance. NaRdR1 transcripts were strongly elicited when puncture wounds were treated with M. sexta oral secretions (OS) to simulate herbivore attack, and with SA and JA, phytohormones that are elicited by herbivore attack. Stably silencing RdR1 by transforming N. attenuata with an inverted-repeat RdR1 construct produced plants (irRdR1) that grew normally but were highly susceptible to both M. sexta larvae and the cell-content-feeder Tupiocoris notatus. When irRdR1 lines were planted into N. attenuata's native habitat in the Great Basin Desert (Utah, USA), they were highly susceptible to herbivore attack, due to deficiencies in direct rather than indirect defenses. Microarray analysis revealed the downregulation of ADC and ODC genes, which supply substrates for synthesizing the chemical defense compound nicotine; irRdR1 lines failed to accumulate nicotine after attack. We conclude that RdR1 mediates herbivore resistance, and infer that the small RNAs produced by RdR1 are probably involved in orchestrating some of the rapid metabolic adjustments required for plants to survive herbivore attack in their natural habitats. The experiment highlights the value of carrying out 'real-world' tests of gene function early in the discovery process.

  2. SGT1 regulates wounding- and herbivory-induced jasmonic acid accumulation and Nicotiana attenuata's resistance to the specialist lepidopteran herbivore Manduca sexta.

    PubMed

    Meldau, Stefan; Baldwin, Ian T; Wu, Jianqiang

    2011-03-01

    • SGT1 (suppressor of G-two allele of SKP1) is a conserved protein in all eukaryotes and is crucial for resisting pathogens in humans and plants. We studied whether SGT1 is involved in the induced defense response of a native tobacco (Nicotiana attenuata) to its natural herbivore, Manduca sexta. • We diminished NaSGT1 transcription in N. attenuata using virus-induced gene silencing (VIGS) and analysed the induced defense responses after wounding and M. sexta elicitation. • Silencing NaSGT1 highly attenuates wounding- and herbivory-induced amounts of jasmonic acid (JA) and JA-isoleucine but elevates the concentration of salicylic acid. Chemical profiling reveals that NaSGT1-silenced plants are also compromised in their ability to accumulate JA precursors produced in chloroplasts. We show that the reduced JA accumulation in NaSGT1-silenced plants is independent of the elevated salicylic acid levels. NaSGT1-silenced plants have decreased contents of defensive metabolites and have compromised resistance to M. sexta larvae. Transcript analyses after methyl jasmonate (MeJA) treatment revealed that NaSGT1 is important for the normal regulation of MeJA-induced transcriptional responses. • This work demonstrates the importance of SGT1 in the regulatory network that deploys defense responses against herbivores, and highlights the significance of SGT1 in plants' responses to JA.

  3. Jasmonate signaling in the field, part II: insect-guided characterization of genetic variations in jasmonate-dependent defenses of transgenic and natural Nicotiana attenuata populations.

    PubMed

    Gaquerel, Emmanuel; Stitz, Michael; Kallenbach, Mario; Baldwin, Ian T

    2013-01-01

    The introduction of genetically modified plants into natural habitats represents a valuable means to determine organismic level functions of a gene and its effects on a plant's interaction with other organisms. Nicotiana attenuata, a wild tobacco species native of the southwestern USA that grows in the immediate postfire environment, is one of the important host plants for herbivore populations recolonizing recently burned habitats in the Great Basin Desert. Here, we provide detailed guidelines for the analysis, under field conditions, of jasmonate-dependent defense and its impact on the plant's native herbivore community. The procedures are based on the field release of transgenic lines silenced for jasmonate biogenesis, metabolism, or perception to conduct association studies between defense trait expression (secondary metabolite and trypsin proteinase inhibitor accumulation) and insect infestations. Additionally, because some insects have evolved mechanisms to "eavesdrop" on jasmonate signaling when selecting their host plants, we describe how leafhoppers of the species Empoasca, which selectively colonize jasmonate-deficient plants, can be used as "bloodhounds" for identifying natural variations in jasmonate signaling among natural N. attenuata populations.

  4. HSPRO acts via SnRK1-mediated signaling in the regulation of Nicotiana attenuata seedling growth promoted by Piriformospora indica

    PubMed Central

    Schuck, Stefan; Baldwin, Ian T.; Bonaventure, Gustavo

    2013-01-01

    Nicotiana attenuata HSPRO (NaHSPRO) is a negative regulator of seedling growth promoted by the fungus Piriformospora indica. Homologs of NaHSPRO in Arabidopsis thaliana (i.e., AtHSPRO1 and AtHSPRO2) are known to physically interact with the AKINβγ subunit of the SnRK1 complex.2 To investigate whether NaHSPRO is associated with SnRK1 function during the stimulation of seedling growth by P. indica, we studied N. attenuata plants silenced in the expression of NaGAL83 (as-gal83 plants)—a gene that encodes for the regulatory β-subunit of SnRK1—and plants silenced in the expression of both NaHSPRO and NaGAL83 (ir-hspro/as-gal83 plants). The results showed that P. indica differentially stimulated the growth of both as-gal83 and ir-hspro/as-gal83 seedlings compared with control seedlings, with a magnitude similar to that observed in ir-hspro seedlings. Thus, we showed that, similar to NaHSPRO, NaGAL83 is a negative regulator of seedling growth stimulated by P. indica. We propose that the effect of NaHSPRO on seedling growth is associated with SnRK1 signaling. PMID:23333980

  5. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis

    PubMed Central

    Groten, Karin; Pahari, Nabin T.; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T.

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  6. Jasmonate and ppHsystemin Regulate Key Malonylation Steps in the Biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides, an Abundant and Effective Direct Defense against Herbivores in Nicotiana attenuata[W

    PubMed Central

    Heiling, Sven; Schuman, Meredith C.; Schoettner, Matthias; Mukerjee, Purba; Berger, Beatrice; Schneider, Bernd; Jassbi, Amir R.; Baldwin, Ian T.

    2010-01-01

    We identified 11 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) that occur in concentrations equivalent to starch (mg/g fresh mass) in aboveground tissues of coyote tobacco (Nicotiana attenuata) and differ in their sugar moieties and malonyl sugar esters (0-2). Concentrations of HGL-DTGs, particularly malonylated compounds, are highest in young and reproductive tissues. Within a tissue, herbivore elicitation changes concentrations and biosynthetic kinetics of individual compounds. Using stably transformed N. attenuata plants silenced in jasmonate production and perception, or production of N. attenuata Hyp-rich glycopeptide systemin precursor by RNA interference, we identified malonylation as the key biosynthetic step regulated by herbivory and jasmonate signaling. We stably silenced N. attenuata geranylgeranyl diphosphate synthase (ggpps) to reduce precursors for the HGL-DTG skeleton, resulting in reduced total HGL-DTGs and greater vulnerability to native herbivores in the field. Larvae of the specialist tobacco hornworm (Manduca sexta) grew up to 10 times as large on ggpps silenced plants, and silenced plants suffered significantly more damage from herbivores in N. attenuata's native habitat than did wild-type plants. We propose that high concentrations of HGL-DTGs effectively defend valuable tissues against herbivores and that malonylation may play an important role in regulating the distribution and storage of HGL-DTGs in plants. PMID:20081114

  7. Silencing two herbivory-activated MAP kinases, SIPK and WIPK, does not increase Nicotiana attenuata's susceptibility to herbivores in the glasshouse and in nature.

    PubMed

    Meldau, Stefan; Wu, Jianqiang; Baldwin, Ian T

    2009-01-01

    Salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) are activated by Manduca sexta attack and elicitors to mediate defense signaling in Nicotiana attenuata. Here, the ecological consequences of SIPK and WIPK silencing for N. attenuata's resistance to M. sexta and its other native herbivores were analyzed. Stably transformed plants with reduced expression of NaSIPK (irNaSIPK) and NaWIPK(irNaWIPK) were generated and characterized in field and glasshouse experiments. Both irNaSIPK and irNaWIPK plants had reduced direct and indirect defenses but were not particularly susceptible in nature. In the glasshouse, M. sexta larvae consumed less and gained the same mass on irNaSIPK and irNaWIPK as on wild-type (WT) plants. Green leaf volatile (GLV) emission was highly attenuated in irNaSIPK and irNaWIPK plants, and complementation with synthetic GLVs increased M. sexta performance. To test the hypothesis that reduced GLV emissions account for the lack of herbivory phenotype, GLV emissions were attenuated by silencing NaHPL in jasmonate-deficient plants (asNaLOX3), which are highly susceptible to herbivores. Reducing GLV emissions in asNaLOX3 plants 'rescued' these plants from being heavily damaged by M. sexta. GLV emissions in irNaSIPK and irNaWIPK plants may compensate for the impaired defenses of NaSIPK- and NaWIPK-silenced plants in nature by reducing their apparency to herbivores.

  8. Dimethyl Disulfide Produced by the Naturally Associated Bacterium Bacillus sp B55 Promotes Nicotiana attenuata Growth by Enhancing Sulfur Nutrition[W

    PubMed Central

    Meldau, Dorothea G.; Meldau, Stefan; Hoang, Long H.; Underberg, Stefanie; Wünsche, Hendrik; Baldwin, Ian T.

    2013-01-01

    Bacillus sp B55, a bacterium naturally associated with Nicotiana attenuata roots, promotes growth and survival of wild-type and, particularly, ethylene (ET)–insensitive 35S-ethylene response1 (etr1) N. attenuata plants, which heterologously express the mutant Arabidopsis thaliana receptor ETR1-1. We found that the volatile organic compound (VOC) blend emitted by B55 promotes seedling growth, which is dominated by the S-containing compound dimethyl disulfide (DMDS). DMDS was depleted from the headspace during cocultivation with seedlings in bipartite Petri dishes, and 35S was assimilated from the bacterial VOC bouquet and incorporated into plant proteins. In wild-type and 35S-etr1 seedlings grown under different sulfate (SO4−2) supply conditions, exposure to synthetic DMDS led to genotype-dependent plant growth promotion effects. For the wild type, only S-starved seedlings benefited from DMDS exposure. By contrast, growth of 35S-etr1 seedlings, which we demonstrate to have an unregulated S metabolism, increased at all SO4−2 supply rates. Exposure to B55 VOCs and DMDS rescued many of the growth phenotypes exhibited by ET-insensitive plants, including the lack of root hairs, poor lateral root growth, and low chlorophyll content. DMDS supplementation significantly reduced the expression of S assimilation genes, as well as Met biosynthesis and recycling. We conclude that DMDS by B55 production is a plant growth promotion mechanism that likely enhances the availability of reduced S, which is particularly beneficial for wild-type plants growing in S-deficient soils and for 35S-etr1 plants due to their impaired S uptake/assimilation/metabolism. PMID:23903320

  9. Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature.

    PubMed

    Schuman, Meredith C; Barthel, Kathleen; Baldwin, Ian T

    2012-10-15

    From an herbivore's first bite, plants release herbivory-induced plant volatiles (HIPVs) which can attract enemies of herbivores. However, other animals and competing plants can intercept HIPVs for their own use, and it remains unclear whether HIPVs serve as an indirect defense by increasing fitness for the emitting plant. In a 2-year field study, HIPV-emitting N. attenuata plants produced twice as many buds and flowers as HIPV-silenced plants, but only when native Geocoris spp. predators reduced herbivore loads (by 50%) on HIPV-emitters. In concert with HIPVs, plants also employ antidigestive trypsin protease inhibitors (TPIs), but TPI-producing plants were not fitter than TPI-silenced plants. TPIs weakened a specialist herbivore's behavioral evasive responses to simulated Geocoris spp. attack, indicating that TPIs function against specialists by enhancing indirect defense.DOI:http://dx.doi.org/10.7554/eLife.00007.001.

  10. Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature

    PubMed Central

    Schuman, Meredith C; Barthel, Kathleen; Baldwin, Ian T

    2012-01-01

    From an herbivore's first bite, plants release herbivory-induced plant volatiles (HIPVs) which can attract enemies of herbivores. However, other animals and competing plants can intercept HIPVs for their own use, and it remains unclear whether HIPVs serve as an indirect defense by increasing fitness for the emitting plant. In a 2-year field study, HIPV-emitting N. attenuata plants produced twice as many buds and flowers as HIPV-silenced plants, but only when native Geocoris spp. predators reduced herbivore loads (by 50%) on HIPV-emitters. In concert with HIPVs, plants also employ antidigestive trypsin protease inhibitors (TPIs), but TPI-producing plants were not fitter than TPI-silenced plants. TPIs weakened a specialist herbivore's behavioral evasive responses to simulated Geocoris spp. attack, indicating that TPIs function against specialists by enhancing indirect defense. DOI: http://dx.doi.org/10.7554/eLife.00007.001 PMID:23066503

  11. NaJAZh Regulates a Subset of Defense Responses against Herbivores and Spontaneous Leaf Necrosis in Nicotiana attenuata Plants[C][W][OA

    PubMed Central

    Oh, Youngjoo; Baldwin, Ian T.; Gális, Ivan

    2012-01-01

    The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development. PMID:22496510

  12. The Sesquiterpenes(E)-ß-Farnesene and (E)-α-Bergamotene Quench Ozone but Fail to Protect the Wild Tobacco Nicotiana attenuata from Ozone, UVB, and Drought Stresses

    PubMed Central

    Palmer-Young, Evan C.; Veit, Daniel; Gershenzon, Jonathan; Schuman, Meredith C.

    2015-01-01

    Among the terpenes, isoprene (C5) and monoterpene hydrocarbons (C10) have been shown to ameliorate abiotic stress in a number of plant species via two proposed mechanisms: membrane stabilization and direct antioxidant effects. Sesquiterpene hydrocarbons (C15) not only share the structural properties thought to lend protective qualities to isoprene and monoterpene hydrocarbons, but also react rapidly with ozone, suggesting that sesquiterpenes may similarly enhance tolerance of abiotic stresses. To test whether sesquiterpenes protect plants against ozone, UVB light, or drought, we used transgenic lines of the wild tobacco Nicotiana attenuata. The transgenic plants expressed a maize terpene synthase gene (ZmTPS10) which produced a blend of (E)-ß-farnesene and (E)-α-bergamotene, or a point mutant of the same gene (ZmTPS10M) which produced (E)-ß-farnesene alone,. (E)-ß-farnesene exerted a local, external, and transient ozone-quenching effect in ozone-fumigated chambers, but we found no evidence that enhanced sesquiterpene production by the plant inhibited oxidative damage, or maintained photosynthetic function or plant fitness under acute or chronic stress. Although the sesquiterpenes (E)-ß-farnesene and (E)-α-bergamotene might confer benefits under intermittent heat stress, which was not tested, any roles in relieving abiotic stress may be secondary to their previously demonstrated functions in biotic interactions. PMID:26030663

  13. Deciphering Herbivory-Induced Gene-to-Metabolite Dynamics in Nicotiana attenuata Tissues Using a Multifactorial Approach1[C][W][OA

    PubMed Central

    Gulati, Jyotasana; Kim, Sang-Gyu; Baldwin, Ian T.; Gaquerel, Emmanuel

    2013-01-01

    In response to biotic stresses, such as herbivore attack, plants reorganize their transcriptomes and reconfigure their physiologies not only in attacked tissues but throughout the plant. These whole-organismic reconfigurations are coordinated by a poorly understood network of signal transduction cascades. To explore tissue-based interdependencies in the resistance of Nicotiana attenuata to insect attack, we conducted time-series transcriptome and metabolome profiling of herbivory-elicited source leaves and unelicited sink leaves and roots. To probe the multidimensionality of these molecular responses, we designed a novel approach of combining an extended self-organizing maps-based dimensionality reduction method with bootstrap-based nonparametric analysis of variance models to identify the onset and context of signaling and metabolic pathway activations. We illustrate the value of this analysis by revisiting dynamic changes in the expression of regulatory and structural genes of the oxylipin pathway and by studying nonlinearities in gene-metabolite associations involved in the acyclic diterpene glucoside pathway after selectively extracting modules based on their dynamic response patterns. This novel dimensionality reduction approach is broadly applicable to capture the dynamic rewiring of gene and metabolite networks in experimental design with multiple factors. PMID:23656894

  14. Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata

    PubMed Central

    2012-01-01

    Background Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies. Results We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs. Conclusions We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata’s responses to herbivore attack. PMID:23134682

  15. A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

    PubMed

    Ferrieri, Abigail P; Arce, Carla C M; Machado, Ricardo A R; Meza-Canales, Ivan D; Lima, Eraldo; Baldwin, Ian T; Erb, Matthias

    2015-10-01

    Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

  16. Tissue specific diurnal rhythms of metabolites and their regulation during herbivore attack in a native tobacco, Nicotiana attenuata.

    PubMed

    Kim, Sang-Gyu; Yon, Felipe; Gaquerel, Emmanuel; Gulati, Jyotasana; Baldwin, Ian T

    2011-01-01

    Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock's role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue-specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant's responses to herbivores and mutualists. We propose that diurnal

  17. Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies1[C][W][OA

    PubMed Central

    Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T.

    2012-01-01

    The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants. PMID:22645069

  18. Increased SA in NPR1-silenced plants antagonizes JA and JA-dependent direct and indirect defenses in herbivore-attacked Nicotiana attenuata in nature.

    PubMed

    Rayapuram, Cbgowda; Baldwin, Ian T

    2007-11-01

    The phytohormone jasmonic acid (JA) is known to mediate herbivore resistance, while salicylic acid (SA) and non-expressor of PR-1 (NPR1) mediate pathogen resistance in many plants. Herbivore attack on Nicotiana attenuata elicits increases in JA and JA-mediated defenses, but also increases SA levels and Na-NPR1 transcripts from the plant's single genomic copy. SA treatment of wild-type plants increases Na-NPR1 and Na-PR1 transcripts. Plants silenced in NPR1 accumulation by RNAi (ir-npr1) are highly susceptible to herbivore and pathogen attack when planted in their native habitat in Utah. They are also impaired in their ability to attract Geocorus pallens predators, due to their decreased ability to release cis-alpha-bergamotene, a JA-elicited volatile 'alarm call'. In the glasshouse, Spodoptera exigua larvae grew better on ir-npr1 plants, which had low levels of JA, JA-isoleucine/leucine, lipoxygenase-3 (LOX3) transcripts and JA-elicited direct defense metabolites (nicotine, caffeoyl putrescine and rutin), but high levels of SA and isochorismate synthase (ICS) transcripts, suggesting de novo biosynthesis of SA. A microarray analysis revealed downregulation of many JA-elicited genes and upregulation of SA biosynthetic genes. JA treatment restored nicotine levels and resistance to S. exigua in ir-npr1 plants. We conclude that, during herbivore attack, NPR1 negatively regulates SA production, allowing the unfettered elicitation of JA-mediated defenses; when NPR1 is silenced, the elicited increases in SA production antagonize JA and JA-related defenses, making the plants susceptible to herbivores.

  19. ISSR and AFLP analysis of the temporal and spatial population structure of the post-fire annual, Nicotiana attenuata, in SW Utah

    PubMed Central

    Bahulikar, Rahul A; Stanculescu, Dominic; Preston, Catherine A; Baldwin, Ian T

    2004-01-01

    Background The native annual tobacco, Nicotiana attenuata, is found primarily in large ephemeral populations (typically for less than 3 growing seasons) after fires in sagebrush and pinyon-juniper ecosystems and in small persistent populations (for many growing seasons) in isolated washes typically along roadsides throughout the Great Basin Desert of the SW USA. This distribution pattern is due to its unusual germination behavior. Ephemeral populations are produced by the germination of dormant seeds from long-lived seed banks which are stimulated to germinate by a combination of unidentified positive cues found in wood smoke and the removal of inhibitors leached from the unburned litter of the dominant vegetation. Persistent populations may result where these inhibitors do not exist, as in washes or along disturbed roadsides. To determine if this germination behavior has influenced population structure, we conducted an AFLP (244 individuals), ISSR (175 individuals) and ISSR+ AFLP (175 individuals) analysis on plants originating from seed collected from populations growing in 11 wash and burns over 11 years from the SW USA. Results Genetic variance as measured by both ISSR and AFLP markers was low among sites and comparatively higher within populations. Cluster analysis of the Utah samples with samples collected from Arizona, California, and Oregon as out-groups also did not reveal patterns. AMOVA analysis of the combined AFLP and ISSR data sets yielded significantly low genetic differentiation among sites (Φct), moderate among populations within sites (Φsc) and higher genetic differentiation within populations (Φst). Conclusions We conclude that the seed dormancy of this post-fire annual and its resulting age structure in conjunction with natural selection processes are responsible for significantly low among sites and comparatively high within-population genetic variation observed in this species. PMID:15350209

  20. Nicotiana attenuata SIPK, WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway1[W][OA

    PubMed Central

    Kallenbach, Mario; Alagna, Fiammetta; Baldwin, Ian Thomas; Bonaventure, Gustavo

    2010-01-01

    Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants. PMID:19897603

  1. Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata: V. Microarray Analysis and Further Characterization of Large-Scale Changes in Herbivore-Induced mRNAs1

    PubMed Central

    Hui, Dequan; Iqbal, Javeed; Lehmann, Katja; Gase, Klaus; Saluz, Hans Peter; Baldwin, Ian T.

    2003-01-01

    We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, is attacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerase chain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome. These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northern analysis on glass slide microarrays, and hybridized with Cy3- and Cy5-labeled probes derived from plants after 2, 6, 12, and 24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of the cloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcription-polymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins (more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defenses used against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, and oxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses. These defense responses require resources that could be made available by decreases in four photosynthetic-related transcripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associated with carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associated transcripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators, chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration that underscores the complexity of response to herbivore attack

  2. R2R3-NaMYB8 Regulates the Accumulation of Phenylpropanoid-Polyamine Conjugates, Which Are Essential for Local and Systemic Defense against Insect Herbivores in Nicotiana attenuata1[W][OA

    PubMed Central

    Kaur, Harleen; Heinzel, Nicolas; Schöttner, Mathias; Baldwin, Ian T.; Gális, Ivan

    2010-01-01

    Although phenylpropanoid-polyamine conjugates (PPCs) occur ubiquitously in plants, their biological roles remain largely unexplored. The two major PPCs of Nicotiana attenuata plants, caffeoylputrescine (CP) and dicaffeoylspermidine, increase dramatically in local and systemic tissues after herbivore attack and simulations thereof. We identified NaMYB8, a homolog of NtMYBJS1, which in BY-2 cells regulates PPC biosynthesis, and silenced its expression by RNA interference in N. attenuata (ir-MYB8), to understand the ecological role(s) of PPCs. The regulatory role of NaMYB8 in PPC biosynthesis was validated by a microarray analysis, which revealed that transcripts of several key biosynthetic genes in shikimate and polyamine metabolism accumulated in a NaMYB8-dependent manner. Wild-type N. attenuata plants typically contain high levels of PPCs in their reproductive tissues; however, NaMYB8-silenced plants that completely lacked CP and dicaffeoylspermidine showed no changes in reproductive parameters of the plants. In contrast, a defensive role for PPCs was clear; both specialist (Manduca sexta) and generalist (Spodoptera littoralis) caterpillars feeding on systemically preinduced young stem leaves performed significantly better on ir-MYB8 plants lacking PPCs compared with wild-type plants expressing high levels of PPCs. Moreover, the growth of M. sexta caterpillars was significantly reduced when neonates were fed ir-MYB8 leaves sprayed with synthetic CP, corroborating the role of PPCs as direct plant defense. The spatiotemporal accumulation and function of PPCs in N. attenuata are consistent with the predictions of the optimal defense theory: plants preferentially protect their most fitness-enhancing and vulnerable parts, young tissues and reproductive organs, to maximize their fitness. PMID:20089770

  3. Alternative Oxidase in Resistance to Biotic Stresses: Nicotiana attenuata AOX Contributes to Resistance to a Pathogen and a Piercing-Sucking Insect But Not Manduca sexta Larvae1[W][OA

    PubMed Central

    Zhang, Lu; Oh, Youngjoo; Li, Hongyu; Baldwin, Ian T.; Galis, Ivan

    2012-01-01

    The role of the alternative respiratory pathway in the protection of plants against biotic stress was examined in transgenic tobacco (Nicotiana attenuata) plants (irAOX) silenced in the expression of ALTERNATIVE OXIDASE (AOX) gene. Wild-type and irAOX plants were independently challenged with (1) chewing herbivores (Manduca sexta), (2) piercing-sucking insects (Empoasca spp.), and (3) bacterial pathogens (Pseudomonas syringae pv tomato DC3000), showing that all these treatments can strongly elicit accumulation of AOX gene transcripts in wild-type plants. When N. attenuata chemical defenses and resistance were examined, irAOX plants showed wild-type levels of defense-related phytohormones, secondary metabolites, and resistance to M. sexta. In contrast, piercing-sucking leafhoppers (Empoasca spp.) caused more leaf damage and induced significantly higher salicylic acid levels in irAOX compared with wild-type plants in the field and/or glasshouse. Subsequently, irAOX plants accumulated lower levels of defense metabolites, 17-hydroxygeranyllinalool diterpene glycosides, caffeoylputrescine, and nicotine compared with wild-type plants under prolonged attack of Empoasca spp. in the glasshouse. Finally, an accelerated cell death phenotype was observed in irAOX plants infected with P. syringae, which correlated with higher levels of salicylic acid and hydrogen peroxide levels in pathogen-infected irAOX compared with wild-type leaves. Overall, the AOX-associated changes in phytohormone and/or redox levels appear to support the resistance of N. attenuata plants against cell piercing-sucking insects and modulate the progression of cell death in pathogen-infected tissues but are not effective against rapidly feeding specialist herbivore M. sexta. PMID:22961128

  4. Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. I. Large-Scale Changes in the Accumulation of Growth- and Defense-Related Plant mRNAs1

    PubMed Central

    Hermsmeier, Dieter; Schittko, Ursula; Baldwin, Ian T.

    2001-01-01

    Plants respond to herbivore attack with a dramatic functional reorganization that involves the activation of direct and indirect defenses and tolerance, which in turn make large demands on primary metabolism. Here we provide the first characterization of the transcriptional reorganization that occurs after insect attack in a model plant-herbivore system: Nicotiana attenuata Torr. ex Wats.-Manduca sexta. We used mRNA differential display to characterize one-twentieth of the insect-responsive transcriptome of N. attenuata and verified differential expression for 27 cDNAs. Northern analyses were used to study the effects of folivory and exposure to airborne methyl jasmonate and for kinetic analyses throughout a 16-h- light/8-h-dark cycle. Sequence similarity searches allowed putative functions to be assigned to 15 transcripts. Genes were related to photosynthesis, electron transport, cytoskeleton, carbon and nitrogen metabolism, signaling, and a group responding to stress, wounding, or invasion of pathogens. Overall, transcripts involved in photosynthesis were strongly down-regulated, whereas those responding to stress, wounding, and pathogens and involved in shifting carbon and nitrogen to defense were strongly up-regulated. The majority of transcripts responded similarly to airborne methyl jasmonate and folivory, and had tissue- and diurnal-specific patterns of expression. Transcripts encoding Thr deaminase (TD) and a putative retrotransposon were absent in control plants, but were strongly induced after herbivory. Full-length sequences were obtained for TD and the pathogen-inducible α-dioxygenase, PIOX. Effects of abiotic and biotic stimuli were investigated for transcripts encoding TD, importin α, PIOX, and a GAL83-like kinase cofactor. PMID:11161026

  5. RuBPCase activase mediates growth-defense tradeoffs: Silencing RCA redirects JA flux from JA-Ile to MeJA to attenuate induced defense responses in Nicotiana attenuata

    PubMed Central

    Mitra, Sirsha; Baldwin, Ian T.

    2014-01-01

    Summary RuBPCase activase (RCA), an abundant photosynthetic protein is strongly down-regulated in response to Manduca sexta’s oral secretion (OS) in Nicotiana attenuata. RCA-silenced plants are impaired not only in photosynthetic capacity and growth, but also in jasmonic acid (JA)-isoleucine (Ile) signaling, and herbivore resistance mediated by JA-Ile dependent defense traits. These responses are consistent with a resource-based growth-defense trade-off. Since JA+Ile-supplementation of OS restored WT levels of JA-Ile, defenses and resistance to M. sexta, but OS supplemented individually with JA- or Ile did not, the JA-Ile deficiency of RCA-silenced plants could not be attributed to lower JA or Ile pools or JAR4/6 conjugating activity. Similar levels of JA-Ile derivatives after OS elicitation indicated unaltered JA-Ile turnover and lower levels of other JA-conjugates ruled out competition from other conjugation reactions. RCA-silenced plants accumulated more methyl jasmonate (MeJA) after OS elicitation, which corresponded with increased jasmonate methyltransferase (JMT) activity. RCA-silencing phenocopies JMT over-expression, wherein elevated JMT activity redirects OS-elicited JA flux towards inactive MeJA, creating a JA sink which depletes JA-Ile and its associated defense responses. Hence RCA plays an additional non-photosynthetic role in attenuating JA-mediated defenses and their associated costs potentially allowing plants to anticipate resource-based constraints on growth before they actually occur. PMID:24491116

  6. Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents

    PubMed Central

    Mhlongo, Msizi I.; Steenkamp, Paul A.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites. PMID

  7. Antihyperglycemic activity of Caralluma attenuata.

    PubMed

    Venkatesh, S; Reddy, G Dayanand; Reddy, B Madhava; Ramesh, M; Rao, A V N Appa

    2003-04-01

    Extracts of Caralluma attenuata are screened for their antihyperglycemic activity. Ethanol, chloroform and butanol extracts were tested on glucose loaded and alloxan-induced diabetic rats. In both tests, the butanol extract, at the oral dose of 250 mg/kg, has shown statistically significant and considerable antihyperglycemic activity.

  8. Phytoplasma SAP11 alters 3-isobutyl-2-methoxypyrazine biosynthesis in Nicotiana benthamiana by suppressing NbOMT1

    PubMed Central

    Tan, Choon Meng; Li, Chia-Hua; Tsao, Nai-Wen; Su, Li-Wen; Lu, Yen-Ting; Chang, Shu Heng; Lin, Yi Yu; Liou, Jyun-Cyuan; Hsieh, Li-Ching; Yu, Jih-Zu; Sheue, Chiou-Rong; Wang, Sheng-Yang; Lee, Chin-Fa; Yang, Jun-Yi

    2016-01-01

    Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana. In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana. These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles. PMID:27279277

  9. Drought stress alters water relations and expression of PIP-type aquaporin genes in Nicotiana tabacum plants.

    PubMed

    Mahdieh, Majid; Mostajeran, Akbar; Horie, Tomoaki; Katsuhara, Maki

    2008-05-01

    Plasma membrane intrinsic proteins (PIPs), a type of aquaporins, mediate water transport in many plant species. In this study, we investigated the relationship between the functions of PIP-type water channels and water relations of tobacco plants (Nicotiana tabacum cv. Samsun) under drought stress. Drought stress treatments have led to reductions in the stomatal conductance, transpiration, water potential and turgor pressure in leaves, and also the sap flow rate and osmotic hydraulic conductance in roots. In contrast, leaf osmotic pressure was increased in response to drought stress. Interestingly, the accumulation of NtPIP1;1 and NtPIP2;1 transcripts was significantly decreased, but only that of the NtAQP1 transcript was increased under drought stress. Functional analysis using Xenopus laevis oocytes revealed that NtPIP2;1 shows marked water transport activity, but the activities of NtAQP1 and NtPIP1;1 are weak or almost negligible, respectively, when expressed alone. However, co-expression of NtPIP1;1 with NtPIP2;1 significantly enhanced water transport activity compared with that of NtPIP1;1- or NtPIP2;1-expressing oocytes, suggesting that these two aquaporins may function as a water channel, forming a heterotetramer. Heteromerization of NtPIP1;1 and NtPIP2;1 was also suggested by co-expression analyses of NtPIP1;1-GFP (green fluorescent protein) and NtPIP2;1 in Xenopus oocytes. Re-watering treatments recovered water relation parameters and the accumulation of the three NtPIP transcripts to levels similar to control conditions. These results suggest that NtPIP1;1 and NtPIP2;1 play an important role in water transport in roots, and that expression of NtPIP1;1 and NtPIP2;1 is down-regulated in order to reduce osmotic hydraulic conductance in the roots of tobacco plants under drought stress.

  10. A new steroidal saponin from Agave attenuata.

    PubMed

    Mendes, Tatiana Paz; Silva, Graziela de Medeiros; da Silva, Bernadete Pereira; Parente, José Paz

    2004-04-01

    A new steroidal saponin was isolated from the leaves of Agave attenuata. Its structure was established as (3beta,beta,25S)-spirostan-3-yl O-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 2)-O-[beta-D-glucopyranosyl-(1 --> 3)]-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside. The structural identification was performed using detailed analyses of 1H- and 13C-NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR, and COLOC) and chemical conversions. The hemolytic activity of the steroidal saponin was evaluated using an in vitro assay.

  11. Organophosphate nerve agent toxicity in Hydra attenuata.

    PubMed

    Lum, Karin T; Huebner, Henry J; Li, Yingchun; Phillips, Timothy D; Raushel, Frank M

    2003-08-01

    The toxicity for analogues of sarin (GB), soman (GD), and VX was evaluated using Hydra attenuata as a model organism. The organophosphate nerve agent analogue simulants used in this investigation included the following: isopropyl p-nitrophenyl methylphosphonate (for GB); pinacolyl p-nitrophenyl methylphosphonate (for GD); and diisopropyl S-(2-diisopropylaminoethyl)phosphorothioate, diethyl S-(2-diisopropylaminoethyl)phosphorothioate, and diethyl S-(2-trimethylaminoethyl)phosphorothioate (for VX). The toxicity of each organophosphate nerve agent was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There is a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate chemical warfare agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogues was 2 orders of magnitude more toxic than the analogue for GD and 4 orders of magnitude more toxic than the analogue for GB.

  12. Ectopic expression of class 1 KNOX genes induce and adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...

  13. Phytochemical and Biological Studies of Agave attenuata

    PubMed Central

    Rizwan, Komal; Zubair, Muhammad; Rasool, Nasir; Riaz, Muhammad; Zia-Ul-Haq, Muhammad; de Feo, Vincenzo

    2012-01-01

    The present study was conducted to examine various biological activities of a methanol extract of Agave attenuata leaves. GC-MS analysis of the n-hexane fraction from the extract revealed the presence of 31 compounds, with mono-2-ethylhexyl phthalate (11.37%), 1,2-benzenedicarboxylic acid (6.33%), n-docosane (6.30%) and eicosane (6.02%) as the major components. The leaves contained appreciable levels of total phenolic contents (10.541–39.35 GAE, mg/100 g) and total flavonoid contents (43.35–304.8 CE, mg/100 g). The extract and some of its fractions showed moderate antimicrobial effects. Leaves extract and fractions also exhibited a good antioxidant potential when measured by DPPH radical scavenging activity and inhibition of lipid peroxidation assays. The hemolytic effect of the plant was found to be in a range of 1.01%–2.64%. From the present study it is concluded that this plant could be used as a source of natural antioxidants and functional food nutraceutical applications. PMID:22754375

  14. Phytochemical and biological studies of Agave attenuata.

    PubMed

    Rizwan, Komal; Zubair, Muhammad; Rasool, Nasir; Riaz, Muhammad; Zia-Ul-Haq, Muhammad; de Feo, Vincenzo

    2012-01-01

    The present study was conducted to examine various biological activities of a methanol extract of Agave attenuata leaves. GC-MS analysis of the n-hexane fraction from the extract revealed the presence of 31 compounds, with mono-2-ethylhexyl phthalate (11.37%), 1,2-benzenedicarboxylic acid (6.33%), n-docosane (6.30%) and eicosane (6.02%) as the major components. The leaves contained appreciable levels of total phenolic contents (10.541-39.35 GAE, mg/100 g) and total flavonoid contents (43.35-304.8 CE, mg/100 g). The extract and some of its fractions showed moderate antimicrobial effects. Leaves extract and fractions also exhibited a good antioxidant potential when measured by DPPH radical scavenging activity and inhibition of lipid peroxidation assays. The hemolytic effect of the plant was found to be in a range of 1.01%-2.64%. From the present study it is concluded that this plant could be used as a source of natural antioxidants and functional food nutraceutical applications.

  15. Sex ratio of mirid populations shifts in response to hostplant co-infestation or altered cytokinin signaling .

    PubMed

    Adam, Nora; Erler, Theresa; Kallenbach, Mario; Kaltenpoth, Martin; Kunert, Grit; Baldwin, Ian T; Schuman, Meredith C

    2017-01-01

    Herbivore species sharing a host plant often compete. In this study, we show that host plant-mediated interaction between two insect herbivores - a generalist and a specialist - results in a sex ratio shift of the specialist's offspring. We studied demographic parameters of the specialist Tupiocoris notatus (Hemiptera: Miridae) when co-infesting the host plant Nicotiana attenuata (Solanaceae) with the generalist leafhopper Empoasca sp. (Hemiptera: Cicadellidae). We show that the usually female-biased sex ratio of T. notatus shifts toward a higher male proportion in the offspring on plants co-infested by Empoasca sp. This sex ratio change did not occur after oviposition, nor is it due differential mortality of female and male nymphs. Based on pyrosequencing and PCR of bacterial 16S rRNA amplicons, we concluded that sex ratio shifts were unlikely to be due to infection with Wolbachia or other known sex ratio-distorting endosymbionts. Finally, we used transgenic lines of N. attenuata to evaluate if the sex ratio shift could be mediated by changes in general or specialized host plant metabolites. We found that the sex ratio shift occurred on plants deficient in two cytokinin receptors (irCHK2/3). Thus, cytokinin-regulated traits can alter the offspring sex ratio of the specialist T. notatus.

  16. Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

    PubMed

    Srinivasan, C; Liu, Zongrang; Scorza, Ralph

    2011-04-01

    Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

  17. Multi-scale biomarker evaluation of the toxicity of a commercial azo dye (Disperse Red 1) in an animal model, the freshwater cnidarian Hydra attenuata.

    PubMed

    de Jong, Laetitia; Pech, Nicolas; de Aragão Umbuzeiro, Gisela; Moreau, Xavier

    2016-06-01

    Acute (24 h, 48 h, 72 h) and chronic (7 days) tests have been performed to evaluate the effects of the commercial azo dye Disperse Red 1 (DR1) using various biomarkers in the freshwater invertebrate Hydra attenuata. Morphological changes have been selected to calculate ecotoxicological thresholds for sublethal and lethal DR1 concentrations. A multinomial logistic model showed that the probability of each morphological stage occurrence was function of concentration, time and interaction between both. Results of oxidative balance parameter measurements (72 h and 7 days) suggest that polyps set up defense mechanisms to limit lipid peroxidation caused by DR1. DR1 exposure at hormetic concentrations induces increase of asexual reproductive rates. This result suggests (1) an impact on the fitness-related phenotypical traits and (2) trade-offs between reproduction and maintenance to allow the population to survive harsher conditions. Changes in serotonin immuno-labeling in polyps showing alterations in feeding behavior suggest that chronic DR1 exposure impaired neuronal processes related to ingesting behavior in H. attenuata. This ecotoxicity study sheds light on the possible serotonin function in Hydra model and reports for the first time that serotonin could play a significant role in feeding behavior. This study used a multi-scale biomarker approach investigating biochemical, morphological, reproductive and behavioral endpoints in Hydra attenuata. This organism is proposed for a pertinent animal model to assess ecotoxicological impact of pollutant mixtures in freshwater environment.

  18. A new bioactive steroidal saponin from Agave attenuata.

    PubMed

    da Silva, Bernadete P; de Sousa, Allyne C; Silva, Graziela M; Mendes, Tatiana P; Parente, José P

    2002-01-01

    A new steroidal saponin was isolated from the leaves of Agave attenuata Salm-Dyck. Its structure was established as (3beta,5beta,22alpha,25S)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurostan-3-yl O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside. The structural identification was performed using detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR and COLOC) and chemical conversions. The haemolytic potential of the steroidal saponin was evaluated and the anti-inflammatory activity was performed using the capillary permeability assay.

  19. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  20. Ecological divergence and evolutionary transition of resprouting types in Banksia attenuata.

    PubMed

    He, Tianhua

    2014-08-01

    Resprouting is a key functional trait that allows plants to survive diverse disturbances. The fitness benefits associated with resprouting include a rapid return to adult growth, early flowering, and setting seed. The resprouting responses observed following fire are varied, as are the ecological outcomes. Understanding the ecological divergence and evolutionary pathways of different resprouting types and how the environment and genetics interact to drive such morphological evolution represents an important, but under-studied, topic. In the present study, microsatellite markers and microevolutionary approaches were used to better understand: (1) whether genetic differentiation is related to morphological divergence among resprouting types and if so, whether there are any specific genetic variations associated with morphological divergence and (2) the evolutionary pathway of the transitions between two resprouting types in Banksia attenuata (epicormic resprouting from aerial stems or branch; resprouting from a underground lignotuber). The results revealed an association between population genetic differentiation and the morphological divergence of postfire resprouting types in B. attenuata. A microsatellite allele has been shown to be associated with epicormic populations. Approximate Bayesian Computation analysis revealed a likely evolutionary transition from epicormic to lignotuberous resprouting in B. attenuata. It is concluded that the postfire resprouting type in B. attenuata is likely determined by the fire's characteristics. The differentiated expression of postfire resprouting types in different environments is likely a consequence of local genetic adaptation. The capacity to shift the postfire resprouting type to adapt to diverse fire regimes is most likely the key factor explaining why B. attenuata is the most widespread member of the Banksia genus.

  1. Functional interactions in the use of direct and indirect defences in native Nicotiana plants.

    PubMed

    Baldwin, I T

    1999-01-01

    Nicotiana attenuata has both direct (induced nicotine production) and indirect (induced release of mono- and sesquiterpenes) defences induced by herbivore attack; both are activated by the jasmonate cascade, albeit in different tissues (roots and shoots, respectively). The fact that both types of defences are induced suggests that their benefits are conditional. Indeed, jasmonate treatment of roots to induce nicotine production increases plant fitness correlates (lifetime viable seed production) when plants are grown in environments with herbivores, but decreases fitness when they are not. Because inducing nicotine production can make 6% of a plant's nitrogen budget unavailable for seed production, it can exact a resource-based cost. Volatile production is likely to be less costly but could make plants more 'apparent' to herbivores and thereby exact an ecological cost. Direct defences could also have ecological costs if they are sequestered by specialist herbivores and used against their enemies. Herbivory by the nicotine-tolerant herbivore Manduca sexta dramatically amplifies the increase in jasmonates and the quantity of volatiles released, but decreases the nicotine response in comparison to mechanical simulations of the wounding that larval feeding causes. The apparent switching from nicotine production to the release of volatiles may reflect incompatibilities in the use of direct and indirect defences with specialist herbivores.

  2. How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by peanut stunt virus in the presence or absence of its satellite RNA.

    PubMed

    Obrępalska-Stęplowska, Aleksandra; Wieczorek, Przemysław; Budziszewska, Marta; Jeszke, Arnika; Renaut, Jenny

    2013-07-01

    Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains associated with a satellite RNA (satRNA) modify the symptoms of infected plants and interfere with plant metabolism. We used PSV-P genomic transcripts (GTs) with and without PSV-P satRNA and a comparative proteomic 2D-DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV-P genomic transcripts-infected (no satRNA present) and mock-inoculated plants were compared 29 differentially regulated proteins were found. When comparisons were made for plants infected with PSV-P-GT in the presence or absence of satRNA, and for mock-infected plants and those infected with the satRNA-associated PSV-P-GT, 40 and 60 such proteins, respectively, were found. The presence of satRNA mostly decreased the amounts of the affected host proteins. Proteins involved in photosynthesis and carbohydrate metabolism, for example ferredoxin-NADP-reductase and malate dehydrogenase, are among the identified affected proteins in all comparisons. Proteins involved in protein synthesis and degradation were also affected. Such proteins include chaperonin 60β--whose abundance of the proteins changed for all comparisons--and aminopeptidase that is a satRNA- or PSV-P-GT/satRNA-responsive protein. Additionally, the levels of the stress-related proteins superoxide dismutase and acidic endochitinase Q increased in the PSV-P-GT- and PSV-P-GT/satRNA-infected plants. This study appears to be the first report on plant proteome changes in response to a satRNA presence during viral infection and, as such, may provide a reference for future studies concerning the influence of satRNAs during viral infections.

  3. Effect of Caralluma attenuata in normal and alloxan induced diabetic rats.

    PubMed

    Jayakar, B; Rajkapoor, B; Suresh, B

    2004-01-01

    The hypoglycemic effect of aqueous and alcoholic extracts of whole plant Caralluma attenuata was investigated in both normal and alloxan induced diabetic rats. The blood glucose levels were measured at 0, 1, 2 and 3 h after the treatment. The aqueous and alcoholic extracts of C. attenuata (100 mg/kg) reduced the blood glucose level of normal rat from 84.5 +/- 2.0 to 68.5 +/- 2.8 mg/dl and 82.6 +/- 1.8 to 67.4 +/- 2.8 mg/dl, respectively, 3h after oral administration (P < 0.001). Blood glucose levels in alloxan induced diabetic rats were also significantly lowered from 316 +/- 3.32 to 162 +/- 2.76 and 324 +/- 4.10 to 150 +/- 3.94 mg/dl, respectively, 3 h after oral administration (P < 0.001).

  4. Ulcer healing potential of ethanolic extract of Caralluma attenuata on experimental diabetic rats

    PubMed Central

    Garg, Sunil; Srivastava, Sajal; Singh, Kisanpal; Sharma, Alok; Garg, Kavita

    2016-01-01

    Introduction: Available data indicated that diabetes mellitus (DM) increases the vulnerability of the gastric ulcers and the need of the hour is to develop effective agents to treat ulcer with diabetes for better patient compliance and cost effectiveness. The ulcer-healing properties of ethanolic extract of Caralluma attenuata (CAEt) against both chemically- and physically induced gastric ulcers in experimental rats are recently studied. Aim: To assess the ulcer healing potential of Ethanolic Extract of Caralluma attenuata on Experimental Diabetic Rats. Material and Methods: The current study aimed to evaluate ulcer healing properties of CAEt on the aspirin induced gastric ulcer in rats with streptozotocin induced DM. The hypothesis is based on the fact that DM results in compromising the mucosal defensive factors associated with delay in gastric ulcer healing, and if these changes can be corrected by using agents known for their antidiabetic and antiulcer properties. Experimental albino rats were divided into six groups. Except for Group I, other groups contained streptozotocin-induced diabetic rats. Group I (normal control) and Group II (diabetic control) were administered vehicle, Groups III and IV (diabetic experimental) were administered CAEt in dose of 100 mg/kg and 250 mg/kg, respectively, and Groups V and VI (positive controls) were respectively administered oral standard drugs omeprazole, 20 mg/kg, and tolbutamide 10 mg/kg. Result: The results confirmed that the CAEt significantly decreases the ulcer index (P < 0.05) in the aspirin-induced gastric ulcers and also significantly exhibit antioxidant and glucose lowering activity in the diabetic ulcer rats. The study showed that C. attenuata has the potential to be used as an antiulcer agent in experimental diabetic rats. PMID:27621520

  5. Acute toxicity assessment of Polish (waste) water with a microplate-based Hydra attenuata assay: a comparison with the Microtox test.

    PubMed

    Pardos, M; Benninghoff, C; Guéguen, C; Thomas, R; Dobrowolski, J; Dominik, J

    1999-12-15

    The use of Hydra attenuata in acute toxicity assessment is a potentially useful tool in (waste) water biomonitoring. The purpose of this study was to compare the sensitivity of H. attenuata with the extensively used Microtox test on 14 (waste) water samples from the Kraków region (South Poland). To this end, specific morphological changes displayed by the freshwater cnidarian Hydra attenuata (lethal LC50s and sublethal EC50s effects) and bioluminescence of the marine bacteria Vibrio fisheri (Microtox) were compared. Clearly, the Hydra assay was the more sensitive indicator of toxicity. No relationship was found among Hydra toxicological responses and water levels of As, Cd, Co, Cu, Pb and Zn. However, it appeared that toxicity to Hydra might be due to ammonia levels. Additional studies to better circumscribe the tolerance of H. attenuata to 'natural' water characteristics are needed.

  6. Pithy Protection: Nicotiana attenuata’s Jasmonic Acid-Mediated Defenses Are Required to Resist Stem-Boring Weevil Larvae1[W][OA

    PubMed Central

    Diezel, Celia; Kessler, Danny; Baldwin, Ian T.

    2011-01-01

    Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C6 aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant’s apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata’s fitness. PMID:21300916

  7. Source parameter estimates of echolocation clicks from wild pygmy killer whales (Feresa attenuata) (L)

    NASA Astrophysics Data System (ADS)

    Madsen, P. T.; Kerr, I.; Payne, R.

    2004-10-01

    Pods of the little known pygmy killer whale (Feresa attenuata) in the northern Indian Ocean were recorded with a vertical hydrophone array connected to a digital recorder sampling at 320 kHz. Recorded clicks were directional, short (25 μs) transients with estimated source levels between 197 and 223 dB re. 1 μPa (pp). Spectra of clicks recorded close to or on the acoustic axis were bimodal with peak frequencies between 45 and 117 kHz, and with centroid frequencies between 70 and 85 kHz. The clicks share characteristics of echolocation clicks from similar sized, whistling delphinids, and have properties suited for the detection and classification of prey targeted by this odontocete. .

  8. Development of the skull of the pantropical spotted dolphin (Stenella attenuata).

    PubMed

    Moran, Meghan M; Nummela, Sirpa; Thewissen, J G M

    2011-10-01

    We describe the bony and cartilaginous structures of five fetal skulls of Stenella attenuata (pantropical spotted dolphin) specimens. The specimens represent early fetal life as suggested by the presence of rostral tactile hairs and the beginnings of skin pigmentation. These specimens exhibit the developmental order of ossification of the intramembranous and endochondral elements of the cranium as well as the functional and morphological development of specific cetacean anatomical adaptations. Detailed observations are presented on telescoping, nasal anatomy, and middle ear anatomy. The development of the middle ear ossicles, ectotympanic bone, and median nasal cartilage is of interest because in the adult these structures are morphologically different from those in land mammals. We follow specific cetacean morphological characteristics through fetal development to provide insight into the form and function of the cetacean body plan. Combining these data with fossil evidence, it is possible to overlie ontogenetic patterns and discern evolutionary patterns of the cetacean skull.

  9. Turritella attenuata (Kasinathan): as biological indicator of marine pollution--a trace metal analytical study.

    PubMed

    Paul, V I; Radhakrishnan, M V; Hemalatha, S

    1999-11-01

    A study to monitor marine pollution with reference to trace elements (Fe, Zn, Mn and Cu) on T. attenuata, commonly called as screw shell over a period of one year on the whole body and various organs, viz. digestive diverticula, foot, mantle and ovary was conducted from the sandy beach of Porto Novo Coast (Lat 11 degrees 29' N Long: 79 degrees 46' E) of Peninsular India using Atomic Absorption Spectrophotometer (AAS). Higher concentration of all the four trace metals analysed were recorded in the digestive diverticula, whereas lower concentration of zinc and manganese were recorded in the ovary during the monsoon period. The higher level of trace metal concentration in the monsoon period may be due to the presence of these pollutants in large amounts in water. The accumulation of selected trace metals varies in different seasons according to the extent of pollution load in the marine environment.

  10. First record of Pantropical spotted dolphins Stenella attenuata in the Yellow Sea, China

    NASA Astrophysics Data System (ADS)

    Wu, Fuxing; Wang, Xianyan; Zhang, Qiuxia; Miao, Xing; Zhang, Ting; Zhu, Qian

    2015-07-01

    On October 1, 2009, sixteen dolphins were obtained from fishermen by incidental catching in the Yellow Sea, China. As the dolphins' skin color was ambiguous, morphological parameters were measured, and mitochondrial DNA cytochrome b (Cyt b) gene sequence was studied to identify the species. Morphological characteristics were consistent with Pantropical spotted dolphins, Stenella attenuata. Furthermore, a partial mitochondrial DNA cytochrome b (Cyt b) gene sequence as long as 328-bp was studied by extracting genomic DNA from the skins, and six haplotypes were detected in the sixteen dolphins. By comparing homologous sequences available in GenBank (www.ncbi.nlm.nih.gov), all the six haplotypes had maximal genetic similarity with Pantropical spotted dolphin. Eight species of cetacean (whales and dolphins) are now recognised in the Yellow Sea. To the best of our knowledge, this is the first record of Pantropical spotted dolphins from this region. Despite this species being listed as a Grade II National Key Protected Animal since 1988, little is known of its biology in Chinese waters. We recommend remedial research be undertaken to ensure appropriate management.

  11. Application of the Hydra attenuata assay for identifying developmental hazards among natural waters and wastewaters

    SciTech Connect

    Fu, L.J.; Staples, R.E.; Stahl, R.G. Jr. )

    1991-12-01

    This study concerns application of the Hydra attenuata assay to detect the developmental toxicity potential of various aqueous samples. First, the assay was modified for testing aqueous samples because water quality has a major impact on aquatic toxicity testing and the results thus obtained. Ranges of sample pH, salinity (conductivity), and hardness were examined for their adverse effects upon the hydra. Adult hydra were unaffected morphologically by pH 5.5-9.5, and the artificial embryo ( embryo') developed normally in a pH range of 6.25 to 8.25. For water hardness, the minimal affective concentration was 1000 mg/liter (as CaCO3) in adults and 625 mg/liter in the embryos; the NOAELs for these were 750 mg/liter in the adult and 250 mg/liter CaCO3 in the embryo. Salinity in excess of 5 ppt was lethal to adults and embryos, indicating the assay may not be applicable to marine or highly saline samples. Finally, grab samples were tested from rivers in Maryland, Pennsylvania, New Jersey, and Delaware, some of which are impacted by industrial and agricultural activities, as well as several samples of industrial wastewaters from one major facility. The assay functioned normally with these diverse samples and yielded results that can be used in assessing the potential developmental hazard of these materials.

  12. The effects of pharmaceuticals on the regeneration of the cnidarian, Hydra attenuata.

    PubMed

    Quinn, Brian; Gagné, François; Blaise, Christian

    2008-08-25

    The Hydra attenuata regeneration assay was used to identify the teratogenic potential of 10 pharmaceuticals identified in effluent from a large city wastewater treatment plant (WWTP). Three types of solvents were used to solubilise the pharmaceuticals (DMSO, acetone and ethanol), at concentrations determined to have no significant effect on measured endpoints. On the one hand, regeneration was significantly inhibited at (nominal) concentrations of 1, 5 and 1 mg/L for gemfibrozil, ibuprofen and naproxen respectively and at the higher concentration of 50 mg/L for bezafibrate and trimethoprim. On the other hand, carbamazepine and the antibiotics sulfapyridine, oxytetracycline and novobiocin significantly increased regeneration at 25, 5, 50 and 50 mg/L respectively. Relatively high IC50 values of 0.9, 3.84, 4.9 and 22.5 mg/L were calculated for gemfibrozil, ibuprofen, naproxen and bezafibrate, respectively. However when subjected to tier two toxicity assessment under EU regulatory guidance using environmentally relevant concentrations a MEC/PNEC value>1 was calculated for gemfibrozil, ibuprofen and naproxen indicating teratogenic potential and the necessity for further tier three assessment. A toxicity index (TI) was also calculated using three different techniques, with TI values>3 (indicating teratogenic potential) found for gemfibrozil, ibuprofen, naproxen and bezafibrate and >1 (indicating a weak teratogenic potential) found for carbamazepine. These results are discussed in the context of their environmental relevance and toxic potential.

  13. Virus infection suppresses Nicotiana benthamiana adaptive phenotypic plasticity.

    PubMed

    Bedhomme, Stéphanie; Elena, Santiago F

    2011-02-17

    Competition and parasitism are two important selective forces that shape life-histories, migration rates and population dynamics. Recently, it has been shown in various pathosystems that parasites can modify intraspecific competition, thus generating an indirect cost of parasitism. Here, we investigated if this phenomenon was present in a plant-potyvirus system using two viruses of different virulence (Tobacco etch virus and Turnip mosaic virus). Moreover, we asked if parasitism interacted with the shade avoidance syndrome, the plant-specific phenotypic plasticity in response to intraspecific competition. Our results indicate that the modification of intraspecific competition by parasitism is not present in the Nicotiana benthamiana--potyvirus system and suggests that this phenomenon is not universal but depends on the peculiarities of each pathosystem. However, whereas the healthy N. benthamiana presented a clear shade avoidance syndrome, this phenotypic plasticity totally disappeared when the plants were infected with TEV and TuMV, very likely resulting in a fitness loss and being another form of indirect cost of parasitism. This result suggests that the suppression or the alteration of adaptive phenotypic plasticity might be a component of virulence that is often overlooked.

  14. Virus Infection Suppresses Nicotiana benthamiana Adaptive Phenotypic Plasticity

    PubMed Central

    Bedhomme, Stéphanie; Elena, Santiago F.

    2011-01-01

    Competition and parasitism are two important selective forces that shape life-histories, migration rates and population dynamics. Recently, it has been shown in various pathosystems that parasites can modify intraspecific competition, thus generating an indirect cost of parasitism. Here, we investigated if this phenomenon was present in a plant-potyvirus system using two viruses of different virulence (Tobacco etch virus and Turnip mosaic virus). Moreover, we asked if parasitism interacted with the shade avoidance syndrome, the plant-specific phenotypic plasticity in response to intraspecific competition. Our results indicate that the modification of intraspecific competition by parasitism is not present in the Nicotiana benthamiana – potyvirus system and suggests that this phenomenon is not universal but depends on the peculiarities of each pathosystem. However, whereas the healthy N. benthamiana presented a clear shade avoidance syndrome, this phenotypic plasticity totally disappeared when the plants were infected with TEV and TuMV, very likely resulting in a fitness loss and being another form of indirect cost of parasitism. This result suggests that the suppression or the alteration of adaptive phenotypic plasticity might be a component of virulence that is often overlooked. PMID:21359142

  15. Jasmonoyl-l-isoleucine hydrolase 1 (JIH1) contributes to a termination of jasmonate signaling in N. attenuata

    PubMed Central

    Woldemariam, Melkamu G; Gális, Ivan; Baldwin, Ian T

    2014-01-01

    The jasmonate signaling pathway is essential for plant development, reproduction, and defense against herbivores and pathogens. When attacked by herbivores, plants elicit defense responses through the rapid accumulation of jasmonates. Although the transduction of the jasmonate burst into downstream responses has been largely resolved in the past decade, how the jasmonate burst is switched off remained unknown. Recently, two mechanisms that involve cytochrome p450-mediated hydroxylation/carboxylation and NaJIH1-mediated hydrolysis of JA-Ile were identified as major termination mechanisms of JA signaling. Due to a lack of hydrolysis, N. attenuata plants silenced in the expression of the JIH1 gene accumulated significantly more JA-Ile than did wild type plants and became more resistant to herbivore attack. Although less likely, additional functions of JIH1, such as contributing to the pool of free Ile and thereby increasing JA-Ile accumulation, remained untested. Here we show that increased isoleucine availability does not explain the observed phenotype in JIH1-deficient N. attenuata plants. PMID:24776843

  16. Genetic variation in alkaloid accumulation in leaves of Nicotiana.

    PubMed

    Sun, Bo; Zhang, Fen; Zhou, Guo-jun; Chu, Guo-hai; Huang, Fang-fang; Wang, Qiao-mei; Jin, Li-feng; Lin, Fu-cheng; Yang, Jun

    2013-12-01

    Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R(2)=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.

  17. Silicon delays tobacco Ringspot virus systemic symptoms in Nicotiana tabacum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the role of Si in viral infections has been elusive. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco rings...

  18. Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance

    PubMed Central

    Beseli, Aydin; Noar, Roslyn; Daub, Margaret E.

    2015-01-01

    The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species’ resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta

  19. Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

    PubMed

    Beseli, Aydin; Noar, Roslyn; Daub, Margaret E

    2015-01-01

    The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

  20. Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids.

    PubMed

    Menczel, L; Nagy, F; Kiss, Z R; Maliga, P

    1981-03-01

    Protoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.

  1. Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

    PubMed

    Chung, K-R; Ehrenshaft, M; Wetzel, D K; Daub, M E

    2003-11-01

    We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

  2. Toxicity of polychlorinated diphenyl ethers in hydra attenuata and in rat whole-embryo culture. Master's thesis

    SciTech Connect

    Becker, M.C.

    1991-05-01

    Polychlorinated diphenyl ethers (PCDEs) are a class of biaryl compounds that have little commercial application, but appear to be widespread in the environment. They have been found in wood preservative waste dumpsites and in fly ash from municipal waste incinerators. They have been detected in bird eggs and tissues, fish, and other edible marine organisms in the United States, Canada, and Europe. There are limited reports in the extant literature on the toxicity of PCDEs. This study was designed to evaluate the toxicity of selected PCDEs in cultures of Hydra attenuata and post-implantation rat whole embryos. The toxicity of several closely related polychlorinated biphenyls (PCBs) was evaluated in both cultures and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was evaluated in whole embryo culture. Embryonic growth and development parameters (yolk sac diameter, crown-rump length, somite count, and DNA and protein content) and gross morphology were determined. Findings indicated that these chemicals were neither embryotoxic nor teratogenic. Thus, the PCDEs, which elicit other diverse toxic and biochemical responses in rodents, are relatively inactive in these bioassays for developmental toxicity.

  3. Cadmium contamination of tissues and organs of delphinids species (Stenella attenuata)--influence of biological and ecological factors

    SciTech Connect

    Andre, J.M.; Amiard, J.C.; Amiard-Triquet, C.; Boudou, A.; Ribeyre, F. )

    1990-12-01

    Based on a sample of 27 dolphins (Stenella attenuata) captured in the Eastern tropical zone of the Pacific Ocean, this study was carried out to analyze the cadmium accumulation levels and distribution in 12 organs or tissue samples. The average cadmium concentrations were between 0.2 mg Cd.kg-1 in the brain and muscle and 48 mg Cd.kg-1 in the kidneys. For most of organs and tissues the average values were between 1 and 5 mg Cd.kg-1. Kidneys, liver, muscle, and intestine contained almost 85% of the total cadmium burden of all tissues considered in this study. Most of the biological and ecological factors taken into account (age, sex, total weight, and length of the dolphins, weight of the organs, place and date of capture) interacted with the cadmium concentrations and burdens in the collected organs or tissues. Three factors appear to be of prime importance: age, body weight, and geographical location of the area of capture.

  4. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot

    NASA Astrophysics Data System (ADS)

    He, Tianhua; D’Agui, Haylee; Lim, Sim Lin; Enright, Neal J.; Luo, Yiqi

    2016-05-01

    Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime.

  5. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot

    PubMed Central

    He, Tianhua; D’Agui, Haylee; Lim, Sim Lin; Enright, Neal J.; Luo, Yiqi

    2016-01-01

    Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime. PMID:27210077

  6. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot.

    PubMed

    He, Tianhua; D'Agui, Haylee; Lim, Sim Lin; Enright, Neal J; Luo, Yiqi

    2016-05-23

    Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime.

  7. [Turkish (Aztec) tobbaco (Nicotiana rustica L.) unnoticeable danger?].

    PubMed

    Modnicki, Daniel

    2007-01-01

    Tobacco harmful influence on men's health is usually linked with smoking cigarettes. Alternative ways of intake tobacco are also dangerous. Nicotine and tobacco-specific N-nitrosamines with carcinogenic activity are not only present in tobacco smoke but also of nicotine-rich product for chewing. There are some internet sources about Turkish (Aztec) tobacco (Nicotiana rustica L.) as easy available material for home-made tobacco mixtures. A some reports about dangerous effects connected with chewing tobacco and similar to smoking-related disorders are present in current literature.

  8. Genetic analysis of Phytophthora nicotianae populations from different hosts using microsatellite markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two hundred thirty-one isolates of P. nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa and Ruta) and other plant genera of lesser econo...

  9. Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana.

    PubMed

    Chen, Ke; Dorlhac de Borne, François; Szegedi, Ernö; Otten, Léon

    2014-11-01

    Nicotiana species carry cellular T-DNA sequences (cT-DNAs), acquired by Agrobacterium-mediated transformation. We characterized the cT-DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT-DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted-repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine-type T-DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine-type orf14-mis fragment and a mannopine-agropine synthesis region (mas2-mas1-ags). The mas2' gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T-DNA, but also carries octopine synthase-like (ocl) and c-like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T-DNA fragments similar to the right end of the A4 TL-DNA, and including an orf14-like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT-DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT-DNAs during the evolution of the genus Nicotiana.

  10. Large-scale detection and application of expressed sequence tag single nucleotide polymorphisms in Nicotiana.

    PubMed

    Wang, Y; Zhou, D; Wang, S; Yang, L

    2015-07-14

    Single nucleotide polymorphisms (SNPs) are widespread in the Nicotiana genome. Using an alignment and variation detection method, we developed 20,607,973 SNPs, based on the expressed sequence tag sequences of 10 Nicotiana species. The replacement rate was much higher than the transversion rate in the SNPs, and SNPs widely exist in the Nicotiana. In vitro verification indicated that all of the SNPs were high quality and accurate. Evolutionary relationships between 15 varieties were investigated by polymerase chain reaction with a special primer; the specific 302 locus of these sequence results clearly indicated the origin of Zhongyan 100. A database of Nicotiana SNPs (NSNP) was developed to store and search for SNPs in Nicotiana. NSNP is a tool for researchers to develop SNP markers of sequence data.

  11. Complete Chloroplast Genome of Nicotiana otophora and its Comparison with Related Species

    PubMed Central

    Asaf, Sajjad; Khan, Abdul L.; Khan, Abdur R.; Waqas, Muhammad; Kang, Sang-Mo; Khan, Muhammad A.; Lee, Seok-Min; Lee, In-Jung

    2016-01-01

    Nicotiana otophora is a wild parental species of Nicotiana tabacum, an interspecific hybrid of Nicotiana tomentosiformis and Nicotiana sylvestris. However, N. otophora is least understood as an alternative paternal donor. Here, we compared the fully assembled chloroplast (cp) genome of N. otophora and with those of closely related species. The analysis showed a cp genome size of 156,073 bp and exhibited a typical quadripartite structure, which contains a pair of inverted repeats separated by small and large single copies, containing 163 representative genes, with 165 microsatellites distributed unevenly throughout the genome. Comparative analysis of a gene with known function across Nicotiana species revealed 76 protein-coding sequences, 20 tRNA sequences, and 3 rRNA sequence shared between the cp genomes. The analysis revealed that N. otophora is a sister species to N. tomentosiformis within the Nicotiana genus, and Atropha belladonna and Datura stramonium are their closest relatives. These findings provide a valuable analysis of the complete N. otophora cp genome, which can identify species, elucidate taxonomy, and reconstruct the phylogeny of genus Nicotiana. PMID:27379132

  12. Acute respiratory failure due to Nicotiana glauca ingestion

    PubMed Central

    Ntelios, D; Kargakis, M; Topalis, T; Drouzas, A; Potolidis, E

    2013-01-01

    Background: A variety of organisms produce potent toxins that impact human health through compromising respiratory function. Case report: We describe a rare case of abrupt respiratory failure afterNicotiana glaucaingestion in a previously healthy sixty years old female patient. She presented complaining for gait instability and malaise after ingestion of cooked leaves of the wild plant and two hours after the onset she developed respiratory failurefor which she was intubated and mechanically ventilated for two days. The patient fully recovered and was discharged from the hospital. Conclusion: Anabasine, the plant’s main active ingredient, can cause severe systemic intoxication due to its nicotinic receptor agonist action with respiratory muscle paralysis being the main effect. PMID:24376330

  13. Changes in photosynthetic metabolism induced by tobamovirus infection in Nicotiana benthamiana studied in vivo by thermoluminescence.

    PubMed

    Sajnani, Carlota; Zurita, Jorge L; Roncel, Mercedes; Ortega, José M; Barón, Matilde; Ducruet, Jean-Marc

    2007-01-01

    * In thylakoids from Nicotiana benthamiana infected with the pepper mild mottle virus (PMMoV), a decreased amount of the PsbP and PsbQ proteins of photosystem II and different proteins of the Calvin cycle have been previously observed. We used thermoluminescence to study the consequences in vivo. * Measurements on unfrozen discs from symptomatic and asymptomatic leaves of plants infected by two tobamovirus PMMoV-S and PMMoV-I strains were compared with homologous samples in control plants. * Thermoluminescence emission did not reveal noticeable alteration of PSII electron transfer activity in infected symptomatic leaves. In these leaves, the relative intensity of the 'afterglow' emission indicated an increase of the NADPH + ATP assimilatory potential, contrasting with its decrease in asymptomatic leaves. High-temperature thermoluminescence, as a result of peroxides, increased in symptomatic and asymptomatic leaves. * In young infected leaves, PSII activity is preserved, producing a high assimilatory potential. Older asymptomatic leaves export more nutrients towards young infected leaves. This depresses their assimilatory potential and weakens their defence mechanisms against reactive oxygen species, resulting in higher peroxide content.

  14. GhWRKY68 Reduces Resistance to Salt and Drought in Transgenic Nicotiana benthamiana

    PubMed Central

    Jia, Haihong; Wang, Chen; Wang, Fang; Liu, Shuchang; Li, Guilin; Guo, Xingqi

    2015-01-01

    The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA) and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS), reduced enzyme activities, elevated malondialdehyde (MDA) content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS. PMID:25793865

  15. RNA-dependent RNA polymerase 1 from Nicotiana tabacum suppresses RNA silencing and enhances viral infection in Nicotiana benthamiana.

    PubMed

    Ying, Xiao-Bao; Dong, Li; Zhu, Hui; Duan, Cheng-Guo; Du, Quan-Sheng; Lv, Dian-Qiu; Fang, Yuan-Yuan; Garcia, Juan Antonio; Fang, Rong-Xiang; Guo, Hui-Shan

    2010-04-01

    Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid-mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

  16. Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis.

    PubMed

    Conley, Andrew J; Zhu, Hong; Le, Linda C; Jevnikar, Anthony M; Lee, Byong H; Brandle, Jim E; Menassa, Rima

    2011-05-01

    Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.

  17. Assessing acute toxicities of pre- and post-treatment industrial wastewaters with Hydra attenuata: A comparative study of acute toxicity with the fathead minnow, Pimephales promelas

    SciTech Connect

    Fu, L.J.; Staples, R.E.; Stahl, R.G. Jr. . Haskell Lab. for Toxicology and Industrial Medicine)

    1994-04-01

    This study was undertaken to (a) determine wastewater treatment effectiveness using two freshwater organisms, (b) compare acute toxicity results from the two species exposed to the wastewaters, and (c) link acute and potential developmental toxicity of wastewaters in one organism. The acute toxicities of several pretreatment and post-treatment industrial waste-water samples wee evaluated with adult Hydra attenuata and fathead minnows. The acute LC50s agreed closely when results in Hydra attenuata were compared with those from fathead minnow tests. Acute LC50s ranged from 3 to >100% of samples with hydra, and from 1.0 to >100% of sample with fathead minnows. The results provided strong evidence of treatment effectiveness because toxicity decreased with progressive stages of treatment. Previously the Hydra Developmental Toxicity Assay was used as a prescreen mainly for in vitro assessment of developmental toxicity with pure compounds and to prioritized toxicants according to selective toxicity to the developing embryo. Recently the authors modified the assay for testing natural waters and wastewaters; hence, some of the wastewater samples also were tested for their developmental toxicity. In this case, the relative selective toxicity of these wastewater samples ranged from 0.7 to 2.1, indicating that no sample was uniquely toxic to the developing embryo, although acute toxicity was manifested. Overall, their results indicate the Hydra Assay functions appropriately in assessments of acute and developmental toxicity of industrial wastewaters and may be a simple and useful tool in a battery of tests for broader scale detection of environmental hazards.

  18. Pathway of phloem unloading in tobacco sink leaves. [Nicotiana tabacum

    SciTech Connect

    Turgeon, R.

    1987-04-01

    Phloem unloading in transition sink leaves of tobacco (Nicotiana tabacum L.) was analyzed by quantitative autoradiography. Source leaves were labeled with /sup 14/CO/sub 2/ and experimental treatments were begun approximately 1 h later when label had entered the sink leaves. Autoradiographs were prepared from rapidly frozen, lyophilized sink tissue at the beginning and end of the treatments and the amount of label in veins and in surrounding cells was determined by microdensitometry. Photoassimilate unloaded from third order and larger, but not smaller, veins. Long-distance import and unloading did not respond the same way to all experimental treatments. Import was completely inhibited by cold, anaerobiosis or steam girdling the sink leaf petiole. Unloading was inhibited by cold but continued in an anaerobic atmosphere and after steam girdling. Uptake of exogenous (/sup 14/C)sucrose was inhibited by anaerobiosis. Since an apoplastic pathway of phloem unloading would involve solute uptake from the apoplast the results are most consistent with passive symplastic unloading of photoassimilates from phloem to surrounding cells.

  19. A tobamovirus expression vector for agroinfection of legumes and Nicotiana.

    PubMed

    Liu, Zun; Kearney, Christopher M

    2010-06-01

    The highest recombinant protein expression levels in plants have been achieved using tobacco mosaic virus (TMV) vectors via agroinoculation of the tobacco, Nicotiana benthamiana. These vectors have been utilized for pharmaceutical protein production and also can serve as rapid gene expression screens for proteonomics. We have constructed a similar vector based on the legume-infecting tobamovirus, sunn hemp mosaic virus (SHMV), by deleting the coat protein gene (SHMV eliminate coat protein gene or SHEC). SHEC/GFP co-agroinoculated with a 35S/p19 binary yielded 600 microg GFP/gfw (25% TSP) in N. benthamiana. In the absence of p19, SHEC/GFP expression was nearly eliminated. SHEC also yielded strong GUS production in agroinoculated Medicago trunculata, Pinto bean, cowpea, pea and lentil even without the aid of systemic infection. A full-length version (SHAC, SHMV alternate coat protein) was created by adding to SHEC the coat protein subgenomic promoter and ORF from the tobamovirus, tobacco mild green mottle virus (TMGMV). SHAC induced a slowly developing, symptomless infection of N. benthamiana and may be of use as a virus induced gene silencing (VIGS) vector.

  20. Reactions of Nicotiana species to inoculation with monopartite and bipartite begomoviruses

    PubMed Central

    2011-01-01

    Background Some Nicotiana species are widely used as experimental hosts for plant viruses. Nicotiana species differ in ploidy levels, chromosome numbers and have diverse geographical origins. Thus, these species are useful model systems to investigate virus-host interactions, co-evolution of pathogens and hosts and the effects of ploidy level on virus resistance/susceptibility. Results Here we have studied the responses of seven Nicotiana species to inoculation with Cotton leaf curl Multan virus (CLCuMV), a monopartite begomovirus, and Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, both from the Indian subcontinent. All Nicotiana species supported the replication of both begomoviruses in inoculated leaves. However, only three Nicotiana species, namely N. benthamiana, N. tabacum and N. sylvestris showed symptoms when inoculated with ToLCNDV, while N. benthamiana was the only species that developed leaf curl symptoms when inoculated with CLCuMV. CLCuMV accumulated to detectable levels in N. tabacum, but plants remained asymptomatic. A previously identified mutation of RNA dependent RNA polymerase 1 was shown to be present only in N. benthamiana. The finding is in line with earlier results showing that the susceptibility of this species to a diverse range of plant viruses correlates with a defective RNA silencing-mediated host defense. Conclusions The results presented show that individual Nicotiana species respond differently to inoculation with begomoviruses. The inability of begomoviruses to systemically infect several Nicotiana species is likely due to inhibition of virus movement, rather than replication, and thus provides a novel model to study virus-host interactions in resistant/susceptible hosts. PMID:22011413

  1. Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum.

    PubMed Central

    Murfett, J; Cornish, EC; Ebert, PR; Bonig, I; McClure, BA; Clarke, AE

    1992-01-01

    In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype. PMID:12297668

  2. Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

    PubMed

    Robert, Stéphanie; Goulet, Marie-Claire; D'Aoust, Marc-André; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

  3. A distinct endogenous pararetrovirus family in Nicotiana tomentosiformis, a diploid progenitor of polyploid tobacco.

    PubMed

    Gregor, Wolfgang; Mette, M Florian; Staginnus, Christina; Matzke, Marjori A; Matzke, Antonius J M

    2004-03-01

    A distinct endogenous pararetrovirus (EPRV) family corresponding to a previously unknown virus has been identified in the genome of Nicotiana tomentosiformis, a diploid ancestor of allotetraploid tobacco (Nicotiana tabacum). The putative virus giving rise to N. tomentosiformis EPRVs (NtoEPRVs) is most similar to tobacco vein clearing virus, an episomal form of a normally silent EPRV family in Nicotiana glutinosa; it is also related to a putative virus giving rise to the NsEPRV family in Nicotiana sylvestris (the second diploid progenitor of tobacco) and in the N. sylvestris fraction of the tobacco genome. The copy number of NtoEPRVs is significantly higher in N. tomentosiformis than in tobacco. This suggests that after the polyploidization event, many copies were lost from the polyploid genome or were accumulated specifically in the diploid genome. By contrast, the copy number of NsEPRVs has remained constant in N. sylvestris and tobacco, indicating that changes have occurred preferentially in the NtoEPRV family during evolution of the three Nicotiana species. NtoEPRVs are often flanked by Gypsy retrotransposon-containing plant DNA. Although the mechanisms of NtoEPRV integration, accumulation, and/or elimination are unknown, these processes are possibly linked to retrotransposon activity.

  4. Efficacy of Chaetomium Species as Biological Control Agents against Phytophthora nicotianae Root Rot in Citrus.

    PubMed

    Hung, Phung Manh; Wattanachai, Pongnak; Kasem, Soytong; Poeaim, Supattra

    2015-09-01

    Thailand is one of the largest citrus producers in Southeast Asia. Pathogenic infection by Phytophthora, however, has become one of major impediments to production. This study identified a pathogenic oomycete isolated from rotted roots of pomelo (Citrus maxima) in Thailand as Phytophthora nicotianae by the internal transcribed spacer ribosomal DNA sequence analysis. Then, we examined the in vitro and in vivo effects of Chaetomium globosum, Chaetomium lucknowense, Chaetomium cupreum and their crude extracts as biological control agents in controlling this P. nicotianae strain. Represent as antagonists in biculture test, the tested Chaetomium species inhibited mycelial growth by 50~56% and parasitized the hyphae, resulting in degradation of P. nicotianae mycelia after 30 days. The crude extracts of these Chaetomium species exhibited antifungal activities against mycelial growth of P. nicotianae, with effective doses of 2.6~101.4 µg/mL. Under greenhouse conditions, application of spores and methanol extracts of these Chaetomium species to pomelo seedlings inoculated with P. nicotianae reduced root rot by 66~71% and increased plant weight by 72~85% compared to that in the control. The method of application of antagonistic spores to control the disease was simple and economical, and it may thus be applicable for large-scale, highly effective biological control of this pathogen.

  5. Current status and prospects for the study of Nicotiana genomics, genetics, and nicotine biosynthesis genes.

    PubMed

    Wang, Xuewen; Bennetzen, Jeffrey L

    2015-02-01

    Nicotiana, a member of the Solanaceae family, is one of the most important research model plants, and of high agricultural and economic value worldwide. To better understand the substantial and rapid research progress with Nicotiana in recent years, its genomics, genetics, and nicotine gene studies are summarized, with useful web links. Several important genetic maps, including a high-density map of N. tabacum consisting of ~2,000 markers published in 2012, provide tools for genetics research. Four whole genome sequences are from allotetraploid species, including N. benthamiana in 2012, and three N. tabacum cultivars (TN90, K326, and BX) in 2014. Three whole genome sequences are from diploids, including progenitors N. sylvestris and N. tomentosiformis in 2013 and N. otophora in 2014. These and additional studies provide numerous insights into genome evolution after polyploidization, including changes in gene composition and transcriptome expression in N. tabacum. The major genes involved in the nicotine biosynthetic pathway have been identified and the genetic basis of the differences in nicotine levels among Nicotiana species has been revealed. In addition, other progress on chloroplast, mitochondrial, and NCBI-registered projects on Nicotiana are discussed. The challenges and prospects for genomic, genetic and application research are addressed. Hence, this review provides important resources and guidance for current and future research and application in Nicotiana.

  6. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  7. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  8. Nicotiana benthamiana as a Production Platform for Artemisinin Precursors

    PubMed Central

    van Herpen, Teun W. J. M.; Cankar, Katarina; Nogueira, Marilise; Bosch, Dirk; Bouwmeester, Harro J.; Beekwilder, Jules

    2010-01-01

    Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg−1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed. PMID:21151979

  9. Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum.

    PubMed

    Dewey, Ralph E; Xie, Jiahua

    2013-10-01

    Alkaloids represent an extensive group of nitrogen-containing secondary metabolites that are widely distributed throughout the plant kingdom. The pyridine alkaloids of tobacco (Nicotiana tabacum L.) have been the subject of particularly intensive investigation, driven largely due to the widespread use of tobacco products by society and the role that nicotine (16) (see Fig. 1) plays as the primary compound responsible for making the consumption of these products both pleasurable and addictive. In a typical commercial tobacco plant, nicotine (16) comprises about 90% of the total alkaloid pool, with the alkaloids nornicotine (17) (a demethylated derivative of nicotine), anatabine (15) and anabasine (5) making up most of the remainder. Advances in molecular biology have led to the characterization of the majority of the genes encoding the enzymes directly responsible the biosynthesis of nicotine (16) and nornicotine (17), while notable gaps remain within the anatabine (15) and anabasine (5) biosynthetic pathways. Several of the genes involved in the transcriptional regulation and transport of nicotine (16) have also been elucidated. Investigations of the molecular genetics of tobacco alkaloids have not only provided plant biologists with insights into the mechanisms underlying the synthesis and accumulation of this important class of plant alkaloids, they have also yielded tools and strategies for modifying the tobacco alkaloid composition in a manner that can result in changing the levels of nicotine (16) within the leaf, or reducing the levels of a potent carcinogenic tobacco-specific nitrosamine (TSNA). This review summarizes recent advances in our understanding of the molecular genetics of alkaloid biosynthesis in tobacco, and discusses the potential for applying information accrued from these studies toward efforts designed to help mitigate some of the negative health consequences associated with the use of tobacco products.

  10. Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers

    PubMed Central

    Brito, Michael S.; Bertolino, Lígia T.; Cossalter, Viviane; Quiapim, Andréa C.; DePaoli, Henrique C.; Goldman, Gustavo H.; Teixeira, Simone P.; Goldman, Maria H. S.

    2015-01-01

    In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil. PMID

  11. Production, partial purification and characterization of xylanase using Nicotiana tabacum leaf dust as substrate.

    PubMed

    Acharya, Komal P; Shilpkar, Prateek

    2016-03-01

    Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.

  12. Nicotiana tabacum as model for ozone - plant surface reactions

    NASA Astrophysics Data System (ADS)

    Jud, Werner; Fischer, Lukas; Wohlfahrt, Georg; Tissier, Alain; Canaval, Eva; Hansel, Armin

    2015-04-01

    Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. The ensuing injuries have been related to the uptake of ozone through the stomatal pores and oxidative effects damaging the internal leaf tissue. A striking question of current research is the environment and plant specific partitioning of ozone loss between gas phase, stomatal or plant surface sink terms. Here we show results from ozone fumigation experiments using various Nicotiana Tabacum varieties, whose surfaces are covered with different amounts of unsaturated diterpenoids exuded by their glandular trichomes. Exposure to elevated ozone levels (50 to 150 ppbv) for 5 to 15 hours in an exceptionally clean cuvette system did neither result in a reduction of photosynthesis nor caused any visible leaf damage. Both these ozone induced stress effects have been observed previously in ozone fumigation experiments with the ozone sensitive tobacco line Bel-W3. In our case ozone fumigation was accompanied by a continuous release of oxygenated volatile organic compounds, which could be clearly associated to their condensed phase precursors for the first time. Gas phase reactions of ozone were avoided by choosing a high enough gas exchange rate of the plant cuvette system. In the case of the Ambalema variety, that is known to exude only the diterpenoid cis-abienol, ozone fumigation experiments yield the volatiles formaldehyde and methyl vinyl ketone (MVK). The latter could be unequivocally separated from isomeric methacrolein (MACR) by the aid of a Selective Reagent Ion Time-of-Flight Mass Spectrometer (SRI-ToF-MS), which was switched every six minutes from H3O+ to NO+ primary ion mode and vice versa. Consistent with the picture of an ozone protection mechanism caused by reactive diterpenoids at the leaf surface are the results from dark-light experiments. The ozone loss obtained from the

  13. Population structure and genetic diversity of Phytophthora nicotianae from tobacco in Georgia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Black shank caused by Phytophthora nicotianae occurs worldwide and is responsible for significant yield loss in tobacco production in Georgia. Management of the disease has primarily relied on utilization of tobacco cultivars with resistance to race 0 of the pathogen and application of the fungicide...

  14. Relationship between Active Oxygen Species, Lipid Peroxidation, Necrosis, and Phytoalexin Production Induced by Elicitins in Nicotiana.

    PubMed Central

    Rusterucci, C.; Stallaert, V.; Milat, M. L.; Pugin, A.; Ricci, P.; Blein, J. P.

    1996-01-01

    Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively. Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment. N. tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin. Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis. Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N. rustica. Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein. Phytoalexin production was lower in response to both elicitins in N. tabacum var Xanthi cells than in N. rustica cells, and capsicein was the most efficient elicitor of this response. In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium. These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses. PMID:12226334

  15. Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana 1

    PubMed Central

    Liu, Shih-Tung; Katz, Charles D.; Knight, C. Arthur

    1978-01-01

    The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid. In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [14C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices. It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants. The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined. PMID:16660376

  16. Genetic Analysis of Phytophthora nicotianae Populations from Different Hosts Using Microsatellite Markers.

    PubMed

    Biasi, Antonio; Martin, Frank N; Cacciola, Santa O; di San Lio, Gaetano Magnano; Grünwald, Niklaus J; Schena, Leonardo

    2016-09-01

    In all, 231 isolates of Phytophthora nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa, and Ruta), and other plant genera were characterized using simple-sequence repeat markers. In total, 99 multilocus genotypes (MLG) were identified, revealing a strong association between genetic grouping and host of recovery, with most MLG being associated with a single host genus. Significant differences in the structure of populations were revealed but clonality prevailed in all populations. Isolates from Citrus were found to be genetically related regardless of their geographic origin and were characterized by high genetic uniformity and high inbreeding coefficients. Higher variability was observed for other populations and a significant geographical structuring was determined for isolates from Nicotiana. Detected differences were related to the propagation and cultivation systems of different crops. Isolates obtained from Citrus spp. are more likely to be distributed worldwide with infected plant material whereas Nicotiana and Lycopersicon spp. are propagated by seed, which would not contribute to the spread of the pathogen and result in a greater chance for geographic isolation of lineages. With regard to ornamental species in nurseries, the high genetic variation is likely the result of the admixture of diverse pathogen genotypes through the trade of infected plant material from various geographic origins, the presence of several hosts in the same nursery, and genetic recombination through sexual reproduction of this heterothallic species.

  17. Anaerobic soil disinfestation reduces survival and infectivity of Phytophthora nicotianae chlamydospores in pepper

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora nicotianae is the principal causal agent of root and crown rot disease of pepper plants in Extremadura (western Spain), a spring-summer crop in this region. Preplant soil treatment by anaerobic soil disinfestation (ASD) may effectively control plant pathogens in many crop production sys...

  18. Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A large number of SSR loci were screened in the genomic assemblies of 14 different isolates of Phytophthora nicotianae and primers were developed for amplification of 17 markers distributed among different contigs. These loci were highly polymorphic and amplified from genetically distant isolates of...

  19. Calcium Efflux as a Component of Hypersensitive Response of Nicotiana benthamiana to Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a model plant Nicotiana benthamiana we have demonstrated that initial calcium uptake in response to HR (hypersensitive response)-causing pathogen, P. syringae pv syringae 61 is followed by the net calcium efflux initiated at about 12 hrs after the bacterial challenge and sustained for at least...

  20. Product variability of the 'cineole cassette' monoterpene synthases of related Nicotiana species.

    PubMed

    Fähnrich, Anke; Krause, Katrin; Piechulla, Birgit

    2011-11-01

    Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the 'cineole cassette' comprising 1,8-cineole, limonene, myrcene, α-pinene, β-pinene, sabinene, and α-terpineol. We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes, which produced the characteristic monoterpenes of this 'cineole cassette' with α-terpineol being most abundant in the volatile spectra. The amino acid sequences of both terpineol synthases were 99% identical. The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum. The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N. alata and N. langsdorfii were less efficient compared to the 'cineole cassette' monoterpene synthases of Arabidopsis thaliana, N. suaveolens, Salvia fruticosa, Salvia officinalis, and Citrus unshiu. The terpineol synthases of N. alata and N. langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals. The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N. alata was highest at the transition from day to night (diurnal rhythm).

  1. Effects of hydrostatic pressure, agitation and CO2 stress on Phytophthora nicotianae zoospore survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora nicotianae Breda de Haan was used as a model pathogen to investigate the effects of hydrostatic pressure, agitation, and aeration with CO2 or breathable air on the survival of Phytophthora zoospores in water. Injecting CO2 into 2 liters of zoospore-infested water for 5 min at 110.4 ml ...

  2. Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells

    SciTech Connect

    Kleinow, Tatjana; Tanwir, Fariha; Kocher, Cornelia; Krenz, Bjoern; Wege, Christina; Jeske, Holger

    2009-09-01

    The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

  3. Screening micro-organisms for cadmium absorption from aqueous solution and cadmium absorption properties of Arthrobacter nicotianae.

    PubMed

    Tsuruta, Takehiko; Umenai, Daishi; Hatano, Tomonobu; Hirajima, Tsuyoshi; Sasaki, Keiko

    2014-01-01

    To obtain basic information on how microbial cells absorb cadmium from aqueous solution, we examined cadmium absorption in various micro-organisms. Of 51 micro-organism strains tested, we found that some Gram-positive bacteria, such as, Arthrobacter nicotianae and Bacillus subtilis, and some actinomycetes, such as, Streptomyces flavoviridis and S. levoris were highly capable of absorbing cadmium from an aqueous solution. A. nicotianae absorbed the largest amount of cadmium, over 800 μmol cadmium per gram of dry wt. cells. However, cadmium absorption by A. nicotianae was affected by the solution pH, cadmium concentration, and cell density. The absorption of cadmium was very rapid. Some factors that affected cadmium absorption by A. nicotianae cells were also discussed.

  4. [Distribution and environmental conditions related to the behavior in the bottlenose dolphin (Tursiops truncatus) and the spotted dolphin (Stenella attenuata) (Cetacea: Delphinidae) in Golfo Dulce, Costa Rica].

    PubMed

    Cubero-Pardo, Priscilla

    2007-06-01

    Habitat characteristics influencing behavior in animal species vary locally. The influence that a particular environmental characteristic can have on a species depends not only on other variables, but on morphological, physiological and social conditions of that species. In this study, developed from June 1996 to July 1997, I studied whether specific behaviors are related to particular distribution areas and environmental factors in the bottlenose (Tursiops truncatus) and the spotted dolphin (Stenella attenuata). The study area was covered along oblicuous linear transects, and the behavior of single groups was observed from 15 min to 5 h. Environmental factors such as depth, temperature, salinity and distance from shore, among others, were considered. For the bottlenose dolphin, foraging/feeding activities showed exclusive coincidence with river mouths, coral reef and mangrove areas, while social and milling activities where seen close to feeding areas. Traveling occurred along different points parallel to the coast, with a low percentage of cases across the gulf (16.56 %), suggesting that the bottlenose rarely crosses from one side to the other. In the spotted dolphin, several behaviors were observed simultaneously in the schools and it was not possible to associate areas with particular behaviors. The lack of significant relationships among activities and particular environmental variables (ANOVA tests) is attributed to three aspects: (a) transitions among activities generally occurred into a low variable area, (b) dolphins often traveled along large areas without changing activities and (c) environmental conditions in Golfo Dulce are homogeneous. In the two species the highest average in the number of individuals per group corresponded to the category of active socializing, followed by traveling, passive socializing and feeding. In the case of the bottlenose dolphin, the smallest group size was associated with feeding activities (ANOVA, F= 2.624, p=0.037, n=156

  5. Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

    PubMed Central

    Hong, Y. P.; Hipkins, V. D.; Strauss, S. H.

    1993-01-01

    The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga. PMID:7905846

  6. BABA and Phytophthora nicotianae Induce Resistance to Phytophthora capsici in Chile Pepper (Capsicum annuum)

    PubMed Central

    Stamler, Rio A.; Holguin, Omar; Dungan, Barry; Schaub, Tanner; Sanogo, Soumaila; Goldberg, Natalie; Randall, Jennifer J.

    2015-01-01

    Induced resistance in plants is a systemic response to certain microorganisms or chemicals that enhances basal defense responses during subsequent plant infection by pathogens. Inoculation of chile pepper with zoospores of non-host Phytophthora nicotianae or the chemical elicitor beta-aminobutyric acid (BABA) significantly inhibited foliar blight caused by Phytophthora capsici. Tissue extract analyses by GC/MS identified conserved change in certain metabolite concentrations following P. nicotianae or BABA treatment. Induced chile pepper plants had reduced concentrations of sucrose and TCA cycle intermediates and increased concentrations of specific hexose-phosphates, hexose-disaccharides and amino acids. Galactose, which increased significantly in induced chile pepper plants, was shown to inhibit growth of P. capsici in a plate assay. PMID:26020237

  7. Nicotiana Roots Recruit Rare Rhizosphere Taxa as Major Root-Inhabiting Microbes.

    PubMed

    Saleem, Muhammad; Law, Audrey D; Moe, Luke A

    2016-02-01

    Root-associated microbes have a profound impact on plant health, yet little is known about the distribution of root-associated microbes among different root morphologies or between rhizosphere and root environments. We explore these issues here with two commercial varieties of burley tobacco (Nicotiana tabacum) using 16S rRNA gene amplicon sequencing from rhizosphere soil, as well as from primary, secondary, and fine roots. While rhizosphere soils exhibited a fairly rich and even distribution, root samples were dominated by Proteobacteria. A comparison of abundant operational taxonomic units (OTUs) between rhizosphere and root samples indicated that Nicotiana roots select for rare taxa (predominantly Proteobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, and Acidobacteria) from their corresponding rhizosphere environments. The majority of root-inhabiting OTUs (~80 %) exhibited habitat generalism across the different root morphological habitats, although habitat specialists were noted. These results suggest a specific process whereby roots select rare taxa from a larger community.

  8. The Ups and Downs of Genome Size Evolution in Polyploid Species of Nicotiana (Solanaceae)

    PubMed Central

    Leitch, I. J.; Hanson, L.; Lim, K. Y.; Kovarik, A.; Chase, M. W.; Clarkson, J. J.; Leitch, A. R.

    2008-01-01

    Background In studies looking at individual polyploid species, the most common patterns of genomic change are that either genome size in the polyploid is additive (i.e. the sum of parental genome donors) or there is evidence of genome downsizing. Reports showing an increase in genome size are rare. In a large-scale analysis of 3008 species, genome downsizing was shown to be a widespread biological response to polyploidy. Polyploidy in the genus Nicotiana (Solanaceae) is common with approx. 40 % of the approx. 75 species being allotetraploid. Recent advances in understanding phylogenetic relationships of Nicotiana species and dating polyploid formation enable a temporal dimension to be added to the analysis of genome size evolution in these polyploids. Methods Genome sizes were measured in 18 species of Nicotiana (nine diploids and nine polyploids) ranging in age from <200 000 years to approx. 4·5 Myr old, to determine the direction and extent of genome size change following polyploidy. These data were combined with data from genomic in situ hybridization and increasing amounts of information on sequence composition in Nicotiana to provide insights into the molecular basis of genome size changes. Key Results and Conclusions By comparing the expected genome size of the polyploid (based on summing the genome size of species identified as either a parent or most closely related to the diploid progenitors) with the observed genome size, four polyploids showed genome downsizing and five showed increases. There was no discernable pattern in the direction of genome size change with age of polyploids, although with increasing age the amount of genome size change increased. In older polyploids (approx. 4·5 million years old) the increase in genome size was associated with loss of detectable genomic in situ hybridization signal, whereas some hybridization signal was still detected in species exhibiting genome downsizing. The possible significance of these results is

  9. The effect of polyploidy and hybridization on the evolution of floral colour in Nicotiana (Solanaceae)

    PubMed Central

    McCarthy, Elizabeth W.; Arnold, Sarah E. J.; Chittka, Lars; Le Comber, Steven C.; Verity, Robert; Dodsworth, Steven; Knapp, Sandra; Kelly, Laura J.; Chase, Mark W.; Baldwin, Ian T.; Kovařík, Aleš; Mhiri, Corinne; Taylor, Lin; Leitch, Andrew R.

    2015-01-01

    Background and Aims Speciation in angiosperms can be accompanied by changes in floral colour that may influence pollinator preference and reproductive isolation. This study investigates whether changes in floral colour can accompany polyploid and homoploid hybridization, important processes in angiosperm evolution. Methods Spectral reflectance of corolla tissue was examined for 60 Nicotiana (Solanaceae) accessions (41 taxa) based on spectral shape (corresponding to pigmentation) as well as bee and hummingbird colour perception in order to assess patterns of floral colour evolution. Polyploid and homoploid hybrid spectra were compared with those of their progenitors to evaluate whether hybridization has resulted in floral colour shifts. Key Results Floral colour categories in Nicotiana seem to have arisen multiple times independently during the evolution of the genus. Most younger polyploids displayed an unexpected floral colour, considering those of their progenitors, in the colour perception of at least one pollinator type, whereas older polyploids tended to resemble one or both of their progenitors. Conclusions Floral colour evolution in Nicotiana is weakly constrained by phylogeny, and colour shifts do occur in association with both polyploid and homoploid hybrid divergence. Transgressive floral colour in N. tabacum has arisen by inheritance of anthocyanin pigmentation from its paternal progenitor while having a plastid phenotype like its maternal progenitor. Potentially, floral colour evolution has been driven by, or resulted in, pollinator shifts. However, those polyploids that are not sympatric (on a regional scale) with their progenitor lineages are typically not divergent in floral colour from them, perhaps because of a lack of competition for pollinators. PMID:25979919

  10. A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae.

    PubMed

    Chung, K R; Jenns, A E; Ehrenshaft, M; Daub, M E

    1999-09-01

    Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.

  11. Resurrection of an ancestral gene: functional and evolutionary analyses of the Ngrol genes transferred from Agrobacterium to Nicotiana.

    PubMed

    Aoki, Seishiro

    2004-08-01

    The Ng rol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca. It is thought that bacterial infection resulted in the transfer of the Ng rol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not. Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes. The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species. This paper focuses on studies of the Ng rol genes in present-day plants and during the evolution of the genus Nicotiana. The functional sequences of several Ng rol genes may have been conserved after their ancient introduction from a bacterium to the plant. Resurrection of an ancestral function of one of the Ng rol genes, as examined by physiological and evolutionary analyses, is also described. The origin of the Ng rol genes is then considered, based on results of molecular phylogenetic analyses. The effects of the horizontal transfer of the Ng rol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.

  12. RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana[W

    PubMed Central

    Ying, Xiao-Bao; Dong, Li; Zhu, Hui; Duan, Cheng-Guo; Du, Quan-Sheng; Lv, Dian-Qiu; Fang, Yuan-Yuan; Garcia, Juan Antonio; Fang, Rong-Xiang; Guo, Hui-Shan

    2010-01-01

    Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid–mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses. PMID:20400679

  13. Involvement of calcium/calmodulin signaling in cercosporin toxin biosynthesis by Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren

    2003-02-01

    Cercosporin is a non-host-selective, perylenequinone toxin produced by many phytopathogenic Cercospora species. The involvement of Ca(2+)/calmodulin (CaM) signaling in cercosporin biosynthesis was investigated by using pharmacological inhibitors. The results suggest that maintaining endogenous Ca(2+) homeostasis is required for cercosporin biosynthesis in Cercospora nicotianae. The addition of excess Ca(2+) to the medium slightly increased fungal growth but resulted in a reduction in cercosporin production. The addition of Ca(2+) chelators [EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] also reduced cercosporin production. Ca(2+) channel blockers exhibited a strong inhibition of cercosporin production only at higher concentrations (>2 mM). Cercosporin production was reduced greatly by Ca(2+) ionophores (A23187 and ionomycin) and internal Ca(2+) blocker [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester]. Phospholipase C inhibitors (lithium, U73122, and neomycin) led to a concentration-dependent inhibition of cercosporin biosynthesis. Furthermore, the addition of CaM inhibitors (compound 48/80, trifluoperazine, W-7, and chlorpromazine) also markedly reduced cercosporin production. In contrast to W-7, W-5, with less specificity for CaM, led to only minor inhibition of cercosporin production. The inhibitory effects of Ca(2+)/CaM inhibitors were partially or completely reversed by the addition of external Ca(2+). As assessed with Fluo-3/AM (a fluorescent Ca(2+) indicator), the Ca(2+) content in the cytoplasm decreased significantly when fungal cultures were grown in a medium containing Ca(2+)/CaM antagonists, confirming the specificity of those Ca(2+)/CaM antagonists in C. nicotianae. Taken together, the results suggest that Ca(2+)/CaM signal transduction may play a pivotal role in cercosporin biosynthesis in C. nicotianae.

  14. Short-term effects of hydrogen fluoride on Nicotiana tabacum L.

    PubMed

    Döğeroğlu, Tuncay; Ciçek, Arzu; Kara, Serap

    2003-09-01

    Data on the short-term effects of fumigation with hydrogen fluoride (HF) on the response behavior of the Nicotiana tabacum L. cv. St. Karabalar 6265 are being presented. Growth rate, fluoride accumulation in leaves and soils, and degradation in chlorophyll and nicotine contents of the plant species against the variations in two experimental factors, namely the exposure concentration and exposure time parameters, were investigated. Resulting data reveals that the variety selected for this study is not so tolerant to fluoride as generally reported in the literature, if only the individual effects of the pollutant is considered.

  15. Influence of selected herbicides on ozone injury in tobacco (Nicotiana tabacum)

    SciTech Connect

    Reilly, J.J.; Moore, L.D.

    1982-01-01

    Field experiments were conducted over a four year period to determine the influence of selected herbicides on ozone injury in tobacco (Nicotiana tabacum L.). Isopropalin (2,6-dinitro-N,N-dipropylcumidine), pebulate (S-propyl butylethylthiocarbamate), and diphenamid (N,N-dimethyl-2,2-diphenylacetamide) were applied at the recommended rates of 1.7, 4.5, and 4.5 kg/ha (ai), respectively. Treatment of tobacco plants with isopropalin or diphenamid reduced oxidant injury for the first two to four weeks after transplanting, but not later in the season. Pebulate has no consistent affect on the sensitivity of tobacco to ozone. 26 references, 3 tables.

  16. Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance.

    PubMed

    Deng, Wei; Luo, Keming; Li, Zhengguo; Yang, Yingwu; Hu, Nan; Wu, Yu

    2009-07-01

    Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.

  17. Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach.

    PubMed

    You, Bang-Jau; Lee, Miin-Huey; Chung, Kuang-Ren

    2009-07-01

    To determine if DNA configuration, gene locus, and flanking sequences will affect homologous recombination in the phytopathogenic fungus Cercospora nicotianae, we evaluated and compared disruption efficiency targeting four cercosporin toxin biosynthetic genes encoding a polyketide synthase (CTB1), a monooxygenase/O-methyltransferase (CTB3), a NADPH-dependent oxidoreductase (CTB5), and a FAD/FMN-dependent oxidoreductase (CTB7). Transformation of C. nicotianae using a circular plasmid resulted in low disruption frequency. The use of endonucleases or a selectable marker DNA fragment flanked by homologous sequence either at one end or at both ends in the transformation procedures, increased disruption efficiency in some but not all CTB genes. A split-marker approach, using two DNA fragments overlapping within the selectable marker, increased the frequency of targeted gene disruption and homologous integration as high as 50%, depending on the target gene and on the length of homologous DNA sequence flanking the selectable marker. The results indicate that the split-marker approach favorably decreased ectopic integration and thus, greatly facilitated targeted gene disruption in this important fungal pathogen.

  18. Some physiological aspects of nitrate reductase-deficient Nicotiana plumbaginifolia plants

    SciTech Connect

    Saux, C.; Morot-Gaudry, J.F.; Lemoine, Y.; Caboche, M.

    1986-04-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv. Viviani) mutants were found to be defective in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild type Nicotiana tabacum. The grafts of NR/sup -/ plants were found to contain less malate but more amino acids, sugars and starch than the wild type. Moreover they were chlorotic, with a slight increase of the proportion of LH Chl a/b protein complexes and they exhibited a lowering of the efficiency of energy transfer between the light-harvesting complexes and the active centers. After /sup 14/CO/sub 2/ pulse and chase experiments. The total /sup 14/C incorporation of the mutant leaves was approximately 20% of that of the control. The NR/sup -/ leaves mainly accumulated /sup 14/C in the whole intermediates of the Calvin-cycle and in sucrose. In the most deficient NR leaves, chloroplasts were stuffed with large starch inclusions disorganizing the lamellar system.

  19. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    SciTech Connect

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-05-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second /sup 14/CO/sub 2/ pulse, the total /sup 14/C incorporation of the mutant leaves was approximately 20/sup 5/ of that of the control. The /sup 14/C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second /sup 14/CO/sub 2/ pulse followed by a 60 second chase with normal CO/sub 2/, /sup 14/C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.

  20. Evolutionary time-scale of the begomoviruses: evidence from integrated sequences in the Nicotiana genome.

    PubMed

    Lefeuvre, Pierre; Harkins, Gordon W; Lett, Jean-Michel; Briddon, Rob W; Chase, Mark W; Moury, Benoit; Martin, Darren P

    2011-01-01

    Despite having single stranded DNA genomes that are replicated by host DNA polymerases, viruses in the family Geminiviridae are apparently evolving as rapidly as some RNA viruses. The observed substitution rates of geminiviruses in the genera Begomovirus and Mastrevirus are so high that the entire family could conceivably have originated less than a million years ago (MYA). However, the existence of geminivirus related DNA (GRD) integrated within the genomes of various Nicotiana species suggests that the geminiviruses probably originated >10 MYA. Some have even suggested that a distinct New-World (NW) lineage of begomoviruses may have arisen following the separation by continental drift of African and American proto-begomoviruses ∼110 MYA. We evaluate these various geminivirus origin hypotheses using Bayesian coalescent-based approaches to date firstly the Nicotiana GRD integration events, and then the divergence of the NW and Old-World (OW) begomoviruses. Besides rejecting the possibility of a<2 MYA OW-NW begomovirus split, we could also discount that it may have occurred concomitantly with the breakup of Gondwanaland 110 MYA. Although we could only confidently narrow the date of the split down to between 2 and 80 MYA, the most plausible (and best supported) date for the split is between 20 and 30 MYA--a time when global cooling ended the dispersal of temperate species between Asia and North America via the Beringian land bridge.

  1. Resistance management in a native plant: nicotine prevents herbivores from compensating for plant protease inhibitors.

    PubMed

    Steppuhn, Anke; Baldwin, Ian T

    2007-06-01

    Plants deploy chemical defenses in complex mixtures, which are thought to be adaptive, but experimental tests have used artificial diets rather than plants. Herbivore attack on Nicotiana attenuata rapidly increases the production and accumulation of trypsin proteinase inhibitors (TPI) and the toxic alkaloid nicotine. By transgenically silencing their respective biosynthetic genes, we were able to abolish TPI activity and reduce inducible nicotine by 85%. Nicotine production was not affected by silencing pi or vice versa, and transformation did not alter levels of other metabolites examined. Spodoptera exigua, a native generalist herbivore that can compensate for heterologous TPI expression, performed better on TPI- or nicotine-deficient plants compared with the wild-type. Because of a compensatory feeding response to TPI when nicotine is absent, larvae performed better on nicotine-deficient plants than they did on plants silenced in both defenses. The antifeedant toxin, nicotine, prevents this compensatory response. We conclude that N. attenuata counters an insect adaptation with a defensive synergism.

  2. [TMV-infection localization and development of induced virus resistance in Nicotiana sanderae Hort., Datura stramonium L. and Datura metel].

    PubMed

    Kovalenko, O H; Kyrychenko, A M

    2004-01-01

    The localization of virus infection and development of local and systemic induced resistance in plants Nicotiana sanderae Hort., Datura stramonium L. and D. metel L. infected by TMV have been studied. It was shown that acquired resistance to the second infection is induced in hypersensitive plants infected by TMV independent of localization mechanism. No definite correlation exists between these defense reactions.

  3. Chronic ozone exacerbates the reduction in photosynthesis and acceleration of senescence caused by limited N availability in Nicotiana sylvestris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elevated ozone (O3) and limiting soil nitrogen (N) availability both negatively affect crop performance. However, little is known about how the combination of elevated O3 and limiting N affect crop growth and metabolism. In this study, we grew tobacco (Nicotiana sylvestris) in ambient and elevated O...

  4. In vitro cytotoxicity of Nicotiana gossei leaves, used in the Australian Aboriginal smokeless tobacco known as pituri or mingkulpa.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Cabot, Peter J; Steadman, Kathryn J

    2016-07-08

    The Aboriginal population of Central Australia use endemic Nicotiana species to make a smokeless tobacco product known usually as pituri or mingkulpa. Nicotiana leaves are masticated with wood ash into a 'quid' that is chewed/sucked for absorption of nicotine. In addition to nicotine, smokeless tobacco products contain a spectrum of biologically active compounds that may contribute to effects on health. The objective of this study was to quantify nicotine, and related alkaloids and tobacco specific nitrosamines (TSNAs), in Nicotiana leaves used in pituri, and compare in vitro toxicity of pure nicotine with Nicotiana leaf extract at the same concentration of nicotine. An aqueous extract of dry leaves of Nicotiana gossei and a reference smokeless tobacco (CORESTA CRP2) were quantified for major pyridine alkaloids and TSNAs using HPLC-UV and LC-MS/MS. A range of extract concentrations and corresponding concentrations of nicotine standard were tested using an MTS assay to measure human lung epithelium cell (A549) survival. Cells treated for 24h with the maximum concentration of 1.5mg/ml of nicotine resulted in 77% viability. In contrast, extracts from N. gossei leaves and CRP2 containing a similar concentration of nicotine (1.3mg/ml) resulted in remarkably lower viability of 1.5 and 6%, respectively. Comparison of cytotoxicity of pure nicotine with that of the extracts revealed that nicotine was not the source of their cytotoxicity. Other biologically active compounds such as the known carcinogens NNK and NNN, derived from nicotine and nornicotine and found to be present in the smokeless tobacco extracts, may be responsible.

  5. Inhibition of in Vitro Pollen Tube Growth by Isolated S-Glycoproteins of Nicotiana alata.

    PubMed Central

    Jahnen, W.; Lush, W. M.; Clarke, A. E.

    1989-01-01

    Pollen from three S-genotypes of Nicotiana alata was grown in vitro in the presence of S-glycoproteins isolated from styles of the same three genotypes. Pollen germination was not affected by the presence of the S-glycoproteins, but pollen tube growth of all genotypes was inhibited. S2 pollen was preferentially inhibited by the S2-glycoprotein and S3 pollen by the S3-glycoprotein. The S6-glycoprotein preferentially inhibited growth of both S2 and S6 pollen over S3 pollen. Heat treatment dramatically increased the inhibitory activity of the S-glycoproteins as inhibitors both of pollen germination and tube growth; after heat treatment, S-allele specificity of pollen tube inhibition was not detected. PMID:12359898

  6. Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana.

    PubMed

    Aslan, Selcuk; Sun, Chuanxin; Leonova, Svetlana; Dutta, Paresh; Dörmann, Peter; Domergue, Frédéric; Stymne, Sten; Hofvander, Per

    2014-09-01

    In a future bio-based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de novo fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl reductases (FAR) and wax ester synthases (WS) were transiently expressed in Nicotiana benthamiana leaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts.

  7. Changes induced by the Pepper mild mottle tobamovirus on the chloroplast proteome of Nicotiana benthamiana.

    PubMed

    Pineda, M; Sajnani, C; Barón, M

    2010-01-01

    We have analyzed the chloroplast proteome of Nicotiana benthamiana using two-dimensional gel electrophoresis and mass spectrometry followed by a database search. In order to improve the resolution of the two-dimensional electrophoresis gels, we have made separate maps for the low and the high pH range. At least 200 spots were detected. We identified 72 polypeptides, some being isoforms of different multiprotein families. In addition, changes in this chloroplast proteome induced by the infection with the Spanish strain of the Pepper mild mottle virus were investigated. Viral infection induced the down-regulation of several chloroplastidic proteins involved in both the photosynthetic electron-transport chain and the Benson-Calvin cycle.

  8. Transcriptomics assisted proteomic analysis of Nicotiana occidentalis infected by Candidatus Phytoplasma mali strain AT.

    PubMed

    Luge, Toni; Kube, Michael; Freiwald, Anja; Meierhofer, David; Seemüller, Erich; Sauer, Sascha

    2014-08-01

    Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant-assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up- and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha-linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.

  9. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae.

    PubMed

    Newman, Adam G; Townsend, Craig A

    2016-03-30

    Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin-a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite's biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product-an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavin-dependent monooxygenases.

  10. Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

    PubMed

    Moore, Lauren; Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

  11. Early events induced by the toxin deoxynivalenol lead to programmed cell death in Nicotiana tabacum cells.

    PubMed

    Yekkour, Amine; Tran, Daniel; Arbelet-Bonnin, Delphine; Briand, Joël; Mathieu, Florence; Lebrihi, Ahmed; Errakhi, Rafik; Sabaou, Nasserdine; Bouteau, François

    2015-09-01

    Deoxynivalenol (DON) is a mycotoxin affecting animals and plants. This toxin synthesized by Fusarium culmorum and Fusarium graminearum is currently believed to play a decisive role in the fungal phytopathogenesis as a virulence factor. Using cultured cells of Nicotiana tabacum BY2, we showed that DON-induced programmed cell death (PCD) could require transcription and translation processes, in contrast to what was observed in animal cells. DON could induce different cross-linked pathways involving (i) reactive oxygen species (ROS) generation linked, at least partly, to a mitochondrial dysfunction and a transcriptional down-regulation of the alternative oxidase (Aox1) gene and (ii) regulation of ion channel activities participating in cell shrinkage, to achieve PCD.

  12. Stable genetic transformation of intact Nicotiana cells by the particle bombardment process

    PubMed Central

    Klein, Theodore M.; Harper, Elisabeth C.; Svab, Zora; Sanford, John C.; Fromm, Michael E.; Maliga, Pal

    1988-01-01

    We show that the genetic transformation of Nicotiana tabacum can be achieved by bombarding intact cells and tissues with DNA-coated particles. Leaves or suspension culture cells were treated with tungsten microprojectiles carrying plasmid DNA containing a neomycin phosphotransferase gene. Callus harboring the foreign gene was recovered from the bombarded tissue by selection on medium containing kanamycin. Kanamycin-resistant plants have subsequently been regenerated from the callus derived from leaves. Transient expression of an introduced β-glucuronidase gene was used to assess the efficiency of DNA delivery by microprojectiles. The frequency of cells that were stably transformed with the neomycin phosphotransferase gene was a few percent of the cells that transiently expressed the β-glucuronidase gene. These results show that gene transfer by high-velocity microprojectiles is a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium. Images PMID:16593993

  13. The transcriptome of Verticillium dahliae-infected Nicotiana benthamiana determined by deep RNA sequencing.

    PubMed

    Faino, Luigi; de Jonge, Ronnie; Thomma, Bart P H J

    2012-09-01

    Verticillium wilt disease is caused by fungi of the Verticillium genus that occur on a wide range of host plants, including Solanaceous species such as tomato and tobacco. Currently, the well characterized Ve1 gene of tomato is the only Verticillium wilt resistance gene cloned. During experiments to identify the Verticillium molecule that activates Ve1 resistance in tomato, RNA sequencing (RNA-Seq) of Verticillium-infected Nicotiana benthamiana was performed. In total, over 99% of the obtained reads were derived from N. benthamiana. Here, we report the assembly and annotation of the N. benthamiana transcriptome. In total, 142,738 transcripts > 100 bp were obtained, amounting to a total transcriptome size of 38.7 Mbp, which is comparable to the Arabidopsis transcriptome. About 30,282 transcripts could be annotated based on homology to Arabidopsis genes. By assembly of the N. benthamiana transcriptome, we provide a catalogue of transcripts of a Solanaceous model plant under pathogen stress.

  14. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    PubMed

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  15. Regulation of catalase activity in leaves of Nicotiana sylvestris by high CO sub 2

    SciTech Connect

    Havir, E.A.; McHale, N.A. )

    1989-03-01

    The effect of high CO{sub 2} (1% CO{sub 2}/21% O{sub 2}) on the activity of specific forms of catalase (CAT-1, -2, and -3) in seedling leaves of tobacco (Nicotiana sylvestris, Nicotiana tabacum) was examined. In high CO{sub 2} total catalase activity decreased by 50% in the first 2 days, followed by a more gradual decline in the next 4 days. The loss of total activity resulted primarily from a decrease in CAT-1 catalase. In contrast, the activity of CAT-3 catalase, a form with enhanced peroxidatic activity, increased 3-fold in high CO{sub 2} relative to air controls after 4 days. Short-term exposure to high CO{sub 2} indicated that the 50% loss of total activity occurs in the firs 12 hours. Catalase levels increased to normal within 12 hours after seedlings were returned to air. When seedlings were transferred to air after prolonged exposure to high CO{sub 2} (13 days), the levels of CAT-1 catalase were partially restored while CAT-3 remained at its elevated level. Levels of superoxide dismutase activity and those of several peroxisomal enzymes were not affected by high CO{sub 2}. Total catalase levels did not decline when seedlings were exposed to atmospheres of 0.04% CO{sub 2}/5% O{sub 2} or 0.04% CO{sub 2}/1% O{sub 2}, indicating that regulation of catalase in high CO{sub 2} is not related directly to suppression of photorespiration. Antibodies prepared against CAT-1 catalase from N. tabacum reacted strongly against CAT-1 catalase from both N. sylvestris and N. tabacum but not against CAT-3 catalase from either species.

  16. The Only African Wild Tobacco, Nicotiana africana: Alkaloid Content and the Effect of Herbivory

    PubMed Central

    Marlin, Danica; Nicolson, Susan W.; Yusuf, Abdullahi A.; Stevenson, Philip C.; Heyman, Heino M.; Krüger, Kerstin

    2014-01-01

    Herbivory in some Nicotiana species is known to induce alkaloid production. This study examined herbivore-induced defenses in the nornicotine-rich African tobacco N. africana, the only Nicotiana species indigenous to Africa. We tested the predictions that: 1) N. africana will have high constitutive levels of leaf, flower and nectar alkaloids; 2) leaf herbivory by the African bollworm Helicoverpa armigera will induce increased alkaloid levels in leaves, flowers and nectar; and 3) increased alkaloid concentrations in herbivore-damaged plants will negatively affect larval growth. We grew N. africana in large pots in a greenhouse and exposed flowering plants to densities of one, three and six fourth-instar larvae of H. armigera, for four days. Leaves, flowers and nectar were analyzed for nicotine, nornicotine and anabasine. The principal leaf alkaloid was nornicotine (mean: 28 µg/g dry mass) followed by anabasine (4.9 µg/g) and nicotine (0.6 µg/g). Nornicotine was found in low quantities in the flowers, but no nicotine or anabasine were recorded. The nectar contained none of the alkaloids measured. Larval growth was reduced when leaves of flowering plants were exposed to six larvae. As predicted by the optimal defense theory, herbivory had a localized effect and caused an increase in nornicotine concentrations in both undamaged top leaves of herbivore damaged plants and herbivore damaged leaves exposed to one and three larvae. The nicotine concentration increased in damaged compared to undamaged middle leaves. The nornicotine concentration was lower in damaged leaves of plants exposed to six compared to three larvae, suggesting that N. africana rather invests in new growth as opposed to protecting older leaves under severe attack. The results indicate that the nornicotine-rich N. africana will be unattractive to herbivores and more so when damaged, but that potential pollinators will be unaffected because the nectar remains alkaloid-free even after herbivory. PMID

  17. Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin.

    PubMed

    Beseli, Aydin; Amnuaykanjanasin, Alongkorn; Herrero, Sonia; Thomas, Elizabeth; Daub, Margaret E

    2015-11-01

    The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants.

  18. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana

    PubMed Central

    Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J.; Phu, My L.; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Rodriguez, Raymond L.; Nandi, Somen; McDonald, Karen A.

    2017-01-01

    Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting. PMID:28054967

  19. Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana

    PubMed Central

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J.; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis. PMID:25343375

  20. Metabolomic analysis of wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses: unraveling metabolic responses.

    PubMed

    Scalabrin, Elisa; Radaelli, Marta; Rizzato, Giovanni; Bogani, Patrizia; Buiatti, Marcello; Gambaro, Andrea; Capodaglio, Gabriele

    2015-08-01

    Nicotiana langsdorffii plants, wild and transgenic for the Agrobacterium rhizogenes rol C gene and the rat glucocorticoid receptor (GR) gene, were exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations). An untargeted metabolomic analysis was carried out in order to investigate the metabolic effects of the inserted genes in response to the applied stresses and to obtain a comprehensive profiling of metabolites induced during abiotic stresses. High-performance liquid chromatography separation (HPLC) coupled to high-resolution mass spectrometry (HRMS) enabled the identification of more than 200 metabolites, and statistical analysis highlighted the most relevant compounds for each plant treatment. The plants exposed to heat stress showed a unique set of induced secondary metabolites, some of which were known while others were not previously reported for this kind of stress; significant changes were observed especially in lipid composition. The role of trichome, as a protection against heat stress, is here suggested by the induction of both acylsugars and glykoalkaloids. Water deficit and Cr(VI) stresses resulted mainly in enhanced antioxidant (HCAs, polyamine) levels and in the damage of lipids, probably as a consequence of reactive oxygen species (ROS) production. Moreover, the ability of rol C expression to prevent oxidative burst was confirmed. The results highlighted a clear influence of GR modification on plant stress response, especially to water deficiency-a phenomenon whose applications should be further investigated. This study provides new insights into the field of system biology and demonstrates the importance of metabolomics in the study of plant functioning. Graphical Abstract Untargeted metabolomic analysis was applied to wild type, GR and RolC modified Nicotiana Langsdorffii plants exposed to heat, water and Cr(VI) stresses. The key metabolites, highly affected by stress application, were identified

  1. Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

    PubMed

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

  2. Necrotic Lesion Resistance Induced by Peronospora tabacina on Leaves of Nicotiana obtusifolia.

    PubMed

    Heist, E P; Zaitlin, D; Funnell, D L; Nesmith, W C; Schardl, C L

    2004-11-01

    ABSTRACT Infection of Nicotiana obtusifolia plant introduction (PI) #555573 by the downy mildew pathogen Peronospora tabacina resulted in a compatible interaction, in which P. tabacina penetrated and freely colonized host leaf tissue. This interaction became incompatible 5 to 6 days later, with the appearance of necrotic lesions (NLs) and inhibition of pathogen growth and subsequent sporulation. NL development depended upon the presence of P. tabacina in host tissue, was not due to the effects of other microbes, and occurred co-incident in time with the pathogen's ability to produce asexual sporangia on a susceptible N. obtusifolia genotype. Inhibition of the necrotic response by CoCl(2) (a calcium channel blocker) and pathogen-induced transcription of a defense-related gene (PR-1a) suggested that necrosis was due to hypersensitive cell death in the host. In contrast, N. obtusifolia PI#555543 did not exhibit hypersensitivity upon infection by P. tabacina, but rather developed characteristic symptoms of tobacco blue mold disease: chlorotic lesions accompanied by abundant pathogen sporulation. Disease reactions scored on PI#555573 x PI#555543 F(2) progeny inoculated with P. tabacina sporangia indicated that the resistance phenotype was due to the action of a single gene from N. obtusifolia PI#555573, which we have named Rpt1. To date, Rpt1 is the only gene known to confer a hypersensitive response (HR) to P. tabacina infection in any species of Nicotiana. A survey of wild N. obtusifolia revealed that the HR to P. tabacina was expressed in the progeny of 7 of 21 (33%) plants collected in southern Arizona, but not in the progeny of plants originating from Death Valley National Park in California and the Big Bend National Park in west Texas.

  3. The transmitting tissue of Nicotiana tabacum is not essential to pollen tube growth, and its ablation can reverse prezygotic interspecific barriers.

    PubMed

    Smith, Alan G; Eberle, Carrie A; Moss, Nicole G; Anderson, Neil O; Clasen, Benjamin M; Hegeman, Adrian D

    2013-12-01

    The Nicotiana tabacum transmitting tissue is a highly specialized file of metabolically active cells that is the pathway for pollen tubes from the stigma to the ovules where fertilization occurs. It is thought to be essential to pollen tube growth because of the nutrients and guidance it provides to the pollen tubes. It also regulates gametophytic self-incompatibility in the style. To test the function of the transmitting tissue in pollen tube growth and to determine its role in regulating prezygotic interspecific incompatibility, genetic ablation was used to eliminate the mature transmitting tissue, producing a hollow style. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue-specific gene expression, self-pollen tubes had growth to the end of the style. Pollen tubes grew at a slower rate in the transmitting-tissue-ablated line during the first 24 h post-pollination. However, pollen tubes grew to a similar length 40 h post-pollination with and without a transmitting tissue. Ablation of the N. tabacum transmitting tissue significantly altered interspecific pollen tube growth. These results implicate the N. tabacum transmitting tissue in facilitating or inhibiting interspecific pollen tube growth in a species-dependent manner and in controlling prezygotic reproductive barriers.

  4. Virus-Induced Gene Silencing of Plastidial Soluble Inorganic Pyrophosphatase Impairs Essential Leaf Anabolic Pathways and Reduces Drought Stress Tolerance in Nicotiana benthamiana1[W][OA

    PubMed Central

    George, Gavin M.; van der Merwe, Margaretha J.; Nunes-Nesi, Adriano; Bauer, Rolene; Fernie, Alisdair R.; Kossmann, Jens; Lloyd, James R.

    2010-01-01

    The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes. PMID:20605913

  5. Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.)

    PubMed Central

    Yin, Guoying; Wang, Wenjing; Niu, Haixia; Ding, Yongqiang; Zhang, Dingyu; Zhang, Jie; Liu, Guanshan; Wang, Sangen; Zhang, Hongbo

    2017-01-01

    Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on JA-sensitivity for screening nicotine mutants from an activation-tagged population of tobacco. In this approach, the mutants were first screened for abnormal JA responses in seed germination and root elongation, and then the levels of nicotine synthesis and expression of nicotine synthetic genes in the mutants with altered JA-response were measured to determine the nicotine-synthetic mutants. We successfully obtained five mutants that maintained stable nicotine contents and JA responses for three generations. This method is simple, effective and low-cost, and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis. PMID:28243248

  6. Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.).

    PubMed

    Yin, Guoying; Wang, Wenjing; Niu, Haixia; Ding, Yongqiang; Zhang, Dingyu; Zhang, Jie; Liu, Guanshan; Wang, Sangen; Zhang, Hongbo

    2017-01-01

    Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on JA-sensitivity for screening nicotine mutants from an activation-tagged population of tobacco. In this approach, the mutants were first screened for abnormal JA responses in seed germination and root elongation, and then the levels of nicotine synthesis and expression of nicotine synthetic genes in the mutants with altered JA-response were measured to determine the nicotine-synthetic mutants. We successfully obtained five mutants that maintained stable nicotine contents and JA responses for three generations. This method is simple, effective and low-cost, and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis.

  7. Rapid metabolic profiling of Nicotiana tabacum defence responses against Phytophthora nicotianae using direct infrared laser desorption ionization mass spectrometry and principal component analysis

    PubMed Central

    2010-01-01

    Background Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection. Results Infrared laser desorption ionization orthogonal time-of-flight mass spectrometry allows rapid and simultaneous detection in both negative and positive ion mode of a wide range of naturally occurring primary and secondary metabolites. An unsupervised principal component analysis was employed to identify correlations between changes in metabolite expression (obtained at different times and sample treatment conditions) and the overall defence response. A one-dimensional projection of the principal components 1 and 2 obtained from positive ion mode spectra was used to generate a Biological Response Index (BRI). The BRI obtained for each sample treatment was compared with the number of dead cells found in the respective tissue. The high correlation between these two values suggested that the BRI provides a rapid assessment of the plant response against the pathogen infection. Evaluation of the loading plots of the principal components (1 and 2) reveals a correlation among three metabolic cascades and the defence response generated in infected leaves. Analysis of selected phytohormones by liquid chromatography electrospray ionization mass spectrometry verified our findings. Conclusion The described methodology allows for rapid assessment of infection-specific changes in the plant metabolism, in particular of phenolics, alkaloids

  8. Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR.

    PubMed

    Li, Mingzhu; Inada, Minoru; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2013-01-01

    Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

  9. Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae.

    PubMed

    Ehrenshaft, M; Chung, K R; Jenns, A E; Daub, M E

    1999-01-01

    The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.

  10. An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines.

    PubMed

    Ling, Huai-Yian; Edwards, Aaron M; Gantier, Michael P; Deboer, Kathleen D; Neale, Alan D; Hamill, John D; Walmsley, Amanda M

    2012-01-01

    The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs.

  11. The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae.

    PubMed

    Choquer, Mathias; Dekkers, Katherine L; Chen, Hui-Qin; Cao, Lihua; Ueng, Peter P; Daub, Margaret E; Chung, Kuang-Ren

    2005-05-01

    Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.

  12. Synthesis of 'cineole cassette' monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis.

    PubMed

    Fähnrich, Anke; Brosemann, Anne; Teske, Laura; Neumann, Madeleine; Piechulla, Birgit

    2012-08-01

    The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, β-myrcene, α- and β-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.

  13. Quantitative Analysis of the Fate of Exogenous DNA in Nicotiana Protoplasts 1

    PubMed Central

    Uchimiya, Hirofumi; Murashige, Toshio

    1977-01-01

    After a 5-hour incubation of protoplasts of Nicotiana tabacum L. `Xanthi' with 3H-DNA (7.26 μg/ml) from N. tabacum L. `Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene. A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli3H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the 3H-DNA entered the protoplasts. Competition experiments between the 3H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded 3H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the “soluble” fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable 3H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 × 10−15g donor DNA, or

  14. Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids

    PubMed Central

    Reynolds, Kyle B.; Taylor, Matthew C.; Zhou, Xue-Rong; Vanhercke, Thomas; Wood, Craig C.; Blanchard, Christopher L.; Singh, Surinder P.; Petrie, James R.

    2015-01-01

    Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation. PMID:25852716

  15. Zoospore density-dependent behaviors of Phytophthora nicotianae are autoregulated by extracellular products.

    PubMed

    Kong, Ping; Hong, Chuanxue

    2010-07-01

    Phytophthora species are destructive fungus-like plant pathogens that use asexual single-celled flagellate zoospores for dispersal and plant infection. Many of the zoospore behaviors are density-dependent although the underlying mechanisms are poorly understood. Here, we use P. nicotianae as a model and demonstrate autoregulation of some zoospore behaviors using signal molecules that zoospores release into the environment. Specifically, zoospore aggregation, plant targeting, and infection required or were enhanced by threshold concentrations of these signal molecules. Below the threshold concentration, zoospores did not aggregate and move toward a cauline leaf of Arabidopsis thaliana (Col-0) and failed to individually attack annual vinca (Catharanthus roseus cv. Little Bright Eye). These processes were reversed when supplemented with zoospore-free fluid (ZFF) prepared from a zoospore suspension above threshold densities but not with calcium chloride at a concentration equivalent to extracellular Ca(2+) in ZFF. These results suggest that Ca(2+) is not a primary signal molecule regulating these communal behaviors. Zoospores coordinated their communal behaviors by releasing, detecting, and responding to signal molecules. This chemical communication mechanism raises the possibility that Phytophthora plant infection may not depend solely on zoospore number in the real world. Single zoospore infection may take place if it is signaled by a common molecule available in the environment which contributes to the destructiveness of these plant pathogens.

  16. Larval Helicoverpa zea Transcriptional, Growth and Behavioral Responses to Nicotine and Nicotiana tabacum

    PubMed Central

    Gog, Linus; Vogel, Heiko; Hum-Musser, Sue M.; Tuter, Jason; Musser, Richard O.

    2014-01-01

    The polyphagous feeding habits of the corn earworm, Helicoverpa zea (Boddie), underscore its status as a major agricultural pest with a wide geographic distribution and host plant repertoire. To study the transcriptomic response to toxins in diet, we conducted a microarray analysis of H. zea caterpillars feeding on artificial diet, diet laced with nicotine and Nicotiana tabacum (L.) plants. We supplemented our analysis with growth and aversion bioassays. The transcriptome reflects an abundant expression of proteases, chitin, cytochrome P450 and immune-related genes, many of which are shared between the two experimental treatments. However, the tobacco treatment tended to elicit stronger transcriptional responses than nicotine-laced diet. The salivary factor glucose oxidase, known to suppress nicotine induction in the plant, was upregulated by H. zea in response to tobacco but not to nicotine-laced diet. Reduced caterpillar growth rates accompanied the broad regulation of genes associated with growth, such as juvenile hormone epoxide hydrolase. The differential expression of chemosensory proteins, such as odorant binding-protein-2 precursor, as well as the neurotransmitter nicotinic-acetylcholine-receptor subunit 9, highlights candidate genes regulating aversive behavior towards nicotine. We suggest that an observed coincidental rise in cannibalistic behavior and regulation of proteases and protease inhibitors in H. zea larvae signify a compensatory response to induced plant defenses. PMID:26462833

  17. The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats.

    PubMed

    Welch, K D; Panter, K E; Lee, S T; Gardner, D R

    2015-01-01

    Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

  18. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Pitino, Marco; Armstrong, Cheryl M.; Cano, Liliana M.; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus “Las” is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  19. Evaluation of hepatoprotective effect of Pistacia lentiscus, Phillyrea latifolia and Nicotiana glauca.

    PubMed

    Janakat, Sana; Al-Merie, Hela

    2002-11-01

    The hepatoprotective effect of the boiled and non-boiled aqueous extracts of Pistacia lentiscus, Phillyrea latifolia, and Nicotiana glauca, that are alleged to be effective in the treatment of jaundice in Jordanian folk medicine, was evaluated in vivo using carbon tetrachloride (CCl(4)) intoxicated rats as an experimental model. Plant extracts were administrated orally at a dose of 4 ml/kg body weight, containing various amounts of solid matter. Only total serum bilirubin level was reduced by treatment with non-boiled aqueous extract of N. glauca leaves, while the boiled and non-boiled aqueous extracts of the N. glauca flowers were non effective. Bilirubin level and the activity of alkaline phosphatase (ALP) were both reduced upon treatment with boiled aqueous extract of P. latifolia without reducing the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Aqueous extract of P. lentiscus (both boiled and non-boiled) showed marked antihepatotoxic activity against CCl(4) by reducing the activity of the three enzymes and the level of bilirubin. The effect of the non-boiled aqueous extract was more pronounced than that of the boiled extract.

  20. Mitochondrial alternative oxidase maintains respiration and preserves photosynthetic capacity during moderate drought in Nicotiana tabacum.

    PubMed

    Dahal, Keshav; Wang, Jia; Martyn, Greg D; Rahimy, Farkhunda; Vanlerberghe, Greg C

    2014-11-01

    The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum 'Petit Havana SR1' wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.

  1. Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Schneider, Jeannine D; Marillonnet, Sylvestre; Castilho, Alexandra; Gruber, Clemens; Werner, Stefan; Mach, Lukas; Klimyuk, Victor; Mor, Tsafrir S; Steinkellner, Herta

    2014-01-01

    Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants. PMID:24618259

  2. Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus.

    PubMed

    Pérez-Bueno, María Luisa; Ciscato, Massimo; VandeVen, Martin; García-Luque, Isabel; Valcke, Roland; Barón, Matilde

    2006-11-01

    We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.

  3. Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants.

    PubMed

    Ryan, Annette C; Hewitt, C Nicholas; Possell, Malcolm; Vickers, Claudia E; Purnell, Anna; Mullineaux, Philip M; Davies, William J; Dodd, Ian C

    2014-01-01

    Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty.

  4. Integration and expression of a rabbit liver cytochrome P-450 gene in transgenic Nicotiana tabacum.

    PubMed Central

    Saito, K; Noji, M; Ohmori, S; Imai, Y; Murakoshi, I

    1991-01-01

    Cytochrome P-450 is involved in the oxidative metabolism of a broad range of substrates. We have made a chimeric construct, pSN002, containing the cDNA for rabbit liver cytochrome P-450 (IIC14) under the control of the TR2' promoter for mannopine synthase in the Agrobacterium Ti plasmid. Nicotiana tabacum was transformed with Agrobacterium tumefaciens harboring a cointegrated plasmid pSN002::pGV2260. The presence of mRNA and of the translated protein from the chimeric cytochrome P-450 gene in transgenic plants was confirmed by RNA blot hybridization and by Western blot and immunohistochemical analyses, respectively. The transformants in which the foreign cytochrome P-450 protein is expressed show marked phenotypic changes, notably a tendency rapidly to senesce. We detected 2-propenylpyrrolidine, a degradative metabolite of nicotine alkaloids, in transgenic tobacco showing this pronounced phenotypic change. Such metabolism is likely to be due to the effect of senescence and not directly to the presence of the cytochrome P-450. Images PMID:1714583

  5. Silencing of the FRO1 gene and its effects on iron partition in Nicotiana benthamiana.

    PubMed

    Gama, Florinda; Saavedra, Teresa; Dandlen, Susana; de Varennes, Amarilis; Correia, Pedro J; Pestana, Maribela; Nolasco, Gustavo

    2017-03-06

    To evaluate the dynamic role of the ferric-chelate reductase enzyme (FCR) and to identify possible pathways of regulation of its activity in different plant organs an investigation was conducted by virus-induced gene silencing (VIGS) using tobacco rattle virus (TRV) to silence the ferric reductase oxidase gene (FRO1) that encodes the FCR enzyme. Half of Nicotiana benthamiana plants received the VIGS vector and the rest remained as control. Four treatments were imposed: two levels of Fe in the nutrient solution (0 or 2.5 μM of Fe), each one with silenced or non-silenced (VIGS-0; VIGS-2.5) plants. Plants grown without iron (0; VIGS-0) developed typical symptoms of iron deficiency in the youngest leaves. To prove that FRO1 silencing had occurred, resupply of Fe (R) was done by adding 2.5 μM of Fe to the nutrient solution in a subset of chlorotic plants (0-R; VIGS-R). Twelve days after resupply, 0-R plants had recovered from Fe deficiency while plants containing the VIGS vector (VIGS-R) remained chlorotic and both FRO1 gene expression and FCR activity were considerably reduced, consequently preventing Fe uptake. With the VIGS technique we were able to silence the FRO1 gene in N. benthamiana and point out its importance in chlorophyll synthesis and Fe partition.

  6. Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

    PubMed

    Betti, Camilla; Lico, Chiara; Maffi, Dario; D'Angeli, Simone; Altamura, Maria Maddalena; Benvenuto, Eugenio; Faoro, Franco; Baschieri, Selene

    2012-02-01

    Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

  7. Crystalline ribulose bisphosphate carboxylase/oxygenase of high integrity and catalytic activity from Nicotiana tabacum.

    PubMed

    Servaites, J C

    1985-04-01

    Crystalline tobacco (Nicotiana tabacum L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was prepared using a procedure which protected the enzyme from hydrolysis by endogenous proteases. Leaves were extracted in a buffered medium containing casein, leupeptin, and high concentrations of MgSO4 and NaHCO3. After filtration through ion-exchange resin to remove contaminants, the enzyme was concentrated by precipitation with polyethylene glycol and crystal formation was induced by low-salt dialysis. The crystalline enzyme had a measured specific activity of 1.7 mumol CO2 mg protein-1 min-1, and about 93% of the enzyme could be activated with Mg2+ and CO2. Crystalline enzyme prepared in the absence of casein exhibited an activity which was only one-third of this rate and only about 70% of the enzyme could be activated with Mg2+ and CO2. Casein-extracted enzyme was resolved into distinct bands corresponding to the large (55,000) and small (14,000) subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of enzyme prepared according to the latter procedure was found to be composed of five different polypeptides of slightly decreasing molecular weight. Only about one-third of the large subunits were of the 55,000 molecular weight type. No differences between the two preparations were observed in the Km (CO2) and apparent Km (ribulose bisphosphate).

  8. Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction.

    PubMed

    Villarroel, Carlos A; Jonckheere, Wim; Alba, Juan M; Glas, Joris J; Dermauw, Wannes; Haring, Michel A; Van Leeuwen, Thomas; Schuurink, Robert C; Kant, Merijn R

    2016-04-01

    Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two-spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector-like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage-specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant-eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses.

  9. Identification of photoperiod-regulated gene in soybean and functional analysis in Nicotiana benthamiana.

    PubMed

    Ai-Hua, Sha; Yin-Hua, Chen; Zhi-Hui, Shan; Xiao-Juan, Zhang; Xue-Jun, Wu; De-Zheng, Qiu; Xin-An, Zhou

    2014-04-01

    Soybean (Glycine max) is a short-day crop and the photoperiod is a crucial factor regulating its flowering time. To investigate the molecular mechanism controlling the flowering time by photoperiod in soybean, cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify photoperiod-regulated genes in leaves of soybean growing under short-day length, neutral photoperiod and long-day length. A total of 36 transcript-derived fragments (TDFs) were identified to be regulated by photoperiod. Among them, 26 TDFs were homologues of genes with known function. These genes are involved in secondary metabolism, cellular metabolism, cell wall components metabolism, ion transport and hormone signalling. Silencing of the homologue genes in Nicotiana benthamiana for 14 TDFs was conducted by virus-induced gene silencing. The flowering time was delayed by silencing of the genes encoding rhodanese and 40S ribosomal protein S4 (RPS4). The results indicated that rhodanese and RPS4 probably play important roles in regulating flowering time.

  10. Metabolic engineering of Nicotiana benthamiana for the increased production of taxadiene.

    PubMed

    Hasan, Md Mohidul; Kim, Hyun-Soon; Jeon, Jae-Heung; Kim, Sung Hong; Moon, BoKyung; Song, Jai-Young; Shim, Sang Hee; Baek, Kwang-Hyun

    2014-06-01

    We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting. Paclitaxel (Taxol(®)) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11-27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

  11. The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in Nicotiana benthamiana

    PubMed Central

    Deng, Xing-Guang; Zhu, Tong; Zhang, Da-Wei; Lin, Hong-Hui

    2015-01-01

    Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana. PMID:26175355

  12. Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2015-01-01

    Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable. PMID:25561925

  13. Metabolomic analysis of isonitrosoacetophenone-induced perturbations in phenolic metabolism of Nicotiana tabacum cells.

    PubMed

    Madala, Ntakadzeni E; Steenkamp, Paul A; Piater, Lizelle A; Dubery, Ian A

    2013-10-01

    Plants have developed biochemical and molecular responses to adapt to different stress environments. One of the characteristics of the multi-component defence response is the production of defence-related metabolites. Plant defences can be triggered by various stimuli, including synthetic or naturally occurring molecules, especially those derived from pathogens. In the current study, Nicotiana tabacum cell suspensions were treated with isonitrosoacetophenone (INAP), a subcomponent of a plant-derived stress metabolite with anti-fungal and anti-oxidant properties, in order to investigate the effect thereof on cellular metabolism. Subsequent metabolomic-based analyses were employed to evaluate changes in the metabolome. UPLC-MS in conjunction with multivariate data analyses was found to be an appropriate approach to study the effect of chemical inducers like INAP on plant metabolism in this model system. Principal component analysis (PCA) indicated that INAP is capable of inducing time-dependent metabolic perturbations in the cultured cells. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) revealed metabolites of which the levels are affected by INAP, and eight of these were tentatively annotated from the mass spectral data and online databases. These metabolites are known in the context of plant stress- and defence responses and include benzoic- or cinnamic acid derivatives that are either glycosylated or quinilated as well as flavonoid derivatives. The results indicate that INAP affects the shikimate-, phenylpropanoid- and flavonoid pathways, the products of which may subsequently lead to an anti-oxidant environment in vivo.

  14. Lysinibacillus tabacifolii sp. nov., a novel endophytic bacterium isolated from Nicotiana tabacum leaves.

    PubMed

    Duan, Yan-Qing; He, Song-Tao; Li, Qing-Qing; Wang, Ming-Feng; Wang, Wen-Yuan; Zhe, Wei; Cao, Yong-Hong; Mo, Ming-He; Zhai, Yu-Long; Li, Wen-Jun

    2013-06-01

    A Gram-positive, catalase- and oxidase-positive, strictly aerobic, endospore-forming rod bacterium, designated K3514(T), was isolated from the leaves of Nicotiana tabacum. The strain was able to grow at temperatures of 8-40°C, pH 5.0-10.0 and NaCl concentrations of 0-7%. The predominant quinones (>30%) of this strain were MK-7(H2) and MK-7. Phylogenetic analysis of 16S rRNA gene sequence showed that strain K3514(T) was affiliated to the genus Lysinibacillus, with its closest relatives being Lysinibacillus mangiferihumi (98.3% sequence similarity), Lysinibacillus sphaericus (97.9% sequence similarity), Lysinibacillus fusiformis (97.4% sequence similarity), and Lysinibacillus xylanilyticus (97.3% sequence similarity). However, low levels of DNA-DNA relatedness values suggested that the isolate was distinct from the other closest Lysinibacillus species. Additionally, based on analysis of morphological, physiological, and biochemical characteristics, the isolate could be differentiated from the closest known relatives. Therefore, based on polyphasic taxonomic data, the novel isolate likely represents a novel species, for which the name Lysinibacillus tabacifolii sp. nov. and the type strain K3514(T) (=KCTC 33042(T) =CCTCC AB 2012050(T)) are proposed.

  15. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  16. Optimization of Engineered Production of the Glucoraphanin Precursor Dihomomethionine in Nicotiana benthamiana

    PubMed Central

    Crocoll, Christoph; Mirza, Nadia; Reichelt, Michael; Gershenzon, Jonathan; Halkier, Barbara Ann

    2016-01-01

    Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we obtained fivefold to eightfold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: (1) coexpression of the large subunit (LSU1) of the heterodimeric isopropylmalate isomerase and (2) coexpression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomomethionine (DHM) levels to a maximum of 432 nmol g−1 fresh weight that is equivalent to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host. PMID:26909347

  17. Molecular analysis of the cercosporin biosynthetic gene cluster in Cercospora nicotianae.

    PubMed

    Chen, Huiqin; Lee, Miin-Huey; Daub, Margret E; Chung, Kuang-Ren

    2007-05-01

    We describe a core gene cluster, comprised of eight genes (designated CTB1-8), and associated with cercosporin toxin production in Cercospora nicotianae. Sequence analysis identified 10 putative open reading frames (ORFs) flanking the previously characterized CTB1 and CTB3 genes that encode, respectively, the polyketide synthase and a dual methyltransferase/monooxygenase required for cercosporin production. Expression of eight of the genes was co-ordinately induced under cercosporin-producing conditions and was regulated by the Zn(II)Cys(6) transcriptional activator, CTB8. Expression of the genes, affected by nitrogen and carbon sources and pH, was also controlled by another transcription activator, CRG1, previously shown to regulate cercosporin production and resistance. Disruption of the CTB2 gene encoding a methyltransferase or the CTB8 gene yielded mutants that were completely defective in cercosporin production and inhibitory expression of the other CTB cluster genes. Similar 'feedback' transcriptional inhibition was observed when the CTB1, or CTB3 but not CTB4 gene was inactivated. Expression of four ORFs located on the two distal ends of the cluster did not correlate with cercosporin biosynthesis and did not show regulation by CTB8, suggesting that the biosynthetic cluster was limited to CTB1-8. A biosynthetic pathway and a regulatory network leading to cercosporin formation are proposed.

  18. Effects of Iron Excess on Nicotiana plumbaginifolia Plants (Implications to Oxidative Stress).

    PubMed Central

    Kampfenkel, K.; Van Montagu, M.; Inze, D.

    1995-01-01

    Fe excess is believed to generate oxidative stress. To contribute to the understanding of Fe metabolism, Fe excess was induced in Nicotiana plumbaginifolia grown in hydroponic culture upon root cutting. Toxicity symptoms leading to brown spots covering the leaf surface became visible after 6 h. Photosynthesis was greatly affected within 12 h; the photosynthetic rate was decreased by 40%. Inhibition of photosynthesis was accompanied by photoinhibition, increased reduction of photosystem II, and higher thylakoid energization. Fe excess seemed to stimulate photorespiration because catalase activity doubled. To cope with cellular damage, respiration rate increased and cytosolic glucose-6-phosphate dehydrogenase activity more than doubled. Simultaneously, the content of free hexoses was reduced. Indicative of generation of oxidative stress was doubling of ascorbate peroxidase activity within 12 h. Contents of the antioxidants ascorbate and glutathione were reduced by 30%, resulting in equivalent increases of dehydroascorbate and oxidized glutathione. Taken together, moderate changes in leaf Fe content have a dramatic effect on plant metabolism. This indicates that cellular Fe concentrations must be finely regulated to avoid cellular damage most probably because of oxidative stress induced by Fe. PMID:12228397

  19. Localization of Barley yellow dwarf virus Movement Protein Modulating Programmed Cell Death in Nicotiana benthamiana.

    PubMed

    Ju, Jiwon; Kim, Kangmin; Lee, Kui-Jae; Lee, Wang Hu; Ju, Ho-Jong

    2017-02-01

    Barley yellow dwarf virus (BYDV) belongs to Luteovirus and is limited only at phloem related tissues. An open reading frame (ORF) 4 of BYDV codes for the movement protein (MP) of BYDV gating plasmodesmata (PD) to facilitate virus movement. Like other Luteoviruses, ORF 4 of BYDV is embedded in the ORF3 but expressed from the different reading frame in leaky scanning manner. Although MP is a very important protein for systemic infection of BYDV, there was a little information. In this study, MP was characterized in terms of subcellular localization and programmed cell death (PCD). Gene of MP or its mutant (ΔMP) was expressed by Agroinfiltration method. MP was clearly localized at the nucleus and the PD, but ΔMP which was deleted distal N-terminus of MP showed no localization to PD exhibited the different target with original MP. In addition to PD localization, MP appeared associated with small granules in cytoplasm whereas ΔMP did not. MP associated with PD and small granules induced PCD, but ΔMP showed no association with PD and small granules did not exhibit PCD. Based on this study, the distal N-terminal region within MP is seemingly responsible for the localization of PD and the induction small granules and PCD induction. These results suggest that subcellular localization of BYDV MP may modulate the PCD in Nicotiana benthamiana.

  20. A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

    PubMed

    Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

    2015-06-03

    Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

  1. Localization of Barley yellow dwarf virus Movement Protein Modulating Programmed Cell Death in Nicotiana benthamiana

    PubMed Central

    Ju, Jiwon; Kim, Kangmin; Lee, Kui-Jae; Lee, Wang Hu; Ju, Ho-Jong

    2017-01-01

    Barley yellow dwarf virus (BYDV) belongs to Luteovirus and is limited only at phloem related tissues. An open reading frame (ORF) 4 of BYDV codes for the movement protein (MP) of BYDV gating plasmodesmata (PD) to facilitate virus movement. Like other Luteoviruses, ORF 4 of BYDV is embedded in the ORF3 but expressed from the different reading frame in leaky scanning manner. Although MP is a very important protein for systemic infection of BYDV, there was a little information. In this study, MP was characterized in terms of subcellular localization and programmed cell death (PCD). Gene of MP or its mutant (ΔMP) was expressed by Agroinfiltration method. MP was clearly localized at the nucleus and the PD, but ΔMP which was deleted distal N-terminus of MP showed no localization to PD exhibited the different target with original MP. In addition to PD localization, MP appeared associated with small granules in cytoplasm whereas ΔMP did not. MP associated with PD and small granules induced PCD, but ΔMP showed no association with PD and small granules did not exhibit PCD. Based on this study, the distal N-terminal region within MP is seemingly responsible for the localization of PD and the induction small granules and PCD induction. These results suggest that subcellular localization of BYDV MP may modulate the PCD in Nicotiana benthamiana. PMID:28167888

  2. Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures.

    PubMed

    Raeymaekers, T; Potters, G; Asard, H; Guisez, Y; Horemans, N

    2003-05-01

    In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H(2)O(2). Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2%. This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90%. The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.

  3. Role of brassinosteroid signaling in modulating Tobacco mosaic virus resistance in Nicotiana benthamiana

    PubMed Central

    Deng, Xing-Guang; Zhu, Tong; Peng, Xing-Ji; Xi, De-Hui; Guo, Hongqing; Yin, Yanhai; Zhang, Da-Wei; Lin, Hong-Hui

    2016-01-01

    Plant steroid hormones, brassinosteroids (BRs), play essential roles in plant growth, development and stress responses. However, mechanisms by which BRs interfere with plant resistance to virus remain largely unclear. In this study, we used pharmacological and genetic approaches in combination with infection experiments to investigate the role of BRs in plant defense against Tobacco Mosaic Virus (TMV) in Nicotiana benthamiana. Exogenous applied BRs enhanced plant resistance to virus infection, while application of Bikinin (inhibitor of glycogen synthase kinase-3), which activated BR signaling, increased virus susceptibility. Silencing of NbBRI1 and NbBSK1 blocked BR-induced TMV resistance, and silencing of NbBES1/BZR1 blocked Bikinin-reduced TMV resistance. Silencing of NbMEK2, NbSIPK and NbRBOHB all compromised BR-induced virus resistance and defense-associated genes expression. Furthermore, we found MEK2-SIPK cascade activated while BES1/BZR1 inhibited RBOHB-dependent ROS production, defense gene expression and virus resistance induced by BRs. Thus, our results revealed BR signaling had two opposite effects on viral defense response. On the one hand, BRs enhanced virus resistance through MEK2-SIPK cascade and RBOHB-dependent ROS burst. On the other hand, BES1/BZR1 inhibited RBOHB-dependent ROS production and acted as an important mediator of the trade-off between growth and immunity in BR signaling. PMID:26838475

  4. Heat shock, with recovery, promotes protection of Nicotiana tabacum during subsequent exposure to Ralstonia solanacearum.

    PubMed

    Byth-Illing, Heather-Anne; Bornman, Liza

    2014-03-01

    Host-pathogen interactions in plants are complex and potentially influenced by heat shock/stress (HS). Host HS proteins (HSPs) induced prior to bacterial exposure may facilitate the folding of newly synthesized defense proteins and promote incompatible host-pathogen interactions. We hypothesized that a non-lethal HS, with recovery, promotes protection of Nicotiana tabacum during subsequent exposure to avirulent soilborne necrotrophic pathogen Ralstonia solanacearum. The objective of this study included investigating the effects of HS with or without recovery on the outcome of bacterial exposure to a virulent and avirulent biovar of R. solanacearum in N. tabacum cell suspensions. This was assessed by quantifying host Hsp70/Hsc70 levels, mitochondrial electron (e (-)) transport activity as a marker of viability, and phosphatidylserine externalization and DNA fragmentation as markers of apoptosis. Our findings support the hypothesis that HS, with recovery, promotes protection of N. tabacum during subsequent exposure to R. solanacearum, suggesting a role for Hsp70/Hsc70 in the observed protection of e (-) transport, increased apoptosis, and DNA fragmentation.

  5. Nicotiana benthamiana: Its History and Future as a Model for Plant-Pathogen Interactions.

    PubMed

    Goodin, Michael M; Zaitlin, David; Naidu, Rayapati A; Lommel, Steven A

    2015-01-01

    Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Addi- tionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus- induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, inter- action, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions cur- rently used by the research community. In addition to ad- dressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

  6. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae

    PubMed Central

    Newman, Adam G.; Townsend, Craig A.

    2016-01-01

    Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin—a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite’s biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product—an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavin-dependent monooxygenases. PMID:26938470

  7. A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style.

    PubMed

    Eberle, Carrie A; Clasen, Benjamin M; Anderson, Neil O; Smith, Alan G

    2012-03-01

    Sexual plant reproduction requires multiple pollen-pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.

  8. In vitro antioxidant properties of flavonoids and polysaccharides extract from tobacco (Nicotiana tabacum L.) leaves.

    PubMed

    Ru, Qiao-Mei; Wang, Li-Juan; Li, Wei-Ming; Wang, Jing-Lu; Ding, Yu-Ting

    2012-09-21

    In the present study, antioxidant properties of flavonoids and polysaccharides from tobacco (Nicotiana tabacum L.) leaves were evaluated in several in vitro systems, e.g., scavenging activities on hydroxyl, superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals, and reducing power. Flavonoids showed much better activity than polysaccharides in scavenging activities on free radicals. When compared to the positive control, ascorbic acid, both showed weaker antioxidant potential. However, flavonoids possessed comparable superoxide anion, DPPH and ABTS radical scavenging abilities to ascorbic acid at high concentration (600 μg/mL). Meanwhile, it was found that flavonoids had prominent effects on the reducing power, which was equivalent to ascorbic acid, and was significantly higher than polysaccharides. These results clearly indicate that flavonoids are effective in scavenging free radicals and have the potential to be powerful antioxidants. Thus, tobacco leaves could be considered as a potential source of natural antioxidants for food, pharmaceutical, cosmetics or nutraceutical industries.

  9. Spectral reflectance, chlorophyll fluorescence and virological investigations of tobacco plants (Nicotiana tabacum L.) infected with Tobacco mosaic virus (TMV)

    NASA Astrophysics Data System (ADS)

    Krezhova, Dora; Hristova, Dimitrina; Iliev, Ilko; Yanev, Tony

    Application of multispectral remote sensing techniques to plant condition monitoring has been adopted for various purposes. Remote sensing is a reliable tool for detecting signs of vege-tation stress and diseases. Spectral reflectance and chlorophyll fluorescence are functions of tissue optical properties and biological status of the plants, and illumination conditions. The mean reflectance spectrum depends on the relative composition of all the pigments in the leaf including chlorophylls, carotenoids etc. Chlorophyll fluorescence results from the primary re-actions of photosynthesis and during the last decade it finds widening application as a means for revelation of stress and diseases. The changes in chlorophyll function take place before the alteration in chlorophyll content to occur so that changes in the fluorescence signal arise before any visible signs are apparent. The aim of our investigations was to study the development and spreading out of a viral infection on the leaves of two cultivars tobacco plants (Nicotiana tabacum L.) infected with Tobacco mosaic virus (TMV). We applied two remote sensing tech-niques (spectral reflectance and chlorophyll fluorescence measurements) for evaluation of the changes in the optical properties of the plants in accordance to their physiological status. The serological analyses via the Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) were made with appropriate kits (Leowe, Germany) for quantitative assessment of the concentration of viruses in the plants. The tobacco plants were grown in green house under controlled conditions. The first cultivar Nevrocop 1146 is known as resistive to the TMV, i.e. it shows hypersensitive response. The second cultivar named Krumovgrad is normally sen-sitive to the TMV. At growth stage 4-6 expanded leaf, up to one leaf from 20 plants for each cultivar were inoculated with TMV. The leaves opposite to the infected ones formed the group of control (untreated) leaves. The

  10. In vitro inhibition of incompatible pollen tubes in Nicotiana alata involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system.

    PubMed

    Roldán, Juan A; Rojas, Hernán J; Goldraij, Ariel

    2015-01-01

    In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S c₁₀-, S₇₀-, and S₇₅-haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed.

  11. Ethylene and the Regulation of Senescence Processes in Transgenic Nicotiana sylvestris Plants

    PubMed Central

    Yang, Thomas F.; Gonzalez-Carranza, Zinnia H.; Maunders, Martin J.; Roberts, Jeremy A.

    2008-01-01

    Background and Aims Exposure of plants to ethylene can influence a spectrum of developmental processes including organ senescence and abscission. The aim of this study was to examine the role of the gaseous regulator in Nicotiana sylvestris plants exhibiting a silenced or constitutive ethylene response. Methods Transgenic N. sylvestris plants were generated that either ectopically expressed the Arabidopsis mutant ethylene receptor ETR1-1 or the tomato EIN3-like (LeEIL1) gene. Highly expressing homozygous lines were selected and the time-course of development, from germination to organ senescence, was studied. Key Results Fifty percent of the homozygous Pro35S:ETR1-1 lines examined showed a high susceptibility to collapse prior to flowering, with plant death occurring within a few days of leaf wilting. The time-course of leaf senescence in the remaining Pro35S:ETR1-1 lines was visibly arrested compared to wild type (negative segregant) plants and this observation was reaffirmed by chlorophyll and protein analysis. Petal necrosis was also delayed in Pro35S:ETR1-1 lines and corolla abscission did not take place. When senescence of Pro35S:ETR1-1 plants did take place this was accompanied by leaf bleaching, but tissues remained fully turgid and showed no signs of collapse. A single Pro35S:LeEIL1 line was found to exhibit consistently accelerated leaf and flower senescence and precocious flower bud shedding. Conclusions These observations support a role for ethylene in regulating a spectrum of developmental events associated with organ senescence and tissue necrosis. Furthermore, the transgenic lines generated during this study may provide a valuable resource for exploring how senescence processes are regulated in plants. PMID:17901061

  12. Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L.

    PubMed Central

    Sheveleva, E.; Chmara, W.; Bohnert, H. J.; Jensen, R. G.

    1997-01-01

    A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present. PMID:12223867

  13. [Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration].

    PubMed

    Hou, Lili; Shi, Heping; Yu, Wu; Tsang, Po Keung Eric; Chow, Cheuk Fai Stephen

    2014-04-01

    By genetic transformation with Agrobacterum rhizogenes and artificial chromosome doubling techniques, we studied the induction of hairy roots and their polyploidization, and subsequent plant regeneration and nicotine determination to enhance the content of nicotine in Nicotiana tabacum. The results show that hairy roots could be induced from the basal surface of leaf explants of N. tabacum 8 days after inoculation with Agrobacterium rhizogenes ATCC15834. The percentage of the rooting leaf explants was 100% 15 days after inoculation. The hairy roots could grow rapidly and autonomously on solid or liquid phytohormones-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and paper electrophoresis of opines from N. tabacum hairy roots. The highest rate of polyploidy induction, more than 64.71%, was obtained after treatment of hairy roots with 0.1% colchicine for 36 h. The optimum medium for plant regeneration from polyploid hairy roots was MS+2.0 mg/L 6-BA +0.2 mg/L NAA. Compared with the control diploid plants, the hairy roots-regenerated plants had weak apical dominance, more axillary buds and more narrow leaves; whereas the polyploid hairy root-regenerated plants had thicker stems, shorter internodes and the colour, width and thickness of leaves were significantly higher than that of the control. Observation of the number of chromosomes in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 96 (4n = 96) chromosomes. Pot-grown experiments showed compared to the control, the flowering was delayed by 21 days in diploid hairy roots-regenerated plants and polyploid hairy root-regenerated plants. GC-MS detection shows that the content of nicotine in polyploid plants was about 6.90 and 4.57 times the control and the diploid hairy roots-regenerated plants, respectively.

  14. Heterologous expression and characterization of choline oxidase from the soil bacterium Arthrobacter nicotianae.

    PubMed

    Ribitsch, D; Karl, W; Wehrschütz-Sigl, E; Tutz, S; Remler, P; Weber, H J; Gruber, K; Stehr, R; Bessler, C; Hoven, N; Sauter, K; Maurer, K H; Schwab, H

    2009-01-01

    In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 degrees C). Specific activities were determined toward choline chloride (4.70 +/- 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 +/- 0.45 x 10(-2) U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 +/- 0.12 x 10(-2) U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K (M) = 1.51 +/- 0.09 mM and V (max) = 42.73 +/- 0.42 mU/min for choline chloride and K (M) = 4.77 +/- 0.76 mM and V (max) = 48.40 +/- 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.

  15. Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum

    PubMed Central

    2013-01-01

    The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of “-ene” reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover “-ene” reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE “-ene” reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme. PMID:27547488

  16. Production of an active anti-CD20-hIL-2 immunocytokine in Nicotiana benthamiana.

    PubMed

    Marusic, Carla; Novelli, Flavia; Salzano, Anna M; Scaloni, Andrea; Benvenuto, Eugenio; Pioli, Claudio; Donini, Marcello

    2016-01-01

    Anti-CD20 murine or chimeric antibodies (Abs) have been used to treat non-Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti-CD20 Abs demonstrated to be effective in inducing regression of B-cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti-CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL-2-based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti-CD20-human interleukin-2 (hIL-2) immunocytokine (2B8-Fc-hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv-Fc-engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS-PAGE and gel filtration. Purification yields using protein-A affinity chromatography were in the range of 15-20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant-type glycosylation. 2B8-Fc-hIL2 and the cognate 2B8-Fc antibody, devoid of hIL-2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody-dependent cell-mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8-Fc-hIL2, IL-2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.

  17. Association of carbonic anhydrase with a Calvin cycle enzyme complex in Nicotiana tabacum.

    PubMed

    Jebanathirajah, J A; Coleman, J R

    1998-02-01

    Chloroplast-localized carbonic anhydrase (CA; EC 4.2.1.1), an enzyme which catalyzes the reversible hydration of CO2, appears to be associated with other enzymes of the Calvin cycle in a large multienzyme complex. Gel-filtration fast protein liquid chromatography (FPLC) of soluble proteins obtained by osmotic lysis of tobacco (Nicotiana tabacum L. cv. Carlson) chloroplasts results in the co-elution of a protein complex of greater than 600 kDa which includes CA, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoribulokinase (PRK), and ribose-5-phosphate isomerase. Anion-exchange FPLC of chloroplast extracts indicates that there is an association of CA with other proteins that modifies its elution profile in a NaCl gradient, and that Rubisco co-elutes with the fractions containing CA. Following a protocol described by Süss et al. (1993, Proc Natl Acad Sci USA 90: 5514-5518), limited protease treatment of chloroplast extracts was used to show that the association of PRK with other chloroplast proteins appears to protect a number of lysine and arginine residues which may be involved in specific protein-protein interactions. A similar treatment of CA indicates some protection of these residues when CA is associated with other chloroplast polypeptides but the level of protection is not as profound as that exhibited by PRK. In concert with previously published immunolocalization studies, these data indicate that CA may be associated with Rubisco at the stromal periphery of a Calvin cycle enzyme complex in which PRK is more centrally located and associated with thylakoid membranes.

  18. Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    PubMed Central

    Yuan, Ning; Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Hu, Qian; Luo, Hong

    2016-01-01

    Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil –SO42−– is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP sulfurylases (ATPS) were the target genes of AthmiR395 (Arabidopsis thaliana miR395). We have cloned a miR395 gene from rice (Oryza sativa) and studied its function in plant nutritional response. Our results indicated that in rice, transcript level of OsamiR395 (Oryza sativa miR395) increased under sulfate deficiency conditions, and the two predicted target genes of miR395 were down-regulated under the same conditions. Overexpression of OsamiR395h in tobacco impaired its sulfate homeostasis, and sulfate distribution was also slightly impacted among leaves of different ages. One sulfate transporter (SULTR) gene NtaSULTR2 was identified to be the target of miR395 in Nicotiana tobacum, which belongs to low affinity sulfate transporter group. Both miR395 and NtaSULTR2 respond to sulfate starvation in tobacco. PMID:27350219

  19. Geographic variation of floral traits in Nicotiana glauca : Relationships with biotic and abiotic factors

    NASA Astrophysics Data System (ADS)

    Nattero, Julieta; Sérsic, Alicia N.; Cocucci, Andrea A.

    2011-09-01

    Geographic pattern of phenotypic variation can appear in a clinal or a mosaic fashion and can evidence adaptive or non-adaptive variation. To shed light on the mechanisms underlying this variation, we studied the relationships between geographic variation of floral traits and both biotic and abiotic factors of the hummingbird-pollinated plant, Nicotiana glauca, across its natural range. We obtained floral measures of 38 populations from an area about 1600 km long and 1050 km wide and an altitude range from 7 to over 3400 m. We used a MANOVA to detect between-population differentiations in flower traits and a DFA to determine the traits that best discriminate between populations. To test for associations between floral traits and climatic variables we used correlation analysis. We explored any possible distance-based pattern of variation (either geographic or altitudinal) in floral traits or bill length of pollinators using Mantel tests. Finally, we used a multiple regression to analyze simultaneously the effects and relative importance of abiotic predictor variables and bill length on corolla length. We found a high variation in flower traits among populations. Morphometric traits were the ones that best discriminated across populations. There was a clinal pattern of floral phenotypic variation explained by climatic factors. Differences in floral phenotypic distances were structured by altitudinal distances but not by geographic distances. Bill length of the hummingbird pollinators was structured both by altitudinal and geographic distances. Differences in bill length of hummingbird pollinators explained differences in corolla length across populations. Our findings support the assumption of flower evolution at a broad geographic scale. Floral traits seem to be structured not only by altitude but also by climatic factors.

  20. Elucidating the physiological and biochemical responses of different tobacco (Nicotiana tabacum) genotypes to lead toxicity.

    PubMed

    Maodzeka, Antony; Hussain, Nazim; Wei, Liquan; Zvobgo, Gerald; Mapodzeke, James Mutemachani; Adil, Muhammad Faheem; Jabeen, Salma; Wang, Feng; Jiang, Lixi; Shamsi, Imran Haider

    2017-01-01

    In the present study, the effects of lead (Pb) uptake and toxicity were investigated in a hydroponic culture using 7 tobacco (Nicotiana tabacum L.) genotypes (Bina 1 [B1], Kutsaga Mammoth 10 [KM10], Nanjing 3 [N3], Kutsaga 35 [K35], Kutsaga E1 [KE1], Cocker 176 [C176], and Kutsaga RK6 [KRK6]) that differed in Pb tolerance. Lead was applied as a solution of Pb nitrate at concentrations of 0 μM, 10 μM, 250 μM, and 500 μM. After 4 wk of Pb treatment, tissue biomass and photosynthetic parameters were measured and elemental analysis was performed. The results showed decreases in growth and photosynthetic parameters with increases in Pb concentration compared with the control. The least reduction in the recorded physiological parameters was noted in K35, whereas the greatest reduction was observed in N3, which is an obvious indication of genotypic differences. Activities of peroxidase, catalase, and malondialdehyde increased significantly with increases in Pb concentration, with genotypes K35 and N3 showing the least and the greatest reduction, respectively. The results demonstrate the phototoxic nature of Pb on plants, and it can be concluded that in Pb-prone areas genotypes K35 and B1 can be used for cultivation because they can grow efficiently in the presence of high Pb concentrations while restricting Pb uptake in the aboveground parts, as seen by the higher Pb tolerance index. Environ Toxicol Chem 2017;36:175-181. © 2016 SETAC.

  1. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

    PubMed

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng

    2016-02-01

    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

  2. Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

    PubMed

    Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

    2014-09-03

    A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

  3. Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthease from suspension-cultured cells of Nicotiana silvestris

    SciTech Connect

    Rubin, J.L.; Gaines, C.G.; Jensen, R.A.

    1984-07-01

    Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg, et Comes with glyphosate (N-(phosphonomethyl)glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK/sub a/ values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO/sup -/CH/sub 2/NH/sub 2//sup +/CH/sub 2/PO/sub 3//sup 2 -/, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K/sub i/ = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K/sub i/ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an (enzyme:shikimate-3-P) complex and ultimately forms the dead-end complex of (enzyme:shikimate-3-P:glyphosate). 36 references, 8 figures, 1 table.

  4. High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

    PubMed Central

    Nausch, Henrik; Mikschofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T0 transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T2 plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein. PMID:23152824

  5. Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

    PubMed Central

    Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

    2011-01-01

    Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and

  6. Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Sack, F. D.

    1989-01-01

    Gravitropism was studied in seedlings of Nicotiana sylvestris Speg. et Comes wild-type (WT) and mutant NS 458 which has a defective plastid phosphoglucomutase (EC 2.7.5.1.). Starch was greatly reduced in NS 458 compared to the WT, but small amounts of starch were detected in rootcap columella cells in NS 458 by light and electron microscopy. The roots of WT are more sensitive to gravity than mutant NS 458 roots since: (1) in mutant roots, curvature was reduced and delayed in the time course of curvature; (2) curvature of mutant roots was 24-56% that of WT roots over the range of induction periods tested; (3) in intermittent-stimulation experiments, curvature of mutant roots was 37% or less than that of WT roots in all treatments tested. The perception time, determined by intermittent-stimulation experiments, was < or = 5 s for WT roots and 30-60 s for mutant roots. The growth rates for WT and NS 458 roots were essentially equal. These results and our previous results with WT and starchless mutant Arabidopsis roots (Kiss et al. 1989, Planta 177, 198-206) support the conclusions that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts function in gravity perception. Since a presumed relatively small increase in plastid buoyant mass (N. sylvestris mutant versus Arabidopsis mutant) significantly improves the orientation of the N. sylvestris mutant roots, we suggest that plastids are the likeliest candidates to be triggering gravity perception in roots of both mutants.

  7. Genetic processes in intergeneric cell hybrids Atropa + Nicotiana : 1. Genetic constitution of cells of different clonal origin grown in vitro.

    PubMed

    Gleba, Y Y; Momot, V P; Okolot, A N; Cherep, N N; Skarzhynskaya, M V; Kotov, V

    1983-06-01

    The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120-150 chromosomes including 50-70 of Atropa and 50-90 of Nicotiana. Other clones are polyploid and possess 200-250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.

  8. Population Structure of Phytophthora nicotianae Reveals Host-Specific Lineages on Brinjal, Ridge Gourd, and Tomato in South India.

    PubMed

    Chowdappa, P; Kumar, B J Nirmal; Kumar, S P Mohan; Madhura, S; Bhargavi, B Reddi; Lakshmi, M Jyothi

    2016-12-01

    Severe outbreaks of Phytophthora fruit rot on brinjal, ridge gourd, and tomato have been observed since 2011 in Andhra Pradesh, Karnataka, Telangana, and Tamil Nadu states of India. Therefore, 76 Phytophthora nicotianae isolates, recovered from brinjal (17), ridge gourd (40), and tomato (19) from different localities in these states during the June to December cropping season of 2012 and 2013, were characterized based on phenotypic and genotypic analyses and aggressiveness on brinjal, tomato, and ridge gourd. All brinjal and ridge gourd isolates were A2, while tomato isolates were both A1 (13) and A2 (6). All isolates were metalaxyl sensitive. In addition, isolates were genotyped for three mitochondrial (ribosomal protein L5-small subunit ribosomal RNA [rpl5-rns], small subunit ribosomal RNA-cytochrome c oxidase subunit 2 [rns-cox2], and cox2+spacer) and three nuclear loci (hypothetical protein [hyp], scp-like extracellular protein [scp], and beta-tubulin [β-tub]). All regions were polymorphic but nuclear regions were more variable than mitochondrial regions. The network analysis of genotypes using the combined dataset of three nuclear regions revealed a host-specific association. However, the network generated using mitochondrial regions limited such host-specific groupings only to brinjal isolates. P. nicotianae isolates were highly aggressive and produced significantly (P ≤ 0.01) larger lesions on their respective host of origin than on other hosts. The results indicate significant genetic variation in the population of P. nicotianae, leading to identification of host-specific lineages responsible for severe outbreaks on brinjal, ridge gourd, and tomato.

  9. Molecular Linkage Mapping and Marker-Trait Associations with NlRPT, a Downy Mildew Resistance Gene in Nicotiana langsdorffii

    PubMed Central

    Zhang, Shouan; Gao, Muqiang; Zaitlin, David

    2012-01-01

    Nicotiana langsdorffii is one of two species of Nicotiana known to express an incompatible interaction with the oomycete Peronospora tabacina, the causal agent of tobacco blue mold disease. We previously showed that incompatibility is due to the hypersensitive response (HR), and plants expressing the HR are resistant to P. tabacina at all stages of growth. Resistance is due to a single dominant gene in N. langsdorffii accession S-4-4 that we have named NlRPT. In further characterizing this unique host-pathogen interaction, NlRPT has been placed on a preliminary genetic map of the N. langsdorffii genome. Allelic scores for five classes of DNA markers were determined for 90 progeny of a “modified backcross” involving two N. langsdorffii inbred lines and the related species N. forgetiana. All markers had an expected segregation ratio of 1:1, and were scored in a common format. The map was constructed with JoinMap 3.0, and loci showing excessive transmission distortion were removed. The linkage map consists of 266 molecular marker loci defined by 217 amplified fragment length polymorphisms (AFLPs), 26 simple-sequence repeats (SSRs), 10 conserved orthologous sequence markers, nine inter-simple sequence repeat markers, and four target region amplification polymorphism markers arranged in 12 linkage groups with a combined length of 1062 cM. NlRPT is located on linkage group three, flanked by four AFLP markers and one SSR. Regions of skewed segregation were detected on LGs 1, 5, and 9. Markers developed for N. langsdorffii are potentially useful genetic tools for other species in Nicotiana section Alatae, as well as in N. benthamiana. We also investigated whether AFLPs could be used to infer genetic relationships within N. langsdorffii and related species from section Alatae. A phenetic analysis of the AFLP data showed that there are two main lineages within N. langsdorffii, and that both contain populations expressing dominant resistance to P. tabacina. PMID

  10. A survey of resistance to Tomato bushy stunt virus in the genus Nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.

    PubMed

    Angel, Carlos A; Schoelz, James E

    2013-02-01

    In this study, we screened 22 Nicotiana spp. for resistance to the tombusviruses Tomato bushy stunt virus (TBSV), Cucumber necrosis virus, and Cymbidium ringspot virus. Eighteen species were resistant, and resistance was manifested in at least two different categories. In all, 13 species responded with a hypersensitive response (HR)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. Three different TBSV proteins were found to trigger HR in Nicotiana spp. in an agroinfiltration assay. The most common avirulence (avr) determinant was the TBSV coat protein P41, a protein that had not been previously recognized as an avr determinant. A mutational analysis confirmed that the coat protein rather than the viral RNA sequence was responsible for triggering HR, and it triggered HR in six species in the Alatae section. The TBSV P22 movement protein triggered HR in two species in section Undulatae (Nicotiana glutinosa and N. edwardsonii) and one species in section Alatae (N. forgetiana). The TBSV P19 RNA silencing suppressor protein triggered HR in sections Sylvestres (N. sylvestris), Nicotiana (N. tabacum), and Alatae (N. bonariensis). In general, Nicotiana spp. were capable of recognizing only one tombusvirus avirulence determinant, with the exceptions of N. bonariensis and N. forgetiana, which were each able to recognize P41, as well as P19 and P22, respectively. Agroinfiltration failed to detect the TBSV avr determinants responsible for triggering HR in N. arentsii, N. undulata, and N. rustica. This study illustrates the breadth and variety of resistance responses to tombusviruses that exists in the Nicotiana genus.

  11. Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation

    SciTech Connect

    Rajeevan, M.S.; Lang, A. )

    1993-05-15

    The capacity to form flower buds in thin-layer explants was studied in Nicotiana of several species, cultivars, and lines of differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied depending on genotype, source tissue and its developmental state, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The capacity to form flow buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression. 14 refs., 5 figs., 3 tabs.

  12. Native Bacterial Endophytes Promote Host Growth in a Species-Specific Manner; Phytohormone Manipulations Do Not Result in Common Growth Responses

    PubMed Central

    Long, Hoang Hoa; Schmidt, Dominik D.; Baldwin, Ian T.

    2008-01-01

    Background All plants in nature harbor a diverse community of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant-growth-promoting (PGP) rhizobacteria which can decrease ethylene (ET) levels enzymatically by 1-aminocyclopropane-1-carboxylate (ACC) deaminase or produce indole acetic acid (IAA). Whether these common PGP mechanisms work similarly for different plant species has not been rigorously tested. Methodology/ Principal Findings We isolated bacterial endophytes from field-grown Solanum nigrum; characterized PGP traits (ACC deaminase activity, IAA production, phosphate solubilization and seedling colonization); and determined their effects on their host, S. nigrum, as well as on another Solanaceous native plant, Nicotiana attenuata. In S. nigrum, a majority of isolates that promoted root growth were associated with ACC deaminase activity and IAA production. However, in N. attenuata, IAA but not ACC deaminase activity was associated with root growth. Inoculating N. attenuata and S. nigrum with known PGP bacteria from a culture collection (DSMZ) reinforced the conclusion that the PGP effects are not highly conserved. Conclusions/ Significance We conclude that natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved. PMID:18628963

  13. Altered sucrose metabolism impacts plant biomass production and flower development.

    PubMed

    Coleman, Heather D; Beamish, Leigh; Reid, Anya; Park, Ji-Young; Mansfield, Shawn D

    2010-04-01

    Nicotiana tabacum (tobacco) was transformed with three genes involved in sucrose metabolism, UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9), sucrose synthase (SuSy, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14). Plants harbouring the single transgenes were subsequently crossed to produce double and triple transgenic lines, including: 2 x 35S::UGPase x SPS, 4CL::UGPase x SPS, 2 x 35S::SuSy x SPS, 4CL::SuSy x SPS, 2 x 35S::UGPase x SuSy x SPS, and 4CL::UGPase x SuSy x SPS. The ultimate aim of the study was to examine whether it is possible to alter cellulose production through the manipulation of sucrose metabolism genes. While altering sucrose metabolism using UGPase, SuSy and SPS does not have an end effect on cellulose production, their simultaneous overexpression resulted in enhanced primary growth as seen in an increase in height growth, in some cases over 50%. Furthermore, the pyramiding strategy of simultaneously altering the expression of multiple genes in combination resulted in increased time to reproductive bud formation as well as altered flower morphology and foliar stipule formation in 4CL lines. Upregulation of these sucrose metabolism genes appears to directly impact primary growth and therefore biomass production in tobacco.

  14. Expression of synthetic human tropoelastin (hTE) protein in Nicotiana tabacum

    PubMed Central

    Abdelghani, Mona; El-Heba, Ghada A Abu; Abdelhadi, Abdelhadi A; Abdallah, Naglaa A

    2015-01-01

    ABSTRACT Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal.  Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5th day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3–10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ∼70 kDa from freshly extracted protein, while two degraded bands of ∼55 and ∼45 kDa, representing a pattern of tropoelastin were appeared with frozen samples

  15. Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana.

    PubMed

    Marín Viegas, Vanesa S; Acevedo, Gonzalo R; Bayardo, Mariela P; Chirdo, Fernando G; Petruccelli, Silvana

    2015-01-01

    Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to

  16. Phytoaccumulation of lead by sunflower (Helianthus annuus), tobacco (Nicotiana tabacum), and vetiver (Vetiveria zizanioides).

    PubMed

    Boonyapookana, Benjaporn; Parkpian, Preeda; Techapinyawat, Sombun; DeLaune, R D; Jugsujinda, Aroon

    2005-01-01

    The ability of three plant species: Helianthus annuus, Nicotiana tabacum, and Vetiveria zizanioides for phytoaccumulation of Pb was studied. Plants were grown in hydroponic solution containing Pb(NO3)2 at concentration of 0.25 and 2.5 mM Pb in the presence or absence of chelating agents (EDTA or DTPA). Lead (Pb) transport and localization within the tissues of the plant species was determined using scanning electron microscope equipped with energy dispersive X-ray spectrometers (SEM-EDS). The addition of chelators increased Pb uptake as compared to plants grown in solution containing Pb alone. Lead taken up by the plant species were concentrated in both leaf and stem at the region of vascular bundles with greater amounts in the leaf portion. Lead granules were also found in the H. annuus root tissue from the epidermis layer to the central axis. After four weeks of growth a 23-fold increase in shoot Pb content for H. annuus and N. tabacum and 17-fold increase in shoot Pb for V. zizanioides resulted from plants grown in the 2.5 mM Pb-EDTA treatment. The higher Pb treatment (2.5 mM Pb containing EDTA) resulted in higher concentrations of Pb in plant tissue at the fourth week of exposure as compared to Pb treatment containing DTPA. Overall, Pb accumulation potential of H. annuus was greater than that of N. tabacum and V. zizanioides as indicated by the bioconcentration factor (171, 70, and 88, respectively). The highest measured Pb concentrations were found in H. annuus roots, stems, and leaves (2668, 843, and 3611 microg/g DW, respectively) grown in the 2.5 mM Pb-EDTA treatment. The addition of chelators caused some reduction in plant growth and biomass. Results showed that the three plant species tested have potential for use in phytoaccumulation of Pb since the Pb was concentrated in leaf and stem as compared to control plants. H. annuus however best meet the prerequisites for a hyperaccumulator plant and would have the potential for use in the restoration of

  17. Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

    PubMed Central

    Marín Viegas, Vanesa S.; Acevedo, Gonzalo R.; Bayardo, Mariela P.; Chirdo, Fernando G.; Petruccelli, Silvana

    2015-01-01

    Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to

  18. Evolution of herbivore-induced early defense signaling was shaped by genome-wide duplications in Nicotiana

    PubMed Central

    Zhou, Wenwu; Brockmöller, Thomas; Ling, Zhihao; Omdahl, Ashton; Baldwin, Ian T; Xu, Shuqing

    2016-01-01

    Herbivore-induced defenses are widespread, rapidly evolving and relevant for plant fitness. Such induced defenses are often mediated by early defense signaling (EDS) rapidly activated by the perception of herbivore associated elicitors (HAE) that includes transient accumulations of jasmonic acid (JA). Analyzing 60 HAE-induced leaf transcriptomes from closely-related Nicotiana species revealed a key gene co-expression network (M4 module) which is co-activated with the HAE-induced JA accumulations but is elicited independently of JA, as revealed in plants silenced in JA signaling. Functional annotations of the M4 module were consistent with roles in EDS and a newly identified hub gene of the M4 module (NaLRRK1) mediates a negative feedback loop with JA signaling. Phylogenomic analysis revealed preferential gene retention after genome-wide duplications shaped the evolution of HAE-induced EDS in Nicotiana. These results highlight the importance of genome-wide duplications in the evolution of adaptive traits in plants. DOI: http://dx.doi.org/10.7554/eLife.19531.001 PMID:27813478

  19. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

    PubMed Central

    Shi, Yanmei; Guo, Jinggong; Zhang, Wei; Jin, Lifeng; Liu, Pingping; Chen, Xia; Li, Feng; Wei, Pan; Li, Zefeng; Li, Wenzheng; Wei, Chunyang; Zheng, Qingxia; Chen, Qiansi; Zhang, Jianfeng; Lin, Fucheng; Qu, Lingbo; Snyder, John Hugh; Wang, Ran

    2015-01-01

    Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum. PMID:26703579

  20. Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

    PubMed

    Lamotte, Olivier; Courtois, Cécile; Dobrowolska, Grazyna; Besson, Angélique; Pugin, Alain; Wendehenne, David

    2006-04-15

    In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

  1. A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Steadman, Kathryn J

    2015-08-01

    A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6μg/mL for nicotine and below 1μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8μg/mL for nornicotine and nicotine respectively, and below 2μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.

  2. Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny.

    PubMed

    Thyssen, Gregory; Svab, Zora; Maliga, Pal

    2012-10-01

    Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

  3. Two faces of Solanaceae telomeres: a comparison between Nicotiana and Cestrum telomeres and telomere-binding proteins.

    PubMed

    Peska, V; Sýkorová, E; Fajkus, J

    2008-01-01

    While most Solanaceae genera (e.g.Solanum, Nicotiana) possess Arabidopsis-type telomeres of (TTTAGGG)n maintained by telomerase, the genera Cestrum, Vestia and Sessea (Cestrum group) lack these telomeres. Here we show that in the Cestrum-group the activity of telomerase has been lost. Nevertheless, proteins binding the single-stranded G-rich strand of the Arabidopsis-type and related human-type (TTAGGG)n telomeric sequences are present in nuclear extracts of both Nicotiana and Cestrum species. These proteins may have a role in telomere function or other cellular activities. In addition to characterizing DNA binding specificity and molecular weights of these proteins, we searched in both N. tabacum (tobacco) and C. parqui for the presence of POT1-like proteins, involved in telomere capping and telomerase regulation. Analysis of POT1-like proteins available on public databases and cloned by us from C. parqui, revealed the N-terminal OB folds typical for this protein family and a novel, plant-specific conserved C-terminal OB-fold domain (CTOB). We propose that CTOB is involved in protein-protein interactions.

  4. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alternanthera mosaic virus (AltMV; Potexvirus) naturally infects several ornamental plants. Differences were previously noted between systemic symptoms induced in Nicotiana benthamiana by different isolates of AltMV, and by four infectious clones derived from AltMV-SP. Virus accumulation was enhance...

  5. Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

    PubMed Central

    Lim, K Y; Skalicka, K; Koukalova, B; Volkov, R A; Matyasek, R; Hemleben, V; Leitch, A R; Kovarik, A

    2004-01-01

    An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions. PMID:15126410

  6. Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains.

    PubMed

    Herrero, Sonia; Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2007-10-01

    Plant pathogens from the genus Cercospora produce cercosporin, a photoactivated fungal toxin that generates toxic reactive oxygen species. Mechanisms governing toxin auto-resistance in Cercospora spp. are poorly understood. In this work, suppressive subtractive hybridization was used to identify genes differentially expressed between the cercosporin-resistant wild-type (WT) Cercospora nicotianae and a sensitive strain lacking a transcription factor (CRG1) that regulates resistance. Out of 338 sequences recovered, 185 unique expressed sequence tags (ESTs) were obtained and classified into functional categories. The majority of genes showed predicted expression differences, and 38.5% were differentially expressed at least twofold between the WT and mutant strain. ESTs were recovered with homology to genes involved in detoxification of noxious compounds, multidrug membrane transporters and antioxidant and polyketide biosynthetic enzymes as well as to ATPases and ATP synthases. The findings suggest that CRG1 regulates genes involved in pH responses in addition to those involved in toxin resistance and biosynthesis.

  7. Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

    PubMed

    Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L

    2014-10-01

    Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

  8. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed Central

    Dalton, Heidi L.; Blomstedt, Cecilia K.; Neale, Alan D.; Gleadow, Ros; DeBoer, Kathleen D.; Hamill, John D.

    2016-01-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID

  9. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  10. The Evolutionary Fate of the Horizontally Transferred Agrobacterial Mikimopine Synthase Gene in the Genera Nicotiana and Linaria

    PubMed Central

    Talianova, Martina; Vyskot, Boris

    2014-01-01

    Few cases of spontaneously horizontally transferred bacterial genes into plant genomes have been described to date. The occurrence of horizontally transferred genes from the T-DNA of Agrobacterium rhizogenes into the plant genome has been reported in the genus Nicotiana and in the species Linaria vulgaris. Here we compare patterns of evolution in one of these genes (a gene encoding mikimopine synthase, mis) following three different events of horizontal gene transfer (HGT). As this gene plays an important role in Agrobacterium, and there are known cases showing that genes from pathogens can acquire plant protection function, we hypothesised that in at least some of the studied species we will find signs of selective pressures influencing mis sequence. The mikimopine synthase (mis) gene evolved in a different manner in the branch leading to Nicotiana tabacum and N. tomentosiformis, in the branch leading to N. glauca and in the genus Linaria. Our analyses of the genus Linaria suggest that the mis gene began to degenerate soon after the HGT. In contrast, in the case of N. glauca, the mis gene evolved under significant selective pressures. This suggests a possible role of mikimopine synthase in current N. glauca and its ancestor(s). In N. tabacum and N. tomentosiformis, the mis gene has a common frameshift mutation that disrupted its open reading frame. Interestingly, our results suggest that in spite of the frameshift, the mis gene could evolve under selective pressures. This sequence may still have some regulatory role at the RNA level as suggested by coverage of this sequence by small RNAs in N. tabacum. PMID:25420106

  11. OBSERVATIONS ON THE SUSCEPTIBILITY OF SOME WILD SPECIES OF THE GENUS NICOTINA AND OF SOME VARIETIES OF NICOTIANA TABACUM L. AND N. RUSTICA L. TO BLUE MOULD (PERONOSPORA TABACINA ADAM) -- PULAWY 1962

    DTIC Science & Technology

    Observations on the susceptibility of some wild species of the genus nicotina and of some varieties of nicotiana tabacum l. and n. rustica l. to blue mould (peronospora tabacina adam) -- pulawy 1962--Translation.

  12. Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

    PubMed

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-08-22

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

  13. Differential Responses to Virus Challenge of Laboratory and Wild Accessions of Australian Species of Nicotiana, and Comparative Analysis of RDR1 Gene Sequences

    PubMed Central

    Zhang, Chao; Long, Vicki; Roossinck, Marilyn J.; Koh, Shu Hui; Jones, Michael G. K.; Iqbal, Sadia

    2015-01-01

    Nicotiana benthamiana is a model plant utilised internationally in plant virology because of its apparent hyper-susceptibility to virus infection. Previously, others showed that all laboratory accessions of N. benthamiana have a very narrow genetic basis, probably originating from a single source. It is unknown if responses to virus infection exhibited by the laboratory accession are typical of the species as a whole. To test this, 23 accessions of N. benthamiana were collected from wild populations and challenged with one to four viruses. Additionally, accessions of 21 other Nicotiana species and subspecies from Australia, one from Peru and one from Namibia were tested for susceptibility to the viruses, and for the presence of a mutated RNA-dependent RNA polymerase I allele (Nb-RDR1m) described previously from a laboratory accession of N. benthamiana. All Australian Nicotiana accessions tested were susceptible to virus infections, although there was symptom variability within and between species. The most striking difference was that plants of a laboratory accession of N. benthamiana (RA-4) exhibited hypersensitivity to Yellow tailflower mild mottle tobamovirus infection and died, whereas plants of wild N. benthamiana accessions responded with non-necrotic symptoms. Plants of certain N. occidentalis accessions also exhibited initial hypersensitivity to Yellow tailflower mild mottle virus resembling that of N. benthamiana RA-4 plants, but later recovered. The mutant Nb-RDR1m allele was identified from N. benthamiana RA-4 but not from any of 51 other Nicotiana accessions, including wild accessions of N. benthamiana, demonstrating that the accession of N. benthamiana used widely in laboratories is unusual. PMID:25822508

  14. NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

    PubMed

    Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

    2013-01-01

    In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

  15. Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

    PubMed Central

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-01-01

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631

  16. Low-temperature derivatization followed by vortex-assisted liquid-liquid microextraction for the analysis of polyamines in Nicotiana Tabacum.

    PubMed

    Cai, Kai; Cai, Bin; Xiang, Zhangmin; Zhao, Huina; Rao, Xingyi; Pan, Wenjie; Lei, Bo

    2016-07-01

    Polyamines are ubiquitous polycationic molecules that play a key role in many biological processes such as nucleic acid metabolism, protein synthesis, cell growth, and nicotine synthesis precursors. This work describes a rapid, sensitive, convenient, green, and cost-effective method for the determination of polyamines in Nicotiana tabacum by ultra high performance liquid chromatography with photodiode array detection. The analytes were derivatized with 3,5-dinitrobenzoyl chloride at low temperature (about 4°C) and then extracted with vortex-assisted liquid-liquid microextraction. The experimental designs based on quarter-fractional factorial design and Doehlert design were used to screen and optimize the important factors in microextraction process. Under the optimal conditions, the method was linear over 0.05-8.00 μg/mL with an r(2) ≥ 0.992 and exhibited good repeatability and reproducibility less than 6.0 and 6.9%, respectively. The limit of detection ranged between 0.013 and 0.029 μg/g. The newly developed method was successfully employed to analyze different leaf samples of Nicotiana tabacum, among which the polyamines contents were found to be very different. Moreover, tyramine, 1,3-diaminopropane, homospermidine, and canavalmine were tentatively identified with the electrospray ionization quadrupole time-of-flight mass spectrometry. To our knowledge, this is the first report of identification of canavalmine in Nicotiana Tabacum.

  17. Molecular cytogenetics and tandem repeat sequence evolution in the allopolyploid Nicotiana rustica compared with diploid progenitors N. paniculata and N. undulata.

    PubMed

    Lim, K Y; Matyasek, R; Kovarik, A; Fulnecek, J; Leitch, A R

    2005-01-01

    Nicotiana rustica (2n = 4x = 48) is a natural allotetraploid composed of P and U genomes which are closely related to genomes of diploid species N. paniculata and N. undulata. Genomic in situ hybridization (GISH) also confirms that the diploid parents, or close relatives, are the ancestors of N. rustica. In order to study genetic interactions between ancestral genomes in the allotetraploid, we isolated three families of repetitive sequences, two from N. paniculata (NPAMBE and NPAMBO) and one from N. undulata (NUNSSP). Southern blot hybridization revealed that the sequences are digested with a range of restriction enzymes into regular ladder patterns indicating a tandem arrangement of high copy repeats possessing monomeric units of about 180 bp. The three-tandem sequences belong to a larger Nicotiana tandem repeat family called here the HRS-60 family. Members of this family are found in all Nicotiana species studied. Fluorescence in situ hybridization (FISH) analysis localized the satellite repeats to subtelomeric regions of most chromosomes of N. paniculata and N. undulata. The pattern of sequence distribution on the P- and U-genomes of N. rustica was similar to the putative parents N. paniculata and N. undulata respectively. However, NPAMBO repeats appear to be reduced and rearranged in N. rustica that may suggest evolution within the P genome. GISH and FISH with the tandem repeat probes failed to reveal intergenomic translocations as might be predicted from the nucleocytoplasmic interaction hypothesis.

  18. Effect of increased UV-B radiation on carotenoid accumulation and total antioxidant capacity in tobacco (Nicotiana tabacum L.) leaves.

    PubMed

    Shen, J; Jiang, C Q; Yan, Y F; Liu, B R; Zu, C L

    2017-03-08

    Carotenoids are important components of plant antioxidant systems, which protect photosystems from photooxidative destruction during ultraviolet-B (UV-B) exposure. The influence of carotenoids on total antioxidant capacity (TAC) of plants has rarely been studied. In this study, tobacco (Nicotiana tabacum L., 'K326') seedlings exposed to UV-B radiation were used in order to evaluate the effects of ambient levels of UV-B radiation on carotenoid accumulation. The aim was to investigate whether carotenoids could enhance TAC as a means of UV protection. Our results showed that leaf carotenoid content in the low UV-B exposure (+9.75 μW/cm(2)) plants was approximately 8% higher than that observed in control plants at 2-8 days of exposure. At high UV-B exposure (+20.76 μW/cm(2)), the carotenoid content increased rapidly after 1 day's exposure (10.41% higher than the control), followed by a return to the content as in control plants. Furthermore, carotenoid content positively correlated with TAC (P = 0.024). These results suggest that carotenoids have antioxidant properties and play an important role in the antioxidant system. UV-B exposure increased the carotenoid synthesis capability of plants. The plants could deplete the carotenoids to scavenge excess ROS at high UV-B radiation levels, which protects the tobacco plant from oxidative damage caused by UV-B stress.

  19. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana[OPEN

    PubMed Central

    Adachi, Hiroaki; Nakano, Takaaki; Miyagawa, Noriko; Ishihama, Nobuaki; Yoshioka, Miki; Katou, Yuri; Yaeno, Takashi

    2015-01-01

    Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB. PMID:26373453

  20. Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes.

    PubMed

    Ahn, Soon Young; Baek, Kwang-Hyun; Moon, Yong Sun; Yun, Hae Keun

    2013-03-01

    Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semi-quantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of pathogenesis-related protein 1a (PR1a), pathogenesis-related protein 1b (PR1b), glutathione S-transferase (GST), allen oxide cyclase (AOC), and lipoxyganase (LOX). All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid.

  1. [The analysis of rbcS gene function by post-transcription gene silencing in Nicotiana benthamiana].

    PubMed

    Zhou, Xiao-Fu; Ma, Peng-Da; Wang, Ren-Hou; Zhu, Xiao-Juan; Liu, Bao; Wang, Xing-Zhi

    2005-06-01

    A system of virus-induced post-transcriptional gene silencing for studying rbcS gene function was established and optimized using tobacco rattle virus vector and Nicotiana benthamiana as experimental materiaes. The following analyses were conducted: phenotypic characterization of rbcS gene silenced plants, transcription levels of rbcS gene by RT-PCR; protein levels of rbcS by the antibodies of rbcS and rbcL and photosynthetic pigments wntents in rbcS silenced plants by HPLC method. The results showed that the seedlings at 21-24-day-old and Agrobacterium concentration at OD600 = 1-1.5 gave the best results for gene silencing. The expression level of rbcL was very likely regulated by rbcS, and rbcS gene did not relate to the collection of photosynthetic energy. Probability analysis showed that the tobacco rattle virus vector system is a useful and effective technique to study rbcS gene function via post-transcriptional gene silencing.

  2. The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

    PubMed Central

    Blackman, LM; Boevink, P; Cruz, SS; Palukaitis, P; Oparka, KJ

    1998-01-01

    The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer. PMID:9548980

  3. Analysis of Nicotiana tabacum PIN genes identifies NtPIN4 as a key regulator of axillary bud growth.

    PubMed

    Xie, Xiaodong; Qin, Guangyong; Si, Ping; Luo, Zhaopeng; Gao, Junping; Chen, Xia; Zhang, Jianfeng; Wei, Pan; Xia, Qingyou; Lin, Fucheng; Yang, Jun

    2017-01-27

    The plant-specific PIN-FORMED (PIN) auxin efflux proteins have been well characterized in many plant species, where they are crucial in the regulation of auxin transport in various aspects of plant development. However, little is known about the exact roles of the PIN genes during plant development in Nicotiana species. This study investigated the PIN genes in tobacco (N. tabacum) and in two ancestral species (N. sylvestris and N. tomentosiformis). Genome-wide analysis of the N. tabacum genome identified 20 genes of the PIN family. An in-depth phylogenetic analysis of the PIN genes of N. tabacum, N. sylvestris and N. tomentosiformis was conducted. NtPIN4 expression was strongly induced by the application of exogenous IAA, but was downregulated by the application of ABA, a strigolactone analogue, and cytokinin, as well as by decapitation treatments, suggesting that the NtPIN4 expression level is likely positively regulated by auxin. Expression analysis indicated that NtPIN4 was highly expressed in tobacco stems and shoots, which was further validated through analysis of the activity of the NtPIN4 promoter. We used CRISPR-Cas9 technology to generate mutants for NtPIN4 and observed that both T0 and T1 plants had a significantly increased axillary bud growth phenotype, as compared with the wild-type plants. Therefore, NtPIN4 offers an opportunity for studying auxin-dependent branching processes.

  4. Temporal and Spatial Resolution of Activated Plant Defense Responses in Leaves of Nicotiana benthamiana Infected with Dickeya dadantii

    PubMed Central

    Pérez-Bueno, María L.; Granum, Espen; Pineda, Mónica; Flors, Víctor; Rodriguez-Palenzuela, Pablo; López-Solanilla, Emilia; Barón, Matilde

    2016-01-01

    The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as experimental host for a very broad range of plant pathogens, is susceptible to infection by D. dadantii. The inoculation with D. dadantii at high dose seems to overcome the plant defense capacity, inducing maceration and death of the tissue, although restricted to the infiltrated area. By contrast, the output of the defense response to low dose inoculation is inhibition of maceration and limitation in the growth, or even eradication, of bacteria. Responses of tissue invaded by bacteria (neighboring the infiltrated areas after 2–3 days post-inoculation) included: (i) inhibition of photosynthesis in terms of photosystem II efficiency; (ii) activation of energy dissipation as non-photochemical quenching in photosystem II, which is related to the activation of plant defense mechanisms; and (iii) accumulation of secondary metabolites in cell walls of the epidermis (lignins) and the apoplast of the mesophyll (phytoalexins). Infiltrated tissues showed an increase in the content of the main hormones regulating stress responses, including abscisic acid, jasmonic acid, and salicylic acid. We propose a mechanism involving the three hormones by which N. benthamiana could activate an efficient defense response against D. dadantii. PMID:26779238

  5. Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector.

    PubMed

    Varsani, Arvind; Williamson, Anna-Lise; Stewart, Debbie; Rybicki, Edward P

    2006-09-01

    A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 microg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector.

  6. Dicer-Like 4 Is Involved in Restricting the Systemic Movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.

    PubMed

    Cordero, Teresa; Cerdán, Lidia; Carbonell, Alberto; Katsarou, Konstantina; Kalantidis, Kriton; Daròs, José-Antonio

    2017-01-01

    Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Ros1 systemic accumulation dramatically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.

  7. Scale-up of Agrobacterium-mediated transient protein expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture.

    PubMed

    O'Neill, Kristin M; Larsen, Jeffrey S; Curtis, Wayne R

    2008-01-01

    The reporter gene beta-glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through co-culture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to transient expression including the timing of Agrobacterium inoculation relative to the plant cell growth phase, plant tissue culture hormonal triggers and plant cell cycle synchronization. The co-culture process was simplified to facilitate implementation in a pilot-scale bioreactor. At the shake flask scale it was determined that elevated concentrations of oxygen in the headspace were detrimental to transient expression levels and the addition of acetosyringone to the co-culture had a negligible effect. The bacterial preparation process was also streamlined, permitting the direct transfer of the Agrobacterium culture from a bench-scale fermentor to the pilot-scale plant cell culture bioreactor. Increasing expression levels and overcoming batch-to-batch variability despite extensive procedure systemization remain the major technical hurdles.

  8. Molecular Characterization of A Novel Effector Expansin-like Protein from Heterodera avenae that Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Liu, Jing; Peng, Huan; Cui, Jiangkuan; Huang, Wenkun; Kong, Lingan; Clarke, Jihong Liu; Jian, Heng; Wang, Guo Liang; Peng, Deliang

    2016-01-01

    Cereal cyst nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands and are secreted into plant tissues through the stylet. To understand the function of nematode effectors in parasitic plants, we cloned predicted effectors genes from Heterodera avenae and transiently expressed them in Nicotiana benthamiana. Infiltration assays showed that HaEXPB2, a predicted expansin-like protein, caused cell death in N. benthamiana. In situ hybridization showed that HaEXPB2 transcripts were localised within the subventral gland cells of the pre-parasitic second-stage nematode. HaEXPB2 had the highest expression levels in parasitic second-stage juveniles. Subcellular localization assays revealed that HaEXPB2 could be localized in the plant cell wall after H. avenae infection.This The cell wall localization was likely affected by its N-terminal and C-terminal regions. In addition, we found that HaEXPB2 bound to cellulose and its carbohydrate-binding domain was required for this binding. The infectivity of H. avenae was significantly reduced when HaEXPB2 was knocked down by RNA interference in vitro. This study indicates that HaEXPB2 may play an important role in the parasitism of H. avenae through targeting the host cell wall. PMID:27808156

  9. Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Giddings, T. H. Jr; Staehelin, L. A.; Sack, F. D.

    1990-01-01

    To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips of Nicotiana and Arabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.

  10. Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

    PubMed

    Fuoco, Roger; Bogani, Patrizia; Capodaglio, Gabriele; Del Bubba, Massimo; Abollino, Ornella; Giannarelli, Stefania; Spiriti, Maria Michela; Muscatello, Beatrice; Doumett, Saer; Turetta, Clara; Zangrando, Roberta; Zelano, Vincenzo; Buiatti, Marcello

    2013-05-01

    Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

  11. Relationship of microbial communities and suppressiveness of Trichoderma fortified composts for pepper seedlings infected by Phytophthora nicotianae

    PubMed Central

    Ros, Margarita; Raut, Iulia; Santisima-Trinidad, Ana Belén; Pascual, Jose Antonio

    2017-01-01

    The understanding of the dynamic of soil-borne diseases is related to the microbial composition of the rhizosphere which is the key to progress in the field of biological control. Trichoderma spp. is commonly used as a biological control agent. The use of next generation sequencing approaches and quantitative PCR are two successful approaches to assess the effect of using compost as substrate fortified with two Trichoderma strains (Trichoderma harzianum or Trichoderma asperellum) on bacterial and fungal communities in pepper rhizosphere infected with Phytophthora nicotianae. The results showed changes in the bacterial rhizosphere community not attributed to the Trichoderma strain, but to the pathogen infection, while, fungi were not affected by pathogen infection and depended on the type of substrate. The Trichoderma asperellum fortified compost was the most effective combination against the pathogen. This could indicate that the effect of fortified composts is greater than compost itself and the biocontrol effect should be attributed to the Trichoderma strains rather than the compost microbiota, although some microorganisms could help with the biocontrol effect. PMID:28346470

  12. Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite*

    PubMed Central

    Huang, Chang-jun; Zhang, Tong; Li, Fang-fang; Zhang, Xin-yue; Zhou, Xue-ping

    2011-01-01

    Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18–32 °C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum. PMID:21265040

  13. The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

    PubMed

    Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

    2015-11-01

    Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production.

  14. Early inhibition of photosynthesis during development of Mn toxicity in tobacco. [Nicotiana tabacum L. cv KY14

    SciTech Connect

    Nable, R.O.; Houtz, R.L.; Cheniae, G.M. )

    1988-04-01

    Early physiological effects of developing Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) were examined in glass-house/water cultured plants grown at high (summer) and low (winter) photon flux. Following transfer of plants to solutions containing 1 millimolar Mn{sup 2+}, sequential samplings were made at various times for the following 9 days, during which Mn accumulation by leaves increased rapidly from {approx} 70 on day 0 to {approx} 1700 and {approx} 5000 microgram per gram dry matter after 1 and 9 days, respectively. In plants grown at high photon flux, net photosynthesis declined by {approx} 20 and {approx} 60% after 1 and 9 days, respectively, and the onset of this decline preceded appearance (after 3 to 4 days) of visible foliar symptoms of Mn toxicity. Intercellular CO{sub 2} concentrations and rates of transpiration were not significantly affected. Though the activity of latent or activated polyphenol oxidase increased in parallel with Mn accumulation, neither leaf respiration nor the activity of catalase (EC 1.11.1.6) and peroxidase (EC 1.10.1.7) were greatly affected. These effects from Mn toxicity could not be explained by any changes in protein or chlorophyll abundance. Additionally, they were not a consequence of Mn induced Fe deficiency. Therefore, inhibition of net photosynthesis and enhancement of polyphenol oxidase activity are early indicators of excess Mn accumulation in tobacco leaves.

  15. Deletion of a MFS transporter-like gene in Cercospora nicotianae reduces cercosporin toxin accumulation and fungal virulence.

    PubMed

    Choquer, Mathias; Lee, Miin-Huey; Bau, Huey-Jiunn; Chung, Kuang-Ren

    2007-02-06

    Many phytopathogenic Cercospora species produce a host-nonselective polyketide toxin, called cercosporin, whose toxicity exclusively relies on the generation of reactive oxygen species. Here, we describe a Cercospora nicotianae CTB4 gene that encodes a putative membrane transporter and provide genetic evidence to support its role in cercosporin accumulation. The predicted CTB4 polypeptide has 12 transmembrane segments with four conserved motifs and has considerable similarity to a wide range of transporters belonging to the major facilitator superfamily (MFS). Disruption of the CTB4 gene resulted in a mutant that displayed a drastic reduction of cercosporin production and accumulation of an unknown brown pigment. Cercosporin was detected largely from fungal hyphae of ctb4 disruptants, but not from the surrounding medium, suggesting that the mutants were defective in both cercosporin biosynthesis and secretion. Cercosporin purified from the ctb4 disruptants exhibited toxicity to tobacco suspension cells, insignificantly different from wild-type, whereas the disruptants formed fewer lesions on tobacco leaves. The ctb4 null mutants retained normal resistance to cercosporin and other singlet oxygen-generating photosensitizers, indistinguishable from the parental strain. Transformation of a functional CTB4 clone into a ctb4 null mutant fully revived cercosporin production. Thus, we propose that the CTB4 gene encodes a putative MFS transporter responsible for secretion and accumulation of cercosporin.

  16. The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis.

    PubMed

    Dekkers, Katherine L; You, Bang-Jau; Gowda, Vivek S; Liao, Hui-Ling; Lee, Miin-Huey; Bau, Huey-Jiunn; Ueng, Peter P; Chung, Kuang-Ren

    2007-05-01

    Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Deltactb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Deltactb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Deltactb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Deltactb3 disruptants drastically reduced lesion formation.

  17. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    PubMed

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  18. Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae.

    PubMed

    Chen, Hui-Qin; Lee, Miin-Huey; Chung, Kuang-Ren

    2007-08-01

    Cercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.

  19. Effects and effectiveness of two RNAi constructs for resistance to Pepper golden mosaic virus in Nicotiana benthamiana plants.

    PubMed

    Medina-Hernández, Diana; Rivera-Bustamante, Rafael Francisco; Tenllado, Francisco; Holguín-Peña, Ramón Jaime

    2013-11-28

    ToChLPV and PepGMV are Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field.

  20. Asymmetric hybridization between Nicotiana tabacum and N. repanda by donor recipient protoplast fusion: transfer of TMV resistance.

    PubMed

    Bates, G W

    1990-10-01

    Genetically asymmetric hybrids were recovered by fusion of Nicotiana tabacum protoplasts with irradiated protoplasts of kanamycin-resistant, nopalineproducing plants of N. repanda. Hybrid calli were selected by culture on media containing kanamycin and were regenerated. These plants were morphologically similar to N. tabacum but produced nopaline, indicating they retained genes from N. repanda. Esterase isozyme profiles also indicated that the plants are somatic hybrids, but are more similar to N. tabacum than N. repanda. Chromosome counts showed most of the hybrids had 55-62 chromosomes, which is consistent with extensive, although incomplete elimination of N. repanda chromosomes. The hybrids were largely male sterile, but about half of them set seed when crossed with N. tabacum. Chromosome numbers of the progeny and the pattern of inheritance of kanamycin resistance indicated the continued elimination of N. repanda genetic material in these backcrosses. The N. repanda parent used in these fusions gave a hypersensitive response to TMV, whereas the N. tabacum parent was TMV sensitive. When inoculated with TMV, plants from two hybrid clones gave a hypersensitive response. Plants from the other clones became systemically infected with the virus.

  1. Relationship of microbial communities and suppressiveness of Trichoderma fortified composts for pepper seedlings infected by Phytophthora nicotianae.

    PubMed

    Ros, Margarita; Raut, Iulia; Santisima-Trinidad, Ana Belén; Pascual, Jose Antonio

    2017-01-01

    The understanding of the dynamic of soil-borne diseases is related to the microbial composition of the rhizosphere which is the key to progress in the field of biological control. Trichoderma spp. is commonly used as a biological control agent. The use of next generation sequencing approaches and quantitative PCR are two successful approaches to assess the effect of using compost as substrate fortified with two Trichoderma strains (Trichoderma harzianum or Trichoderma asperellum) on bacterial and fungal communities in pepper rhizosphere infected with Phytophthora nicotianae. The results showed changes in the bacterial rhizosphere community not attributed to the Trichoderma strain, but to the pathogen infection, while, fungi were not affected by pathogen infection and depended on the type of substrate. The Trichoderma asperellum fortified compost was the most effective combination against the pathogen. This could indicate that the effect of fortified composts is greater than compost itself and the biocontrol effect should be attributed to the Trichoderma strains rather than the compost microbiota, although some microorganisms could help with the biocontrol effect.

  2. Immediate effects of nectar robbing by Palestine sunbirds (Nectarinia osea) on nectar alkaloid concentrations in tree tobacco (Nicotiana glauca).

    PubMed

    Kaczorowski, Rainee L; Koplovich, Avi; Sporer, Frank; Wink, Michael; Markman, Shai

    2014-04-01

    Plant secondary metabolites (PSMs), such as alkaloids, are often found in many parts of a plant, including flowers, providing protection to the plant from various types of herbivores or microbes. PSMs are also present in the floral nectar of many species, but typically at lower concentrations than in other parts of the plant. Nectar robbers often damage floral tissue to access the nectar. By doing so, these nectar robbers may initiate an increase of PSMs in the floral nectar. It is often assumed that it takes at least a few hours before the plant demonstrates an increase in PSMs. Here, we addressed the question of whether PSMs in the floral tissue are immediately being released into the floral nectar following nectar robbing. To address this research question, we investigated whether there was an immediate effect of nectar robbing by the Palestine Sunbird (Nectarinia osea) on the concentration of nectar alkaloids, nicotine and anabasine, in Tree Tobacco (Nicotiana glauca). We found that the concentration of anabasine, but not nicotine, significantly increased in floral nectar immediately following simulated nectar robbing. These findings suggest that nectar robbers could be ingesting greater amounts of PSMs than they would if they visit flowers legitimately. As a consequence, increased consumption of neurotoxic nectar alkaloids or other PSMs could have negative effects on the nectar robber.

  3. Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

    PubMed

    Yan, Ning; Zhang, Hongbo; Zhang, Zhongfeng; Shi, John; Timko, Michael P; Du, Yongmei; Liu, Xinmin; Liu, Yanhua

    2016-11-15

    Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

  4. A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

    PubMed

    Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash

    2017-03-02

    A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.

  5. The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana.

    PubMed

    Wang, Hongyang; Sun, Chunlian; Jiang, Rui; He, Qin; Yang, Yu; Tian, Zhejuan; Tian, Zhendong; Xie, Conghua

    2014-07-01

    Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans-potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H2O2). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a(KI) pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H2O2 production.

  6. The Sclerophyllous Eucalyptus camaldulensis and Herbaceous Nicotiana tabacum Have Different Mechanisms to Maintain High Rates of Photosynthesis

    PubMed Central

    Huang, Wei; Tong, You-Gui; Yu, Guo-Yun; Yang, Wei-Xian

    2016-01-01

    It is believed that high levels of mesophyll conductance (gm) largely contribute to the high rates of photosynthesis in herbaceous C3 plants. However, some sclerophyllous C3 plants that display low levels of gm have high rates of photosynthesis, and the underlying mechanisms responsible for high photosynthetic rates in sclerophyllous C3 plants are unclear. In the present study, we examined photosynthetic characteristics in two high-photosynthesis plants (the sclerophyllous Eucalyptus camaldulensis and the herbaceous Nicotiana tabacum) using measurements of gas exchange and chlorophyll fluorescence. Under saturating light intensities, both species had similar rates of CO2 assimilation at 400 μmol mol−1 CO2 (A400). However, E. camaldulensis exhibited significantly lower gm and chloroplast CO2 concentration (Cc) than N. tabacum. A quantitative analysis revealed that, in E. camaldulensis, the gm limitation was the most constraining factor for photosynthesis. By comparison, in N. tabacum, the biochemical limitation was the strongest, followed by gm and gs limitations. In conjunction with a lower Cc, E. camaldulensis up-regulated the capacities of photorespiratory pathway and alternative electron flow. Furthermore, the rate of alternative electron flow was positively correlated with the rates of photorespiration and ATP supply from other flexible mechanisms, suggesting the important roles of photorespiratory pathway, and alternative electron flow in sustaining high rate of photosynthesis in E. camaldulensis. These results highlight the different mechanisms used to maintain high rates of photosynthesis in the sclerophyllous E. camaldulensis and the herbaceous N. tabacum. PMID:27933083

  7. Complex I impairment, respiratory compensations, and photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.

    PubMed

    Sabar, M; De Paepe, R; de Kouchkovsky, Y

    2000-11-01

    We have previously shown that in Nicotiana sylvestris cytoplasmic male-sterile (CMS) mutants where the mtDNA lacks the nad7 gene coding for a subunit of respiratory Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), glycine (Gly) oxidation was lower than in the wild type and insensitive to rotenone, suggesting Complex I dysfunction. In contrast, the oxidation rate of exogenous NADH and the capacity of the cyanide-resistant respiration (AOX) were enhanced. Here we report that, in contrast to Gly, the rate of malate oxidation was not affected, but proceeded totally in a rotenone-insensitive pathway, strongly suggesting that survival of CMS plants depends on the activation of internal and external alternative NAD(P) H dehydrogenases and that Gly decarboxylase activity depends on Complex I functioning. A similar defect in Complex I activity and Gly oxidation was found in the NMS1 nuclear mutant, defective in the processing of the nad4 transcript, but alternative NAD(P) H dehydrogenases were less activated. In CMS and NMS1, the fraction of the AOX pathway was increased, as compared to wild type, associated with higher amounts of aox transcripts, AOX protein, and plant resistance to cyanide. Non-phosphorylating respiratory enzymes maintained normal in vivo respiration levels in both mutants, but photosynthesis was decreased, in correlation with lower leaf conductance, emphasizing mitochondrial control on photosynthesis.

  8. Mitochondrial alternative oxidase is involved in both compatible and incompatible host-virus combinations in Nicotiana benthamiana.

    PubMed

    Zhu, Feng; Deng, Xing-Guang; Xu, Fei; Jian, Wei; Peng, Xing-Ji; Zhu, Tong; Xi, De-Hui; Lin, Hong-Hui

    2015-10-01

    The alternative oxidase (AOX) functions in the resistance to biotic stress. However, the mechanisms of AOX in the systemic antiviral defense response and N (a typical resistance gene)-mediated resistance to Tobacco mosaic virus (TMV) are elusive. A chemical approach was undertaken to investigate the role of NbAOX in the systemic resistance to RNA viruses. Furthermore, we used a virus-induced gene-silencing (VIGS)-based genetics approach to investigate the function of AOX in the N-mediated resistance to TMV. The inoculation of virus significantly increased the NbAOX transcript and protein levels and the cyanide-resistant respiration in the upper un-inoculated leaves. Pretreatment with potassium cyanide greatly increased the plant's systemic resistance, whereas the application of salicylhydroxamic acid significantly compromised the plant's systemic resistance. Additionally, in NbAOX1a-silenced N-transgenic Nicotiana benthamiana plants, the inoculated leaf collapsed and the movement of TMV into the systemic tissue eventually led to the spreading of HR-PCD and the death of the whole plant. The hypersensitive response marker gene HIN1 was significantly increased in the NbAOX1a-silenced plants. Significant amounts of TMV-CP mRNA and protein were detected in the NbAOX1a-silenced plants but not in the control plants. Overall, evidence is provided that AOX plays important roles in both compatible and incompatible plant-virus combinations.

  9. Severely reduced gravitropism in dark-grown hypocotyls of a starch-deficient mutant of Nicotiana sylvestris

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Sack, F. D.

    1990-01-01

    Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10 degrees for NS 458 and about 70 degrees for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots.

  10. Effects and Effectiveness of Two RNAi Constructs for Resistance to Pepper golden mosaic virus in Nicotiana benthamiana Plants

    PubMed Central

    Medina-Hernández, Diana; Rivera-Bustamante, Rafael Francisco; Tenllado, Francisco; Holguín-Peña, Ramón Jaime

    2013-01-01

    ToChLPV and PepGMV are Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field. PMID:24287597

  11. Pretreatment with alternation of light/dark periods improves the tolerance of tobacco (Nicotiana tabacum) to clomazone herbicide.

    PubMed

    Darwish, Majd; Lopez-Lauri, Félicie; El Maataoui, Mohamed; Urban, Laurent; Sallanon, Huguette

    2014-05-05

    This work analyses the effects of alternation of light/dark periods pretreatment (AL) in tobacco plantlets (Nicotiana tabacum L. cv.Virginie vk51) growing in solution with low concentration of the clomazone herbicide. The experimentation has been carried out by exposing the plantlets to successive and regulated periods of light (16min light/8min dark cycles, PAR 50μmolm(-2)s(-1)) for three days. The photosynthesis efficiency was determined by mean of the chlorophyll fluorescence and JIP-test. The AL pretreatment improved the clomazone tolerance; this has been observed by the increase in the leaf area of the plant, the maximal photochemical quantum efficiency of PSII (Fv/Fm), the actual PSII efficiency (ФPSII), the performance index (PIabs), the electron flux beyond Quinone A (1-VJ), and also by the diminution of the energy dissipating into heat (DI0/RC). Furthermore, AL pretreatment led to low accumulation of hydrogen peroxide (H2O2) which proves that the scavenging enzymatic system have been activated before clomazone treatment. In the plantlets pretreated with AL, with regard to the ascorbate content, some of antioxidant enzyme whose function is associated with it have continued to scavenge reactive oxygen species (ROS) induced by clomazone, such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). So, the observed photooxidative damages induced by clomazone herbicide were noticeably reduced.

  12. Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana.

    PubMed

    Bi, Huiping; Zhang, Peng

    2012-03-01

    Sweepoviruses are important begomoviruses that infect Ipomoea plants worldwide and cause sweet potato yield losses and cultivar decline. Two sweepoviruses, sweet potato leaf curl virus-Jiangsu (SPLCV-JS) and sweet potato leaf curl China virus-Zhejiang (SPLCCNV-ZJ), were cloned from diseased sweet potato plants collected in the Jiangsu and Zhejiang provinces of China. Sequence characterization and phylogenetic analysis demonstrated that both are typical monopartite begomoviruses and have close relationships to several reported SPLCV and SPLCCNV isolates, respectively, from Asian countries. Analysis of the protein alignments and subcellular localizations of the six SPLCV-JS proteins was also conducted to verify their putative functions. In Nicotiana benthamiana, an infectivity assay of the infectious SPLCV-JS clone resulted in mild symptoms and weak viral DNA accumulation. Interestingly, SPLCV-JS, together with a heterologous betasatellite DNA (tomato yellow leaf curl China virus isolate Y10 [TYLCCNV-Y10] DNA-β), showed a synergistic effect on enhanced symptom severity and viral DNA accumulation. This is the first reported infectious SPLCV clone.

  13. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

    PubMed Central

    Mathew, Lolita George; Herbst-Kralovetz, Melissa M.; Mason, Hugh S.

    2014-01-01

    Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs. PMID:24949472

  14. Overexpression of Cotton GhMPK11 Decreases Disease Resistance through the Gibberellin Signaling Pathway in Transgenic Nicotiana benthamiana

    PubMed Central

    Wang, Fang; Wang, Chen; Yan, Yan; Jia, Haihong; Guo, Xingqi

    2016-01-01

    Many changes in development, growth, hormone activity and environmental stimuli responses are mediated by mitogen-activated protein kinase (MAPK) cascades. However, in plants, studies on MAPKs have mainly focused on MPK3, MPK4 and MPK6. Here, a novel group B MAPK gene, GhMPK11, was isolated from cotton (Gossypium hirsutum L.) and characterized. Both promoter and expression pattern analyses revealed that GhMPK11 is involved in defense responses and signaling pathways. GhMPK11 overexpression in Nicotiana benthamiana plants could increase gibberellin 3 (GA3) content through the regulation of GA-related genes. Interestingly, either GhMPK11 overexpression or exogenous GA3 treatment in N. benthamiana plants could enhance the susceptibility of these plants to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani. Moreover, reactive oxygen species (ROS) accumulation was increased after pathogen infiltration due to the increased expression of ROS-related gene respiratory burst oxidative homologs (RbohB) and the decreased expression or activity of ROS detoxification enzymes regulated by GA3, such as superoxide dismutases (SODs), peroxidases (PODs), catalase (CAT) and glutathione S-transferase (GST). Taken together, these results suggest that GhMPK11 overexpression could enhance the susceptibility of tobacco to pathogen infection through the GA3 signaling pathway via down-regulation of ROS detoxification enzymes. PMID:27242882

  15. Isonitrosoacetophenone Drives Transcriptional Reprogramming in Nicotiana tabacum Cells in Support of Innate Immunity and Defense

    PubMed Central

    Djami-Tchatchou, Arnaud T.; Maake, Mmapula P.; Piater, Lizelle A.; Dubery, Ian A.

    2015-01-01

    Plants respond to various stress stimuli by activating broad-spectrum defense responses both locally as well as systemically. As such, identification of expressed genes represents an important step towards understanding inducible defense responses and assists in designing appropriate intervention strategies for disease management. Genes differentially expressed in tobacco cell suspensions following elicitation with isonitrosoacetophenone (INAP) were identified using mRNA differential display and pyro-sequencing. Sequencing data produced 14579 reads, which resulted in 198 contigs and 1758 singletons. Following BLAST analyses, several inducible plant defense genes of interest were identified and classified into functional categories including signal transduction, transcription activation, transcription and protein synthesis, protein degradation and ubiquitination, stress-responsive, defense-related, metabolism and energy, regulation, transportation, cytoskeleton and cell wall-related. Quantitative PCR was used to investigate the expression of 17 selected target genes within these categories. Results indicate that INAP has a sensitising or priming effect through activation of salicylic acid-, jasmonic acid- and ethylene pathways that result in an altered transcriptome, with the expression of genes involved in perception of pathogens and associated cellular re-programming in support of defense. Furthermore, infection assays with the pathogen Pseudomonas syringae pv. tabaci confirmed the establishment of a functional anti-microbial environment in planta. PMID:25658943

  16. Dynamic metabonomic responses of tobacco (Nicotiana tabacum) plants to salt stress.

    PubMed

    Zhang, Jingtao; Zhang, Yong; Du, Yuanyuan; Chen, Shiyun; Tang, Huiru

    2011-04-01

    Metabolic responses are important for plant adaptation to osmotic stresses. To understand the dosage and duration dependence of salinity effects on plant metabolisms, we analyzed the metabonome of tobacco plants and its dynamic responses to salt treatments using NMR spectroscopy in combination with multivariate data analysis. Our results showed that the tobacco metabonome was dominated by 40 metabolites including organic acids/bases, amino acids, carbohydrates and choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic responses to the dosage of salinity. Short-term low-dose salt stress (50 mM NaCl, 1 day) caused metabolic shifts toward gluconeogenesis with depletion of pyrimidine and purine metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach for understanding the osmotic effects on plant biochemistry.

  17. Overexpression of mitochondrial uncoupling protein 1 (UCP1) induces a hypoxic response in Nicotiana tabacum leaves

    PubMed Central

    Barreto, Pedro; Okura, Vagner; Pena, Izabella A.; Maia, Renato; Maia, Ivan G.; Arruda, Paulo

    2016-01-01

    Mitochondrial uncoupling protein 1 (UCP1) decreases reactive oxygen species production under stress conditions by uncoupling the electrochemical gradient from ATP synthesis. This study combined transcriptome profiling with experimentally induced hypoxia to mechanistically dissect the impact of Arabidopsis thaliana UCP1 (AtUCP1) overexpression in tobacco. Transcriptomic analysis of AtUCP1-overexpressing (P07) and wild-type (WT) plants was carried out using RNA sequencing. Metabolite and carbohydrate profiling of hypoxia-treated plants was performed using 1H-nuclear magnetic resonance spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection. The transcriptome of P07 plants revealed a broad induction of stress-responsive genes that were not strictly related to the mitochondrial antioxidant machinery, suggesting that overexpression of AtUCP1 imposes a strong stress response within the cell. In addition, transcripts that mapped into carbon fixation and energy expenditure pathways were broadly altered. It was found that metabolite markers of hypoxic adaptation, such as alanine and tricarboxylic acid intermediates, accumulated in P07 plants under control conditions at similar rates to WT plants under hypoxia. These findings indicate that constitutive overexpression of AtUCP1 induces a hypoxic response. The metabolites that accumulated in P07 plants are believed to be important in signalling for an improvement in carbon assimilation and induction of a hypoxic response. Under these conditions, mitochondrial ATP production is less necessary and fermentative glycolysis becomes critical to meet cell energy demands. In this scenario, the more flexible energy metabolism along with an intrinsically activated hypoxic response make these plants better adapted to face several biotic and abiotic stresses. PMID:26494730

  18. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    PubMed

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  19. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

    PubMed Central

    2011-01-01

    Background Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses. Results All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct. Conclusions Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case. PMID

  20. Amazing Altered Books

    ERIC Educational Resources Information Center

    Kieling, Linda W.

    2006-01-01

    Linda Kieling, an art teacher at Rosemont Ridge Middle school in West Linn, Oregon, describes an altered book art project she introduced to her students. Alteration of books is a form of recycling that started in the eleventh century when Italian monks recycled old manuscripts written on vellum by scraping off the ink and adding new text and…

  1. Transgenic production of cytokinin suppresses bacterially induced hypersensitive response symptoms and increases antioxidative enzyme levels in Nicotiana spp.

    PubMed

    Barna, B; Smigocki, A C; Baker, J C

    2008-11-01

    Responses of cytokinin overproducing transgenic Nicotiana plants to infections with compatible and incompatible Pseudomonas syringae pathovars were compared. Plants used were transformed with the ipt(isopentenyl transferase) gene that catalyzes the synthesis of cytokinin. In cytokinin overproducing lines that carry the ipt gene fused to the CaMV 35S (Nt+ipt), the wound-inducible proteinase inhibitor II (Ntx+ipt), or the light-inducible Rubisco small subunit protein (Npl+ipt) promoter, development of the hypersensitive response (HR) after infection with incompatible bacteria (P. syringae pv. tomato) was significantly inhibited as compared to the untransformed (Nt) controls. Over a 12 h period following inoculation, P. syrinage pv. tomato populations were slightly reduced in leaves of the cytokinin-overproducing Nt-ipt line compared with the Nt control. When the compatible P. syringae. pv. tabaci was used to infect the ipt transformed lines, slight or no significant differences in necrosis development were observed. Following infection, the titer of P. syringae pv. tabaci increased rapidly in both the transgenic and control lines but was higher in Nt+ipt plants. Leaf superoxide dismutase and catalase enzyme activities were about 60% higher in ipt leaf extracts than in the controls. This augmented antioxidant capacity likely decreased the amount of H(2)O(2) that may be associated with the higher tolerance of plants to pathogen-induced necrosis. In addition, the Nt+ipt lines had a significantly lower molar ratio of free sterols to phospholipids. The more stable membrane lipid composition and the higher antioxidant capacity likely contributed to the suppressed HR symptoms in the cytokinin overproducing Nt+ipt plants. In conclusion, the overproduction of cytokinins in tobacco appears to suppress the HR symptoms induced by incompatible bacteria.

  2. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence

    PubMed Central

    Lorenc, Michał T.; Dudley, Kevin J.; Hellens, Roger P.

    2017-01-01

    Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5’ terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker. PMID:28231340

  3. Induction of UDP-glucose:salicylic acid glucosyltransferase activity in tobacco mosaic virus-inoculated tobacco (Nicotiana tabacum) leaves

    SciTech Connect

    Enyedi, A.J.; Raskin, I. )

    1993-04-01

    Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco masaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g[sup [minus]1] fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-GTase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7--27.0 [mu]g g[sup [minus]1] fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity. 21 refs., 5 figs.

  4. Enhancement of cadmium tolerance and accumulation by introducing Perilla frutescens (L.) Britt var. frutescens genes in Nicotiana tabacum L. plants.

    PubMed

    Wei, Keqiang; Pang, Shengxi; Yang, Junxian; Wei, Zhizhong

    2015-04-01

    The tobacco has the genetic potential to remove toxic metals from the soil. To develop hyperaccumulating tobacco plants, distant hybridization between tobacco (Nicotiana tabacum L.), a high-biomass crop, and Perilla frutescens (L.) Britt var. frutescens, a newfound Cd-hyperaccumulator species, was carried out using a novel method viz. pollination following grafting. Their hybrid nature was preliminarily confirmed by phenotype, isozyme pattern, random amplified polymorphic DNA (RAPD) and metabolites analysis. About 120 putative F2 hybrids derived from the cross-combination [(N. sylvestris Speg. & Comes rootstock + N. tabacum L. var. 78-04 scion) × P. frutescens (L.) Britt var. frutescens] were then subjected to up to 300 μM CdCl2 in hydroponic conditions for 10 days. Results showed five seedlings were more resistant to Cd than female parent and accumulated 314.6 ± 99.9 mg kg(-1) Cd in their aerial biomass, which was 5.7 times greater than that in "78-04" tobacco (47.2 ± 3.56 mg kg(-1)) (P ≤ 0.05). Two of these seedlings exceeded male parent P. frutescens in the Cd concentration of shoots and reached 424 and 396 mg kg(-1), which was 13% and 6% greater for that of perilla (374.2 ± 10.38 mg kg(-1)), respectively. Compared with parents, two other F2 hybrids tended to accumulate more Cd in the root with bioconcentration factor (BCF) 7.05 and 5.17, respectively. Only one hybrid showed lower Cd concentration but transferred Cd more effectively from the root to the shoot than parents and other F2 hybrids, with the maximum translocation factor (TF) value 1.37. These indicated that the introduction of P. frutescens genes could obviously enhance the cadmium tolerance and accumulation of superior individuals.

  5. Bensulfuron-Methyl Treatment of Soil Affects the Infestation of Whitefly, Aphid, and Tobacco Mosaic Virus on Nicotiana tabacum

    PubMed Central

    Li, Renyi; Islam, Saif Ul; Wu, Zujian; Ye, Xiujuan

    2016-01-01

    Bensulfuron-methyl (BSM) is widely used in paddy soil for weed control. BSM residue in the soil has been known to inhibit the growth of sensitive crop plants. However, it is unknown whether BSM residue can affect the agrosystem in general. In this study, we have found significant effects of BSM on the infestation of Bemisia tabaci, Myzus persicae, and Tobacco mosaic virus (TMV) in Nicotiana tabacum. The soil was treated with BSM before the pest inoculation. The herbicide-treated tobaccos showed resistance to B. tabaci, but this resistance could not be detected until 15-day post-infestation when smaller number of adults B. tabaci appeared. In M. persicae assay, the longevity of all development stages of insects, and the fecundity of insects were not significantly affected when feeding on BSM-treated plants. In TMV assay, the BSM treatment also reduced virus-induced lesions in early infection time. However, the titer of TMV in BSM treated plants increased greatly over time and was over 40-fold higher than the mock-infected control plants after 20 days. Further studies showed that BSM treatment increased both jasmonic acid (JA) and salicylic acid (SA) levels in tobacco, as well as the expression of target genes in the JA and SA signaling pathways, such as NtWIPK, NtPR1a, and NtPAL. NtPR1a and NtPAL were initially suppressed after virus-inoculation, while NtRDR1 and NtRDR6, which play a key role in fighting virus infection, only showed up- or were down-regulated 20 days post virus-inoculation. Taken together, our results suggested that BSM residue in the soil may affect the metabolism of important phytohormones such as JA and SA in sensitive plants and consequently affect the plant immune response against infections such as whitefly, aphids, and viruses. PMID:28083007

  6. Jasmonate ZIM-domain (JAZ) protein regulates host and nonhost pathogen-induced cell death in tomato and Nicotiana benthamiana.

    PubMed

    Ishiga, Yasuhiro; Ishiga, Takako; Uppalapati, Srinivasa Rao; Mysore, Kirankumar S

    2013-01-01

    The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000), and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP), INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

  7. NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana.

    PubMed

    Saur, Isabel M L; Kadota, Yasuhiro; Sklenar, Jan; Holton, Nicholas J; Smakowska, Elwira; Belkhadir, Youssef; Zipfel, Cyril; Rathjen, John P

    2016-03-22

    Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.

  8. NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana

    PubMed Central

    Saur, Isabel M. L.; Kadota, Yasuhiro; Sklenar, Jan; Holton, Nicholas J.; Smakowska, Elwira; Belkhadir, Youssef; Zipfel, Cyril; Rathjen, John P.

    2016-01-01

    Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined. PMID:26944079

  9. Tomato Pto encodes a functional N-myristoylation motif that is required for signal transduction in Nicotiana benthamiana.

    PubMed

    de Vries, Jeroen S; Andriotis, Vasilios M E; Wu, Ai-Jiuan; Rathjen, John P

    2006-01-01

    Pto kinase of tomato (Lycopersicon esculentum) confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato expressing avrPto or avrPtoB. Pto interacts directly with these type-III secreted effectors, leading to induction of defence responses including the hypersensitive response (HR). Signalling by Pto requires the nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein Prf. Little is known of how Pto is controlled prior to or during stimulation, although kinase activity is required for Avr-dependent activation. Here we demonstrate a role for the N-terminus in signalling by Pto. N-terminal residues outside the kinase domain were required for induction of the HR in Nicotiana benthamiana. The N-terminus also contributed to both AvrPto-binding and phosphorylation abilities. Pto residues 1-10 comprise a consensus motif for covalent attachment of myristate, a hydrophobic 14-carbon saturated fatty acid, to the Gly-2 residue. Several lines of evidence indicate that this motif is important for Pto function. A heterologous N-myristoylation motif complemented N-terminal deletion mutants of Pto for Prf-dependent signalling. Signalling by wild-type and mutant forms of Pto was strictly dependent on the Gly-2 residue. The N-myristoylation motif of Pto complemented the cognate motif of AvrPto for avirulence function and membrane association. Furthermore, Pto was myristoylated in vivo dependent on the presence of Gly-2. The subcellular localization of Pto was independent of N-myristoylation, indicating that N-myristoylation is required for some function other than membrane affinity. Consistent with this idea, AvrPtoB was also found to be a soluble protein. The data indicate an important role(s) for the myristoylated N-terminus in Pto signalling.

  10. Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: functional characterization of the CYP82E10 gene.

    PubMed

    Lewis, Ramsey S; Bowen, Steven W; Keogh, Matthew R; Dewey, Ralph E

    2010-12-01

    In most tobacco (Nicotiana tabacum L.) plants, nornicotine is a relatively minor alkaloid, comprising about 2-5% of the total pyridine alkaloid pool in the mature leaf. Changes in gene expression at an unstable locus, however, can give rise to plants that produce high levels of nornicotine, specifically during leaf senescence and curing. Minimizing the nornicotine content in tobacco is highly desirable, because this compound serves as the direct precursor in the synthesis of N'-nitrosonornicotine, a potent carcinogen in laboratory animals. Nornicotine is likely produced almost entirely via the N-demethylation of nicotine, in a process called nicotine conversion that is catalyzed by the enzyme nicotine N-demethylase (NND). Previous studies have identified CYP82E4 as the specific NND gene responsible for the unstable conversion phenomenon, and CYP82E5v2 as a putative minor NND gene. Here, by discovery and characterization of CYP82E10, a tobacco NND gene, is reported. PCR amplification studies showed that CYP82E10 originated from the N. sylvestris ancestral parent of modern tobacco. Using a chemical mutagenesis strategy, knockout mutations were induced and identified in all three tobacco NND genes. By generating a series of mutant NND genotypes, the relative contribution of each NND gene toward the nornicotine content of the plant was assessed. Plants possessing knockout mutations in all three genes displayed nornicotine phenotypes that were much lower (∼0.5% of total alkaloid content) than that found in conventional tobacco cultivars. The introduction of these mutations into commercial breeding lines promises to be a viable strategy for reducing the levels of one of the best characterized animal carcinogens found in tobacco products.

  11. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

    PubMed

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

    2014-10-01

    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

  12. Silencing of DS2 aminoacylase-like genes confirms basal resistance to Phytophthora infestans in Nicotiana benthamiana.

    PubMed

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2014-01-01

    Nicotiana benthamiana is a potential host to several plant pathogens, and immature leaves of N. benthamiana are susceptible to Phytophthora infestans. In contrast, mature leaves of N. benthamiana are weakly susceptible and show basal resistance to P. infestans. We screened a gene-silenced mature plant showing high resistance to P. infestans, designated as DS2 (Disease suppression 2). The deduced amino acid sequence of cDNA responsible for DS2 encoded a putative aminoacylase. Growth of P. infestans decreased in DS2 plants. Trypan blue staining revealed inhibited hyphae growth of P. infestans with an increased number of dead cells under the penetration site in DS2 plants. Consistent with growth inhibition of P. infestans, defense responses such as reactive oxygen generation and expression of a salicylic acid-dependent PR-1a increased markedly in DS2 plants compared with that of control plants. DS2 phenotype was compromised in NahG plants, suggesting DS2 phenotype depends on the salicylic acid signaling pathway. Accelerated defense response was observed in DS2 plants elicited by INF1 elicitin as well as by NbMEK2(DD), which is the constitutive active form of NbMEK2, and act as a downstream regulator of INF1 perception. On the other hand, INF1- and NbMEK2(DD)-induced defense responses were prevented by DS2-overexpressing transgenic tobacco. These results suggest that DS2 negatively regulates plant defense responses against P. infestans via NbMEK2 and SA-dependent signaling pathway in N. benthamiana.

  13. Comparative study on macro- and micro-elements concentration in Nicotiana tabacum and Faba siliquis plants by ICP-MS

    NASA Astrophysics Data System (ADS)

    Balazs, Zoltan; Voica, Cezara; Dehelean, Adriana; Magdas, Dana Alina; Ristoiu, Dumitru

    2015-12-01

    Plants are important components of ecosystems as they transfer elements from abiotic into biotic environments. The concentration of macro and micro-elements in tobacco leaves (Nicotiana tabacum) and bean (Faba siliquis) was analyzed using ICP-MS technique. The results obtained indicated that the mean concentration of Mg, P, K and Ca in tobacco leaves was 0.965, 0.812, 4.412 and 2.694 g.kg-1, respectively, while in bean samples were 0.899, 2.024, 6.725 and 1.387 g.kg-1, respectively. Mn concentration ranged from 156.835 mg.kg-1 to 234.593 mg.kg-1 in tobacco leaves and from 116.174 mg.kg-1 to 440.423 mg.kg-1 in bean samples. The results for Cu and Zn were between 7.262 mg.kg-1 and 105.738 mg.kg-1, 68.549 mg.kg-1 and 113.720 mg.kg-1 (tobacco leaves); and 6.830 mg.kg-1 and 46.034 mg.kg-1, 50.166 mg.kg-1 and 77.242 mg.kg-1 (bean samples), respectively. In analyzed samples, Pb, Cd and As concentrations ranged between <0.001-0.717 mg.kg-1, 0.046 mg.kg-1 -6.218 mg.kg-1, <0.001-0.381 mg.kg-1. The paper discusses the transfer of metal ions (Mn, As, Cd, Cu, Pb and Zn, respectively) from soil to these plants in terms of transfer factors (TF).

  14. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence.

    PubMed

    Philips, Joshua G; Naim, Fatima; Lorenc, Michał T; Dudley, Kevin J; Hellens, Roger P; Waterhouse, Peter M

    2017-01-01

    Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5' terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker.

  15. Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

    PubMed

    Zhu, Feng; Xi, De-Hui; Yuan, Shu; Xu, Fei; Zhang, Da-Wei; Lin, Hong-Hui

    2014-06-01

    Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

  16. Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by sulphur starvation.

    PubMed

    Wawrzyńska, Anna; Lewandowska, Małgorzata; Sirko, Agnieszka

    2010-03-01

    Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3'-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants.

  17. Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

    PubMed

    Sticher, L; Hofsteenge, J; Neuhaus, J M; Boller, T; Meins, F

    1993-04-01

    The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

  18. Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1

    PubMed Central

    Adlung, Norman; Prochaska, Heike; Thieme, Sabine; Banik, Anne; Blüher, Doreen; John, Peter; Nagel, Oliver; Schulze, Sebastian; Gantner, Johannes; Delker, Carolin; Stuttmann, Johannes; Bonas, Ulla

    2016-01-01

    Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement. PMID:27965697

  19. Plant defense phenotypes determine the consequences of volatile emission for individuals and neighbors.

    PubMed

    Schuman, Meredith C; Allmann, Silke; Baldwin, Ian T

    2015-04-15

    Plants are at the trophic base of terrestrial ecosystems, and the diversity of plant species in an ecosystem is a principle determinant of community structure. This may arise from diverse functional traits among species. In fact, genetic diversity within species can have similarly large effects. However, studies of intraspecific genetic diversity have used genotypes varying in several complex traits, obscuring the specific phenotypic variation responsible for community-level effects. Using lines of the wild tobacco Nicotiana attenuata genetically altered in specific well-characterized defense traits and planted into experimental populations in their native habitat, we investigated community-level effects of trait diversity in populations of otherwise isogenic plants. We conclude that the frequency of defense traits in a population can determine the outcomes of these traits for individuals. Furthermore, our results suggest that some ecosystem-level services afforded by genetically diverse plant populations could be recaptured in intensive monocultures engineered to be functionally diverse.

  20. Properties of tobacco (Nicotiana tabacum) cadmium-binding peptide(s). Unique non-metallothionein cadmium ligands.

    PubMed Central

    Reese, R N; Wagner, G J

    1987-01-01

    The chemical and physical characteristics of Cd-binding peptides isolated from tobacco (Nicotiana tabacum) leaves and suspension-cultured tobacco cells were determined and compared with properties of rat liver Cd,Zn-thionein. Some emphasis was placed on metal-binding and specificity properties. Cd-peptides of apparent Mr 6000 and 2000 were induced in tobacco leaves by growth of plants with 90 microM-Cd. Only the apparent-Mr-2000 Cd-peptide was induced in the leaves of tobacco plants grown in the presence of 3 microM-Cd. In cultured tobacco cells exposed to a wide range of Cd levels (3-180 microM), a peptide of apparent Mr 2000 was observed. Under denaturing conditions [6 M-guanidinium chloride (GdmCl) with or without 100 mM-2-mercaptoethanol], all of the above forms were shown to have an Mr of approx. 1300, compared with an Mr of 6000 for Cd,Zn-thionein. The apparent disaggregation of the Mr-6000 form by GdmCl to what appears to be the unit Cd-binding peptide was reversible. Tobacco-derived Cd-peptide contained approx. 40, 35 and 15 residues of glutamate/glutamine, cysteine and glycine respectively, with serine, lysine, and aromatic residues being absent. Tobacco Cd-peptide had an isoelectric point (pI) of 3.15, which is lower than the pI greater than or equal to 4 reported for metallothionein. A 50% dissociation of Cd occurred at pH 5 and 3.5 for the tobacco Cd-peptide and Cd,Zn-thionein respectively, and GdmCl was shown to cause Cd dissociation from tobacco peptide, but not from metallothionein. No evidence was obtained for Zn induction in vivo of, or Zn binding in vitro to, tobacco Cd-peptide. Copper induced a low-Mr metal-binding component in cultured tobacco cells which did not appear to be identical with the peptide induced by Cd. Properties of tobacco Cd-peptide and Cd,Zn-thionein, including metal affinity and selectivity, are greatly different, except for the common presence of 30 residues of cysteine/100 residues. PMID:3593213

  1. The role of mesophyll conductance during water stress and recovery in tobacco (Nicotiana sylvestris): acclimation or limitation?

    PubMed

    Galle, Alexander; Florez-Sarasa, Igor; Tomas, Magdalena; Pou, Alicia; Medrano, Hipolito; Ribas-Carbo, Miquel; Flexas, Jaume

    2009-01-01

    While the responses of photosynthesis to water stress have been widely studied, acclimation to sustained water stress and recovery after re-watering is poorly understood. In particular, the factors limiting photosynthesis under these conditions, and their possible interactions with other environmental conditions, are unknown. To assess these issues, changes of photosynthetic CO(2) assimilation (A(N)) and its underlying limitations were followed during prolonged water stress and subsequent re-watering in tobacco (Nicotiana sylvestris) plants growing under three different climatic conditions: outdoors in summer, outdoors in spring, and indoors in a growth chamber. In particular, the regulation of stomatal conductance (g(s)), mesophyll conductance to CO(2) (g(m)), leaf photochemistry (chlorophyll fluorescence), and biochemistry (V(c,max)) were assessed. Leaf gas exchange and chlorophyll fluorescence data revealed that water stress induced a similar degree of stomatal closure and decreased A(N) under all three conditions, while V(c,max) was unaffected. However, the behaviour of g(m) differed depending on the climatic conditions. In outdoor plants, g(m) strongly declined with water stress, but it recovered rapidly (1-2 d) after re-watering in spring while it remained low many days after re-watering in summer. In indoor plants, g(m) initially declined with water stress, but then recovered to control values during the acclimation period. These differences were reflected in different velocities of recovery of A(N) after re-watering, being the slowest in outdoor summer plants and the fastest in indoor plants. It is suggested that these differences among the experiments are related to the prevailing climatic conditions, i.e. to the fact that stress factors other than water stress have been superimposed (e.g. excessive light and elevated temperature). In conclusion, besides g(s), g(m) contributes greatly to the limitation of photosynthesis during water stress and during

  2. Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren; Ehrenshaft, Marilyn; Daub, Margaret E

    2002-06-01

    The Cercospora nicotianae pdx1 and crg1 genes were previously identified as genes required for resistance to the singlet oxygen ((1)O(2))-generating toxin cercosporin. The pdx1 gene has subsequently been shown to be required for pyridoxine biosynthesis, but both the precise biochemical function of the PDX1 protein and the function of the CRG1 protein remain undefined, as both sequences lack defined enzymatic domains or cofactor-binding sites. The gfp gene encoding green fluorescent protein was translationally fused with pdx1 and crg1. Transformation of these constructs into strains mutant in these respective genes resulted in green-fluorescent transformants complemented for the mutant phenotype. Microscopic studies revealed that in transformants transformed with gfp alone, fluorescence was distributed evenly throughout the cytoplasm and excluded from the vacuoles. Expression of PDX1::GFP either under the constitutive Aspergillus nidulans gpdA promoter or its own native promoter was visualized as distinct fluorescent circular structures in the cytoplasm, suggesting that PDX1::GFP was probably localized in the intracellular vesicles. Expression of CRG1 fused with GFP at either its N- or C-terminus resulted in low green fluorescence, compared with that of GFP alone or PDX1::GFP. The green fluorescence of either of the CRG1::GFP fusion proteins was barely observable in transformants and was generally seen as a few scattered regions of fluorescence in the hyphae. Southern blot analysis indicated multiple copies of the constructs were integrated into the fungal genome. Northern analysis revealed that pdx1:: gfp and crg1:: gfp were each expressed as an intact transcriptional unit. Cell fractionation followed by immunoblotting against a GFP antibody showed that GFP alone and PDX1::GFP were detected exclusively in the cytoplasmic fraction. The two CRG1::GFP proteins were barely detected in the cytoplasmic fraction and not at all from the membrane fraction, a result

  3. A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update

    PubMed Central

    Ambrós, Silvia; Ruiz-Ruiz, Susana; Peña, Leandro; Moreno, Pedro

    2013-01-01

    In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system. PMID

  4. Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid-mediated systemic virus resistance in Nicotiana benthamiana.

    PubMed

    Deng, Xing-Guang; Zhu, Tong; Zou, Li-Juan; Han, Xue-Ying; Zhou, Xue; Xi, De-Hui; Zhang, Da-Wei; Lin, Hong-Hui

    2016-02-01

    Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H2O2 and NO. Scavenging of H2O2 or NO in upper leaves blocked BR-induced systemic virus resistance. BR-induced systemic H2O2 accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite-dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR-triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR-activated H2O2 is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H2O2 generation blocked BR-induced systemic NO production, but BR-induced H2O2 production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR-induced systemic virus defense in NbRBOHB-silenced plants, but H2O2 did not reverse the effect of NbNR silencing on BR-induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR-mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR-induced H2O2 and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR-mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)-dependent H2O2 production and subsequent systemic NR-dependent NO generation.

  5. Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

    PubMed Central

    Zha, Hong-Guang; Flowers, V. Lynn; Yang, Min; Chen, Ling-Yang; Sun, Hang

    2012-01-01

    Background and Aims To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco

  6. Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

    PubMed

    Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

    2016-08-01

    Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG.

  7. Effect of UV irradiation, toluidine blue, and environment on maternal haploid frequencies from the cross between Nicotiana tabacum and N. africana

    SciTech Connect

    Chimoyo, H.M.

    1988-01-01

    Treating Nicotiana africana Merxm. pollen with three levels UV radiation prior to pollinating four cultivars of flue-cured tobacco (Coker 176, NC95, McN944 and PD4), Nicotiana tabacum produced 1,953 viable seedlings from an estimated total of 170,248 seeds, of which 1,667 were haploid and 286 were hybrids. Drenching N. tabacum flowers with toluidine blue 18 hours after pollination with normal N. africana pollen, yielded 511 viable seedlings from 70,613 seeds, of which 346 were haploid and 165 hybrids. Untreated pollen gave 548 viable seedlings from 56,291 seeds, comprising 341 haploids and 208 hybrids. Contrary to results from a previous histological study, in vivo pollen tube growth rate appears to be similar irrespective of pollen source or treatment, and fertilization seems to occur at about the same time as in the selfed control. From an estimated total of 803,854 seeds sown, 3,014 viable seedlings were obtained. Coker 176 gave significantly higher yields of haploids than the other three cultivars. Field grown plants produced more haploids than greenhouse grown plants. Further evidence was obtained to support selective chromosomal elimination as the mechanism governing the development of maternal haploids from this interspecific cross.

  8. The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

    PubMed

    Shibata, Yusuke; Ojika, Makoto; Sugiyama, Akifumi; Yazaki, Kazufumi; Jones, David A; Kawakita, Kazuhito; Takemoto, Daigo

    2016-05-01

    The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

  9. Non-host Plant Resistance against Phytophthora capsici Is Mediated in Part by Members of the I2 R Gene Family in Nicotiana spp.

    PubMed

    Vega-Arreguín, Julio C; Shimada-Beltrán, Harumi; Sevillano-Serrano, Jacobo; Moffett, Peter

    2017-01-01

    The identification of host genes associated with resistance to Phytophthora capsici is crucial to developing strategies of control against this oomycete pathogen. Since there are few sources of resistance to P. capsici in crop plants, non-host plants represent a promising source of resistance genes as well as excellent models to study P. capsici - plant interactions. We have previously shown that non-host resistance to P. capsici in Nicotiana spp. is mediated by the recognition of a specific P. capsici effector protein, PcAvr3a1 in a manner that suggests the involvement of a cognate disease resistance (R) genes. Here, we have used virus-induced gene silencing (VIGS) and transgenic tobacco plants expressing dsRNA in Nicotiana spp. to identify candidate R genes that mediate non-host resistance to P. capsici. Silencing of members of the I2 multigene family in the partially resistant plant N. edwardsonii and in the resistant N. tabacum resulted in compromised resistance to P. capsici. VIGS of two other components required for R gene-mediated resistance, EDS1 and SGT1, also enhanced susceptibility to P. capsici in N. edwardsonii, as well as in the susceptible plants N. benthamiana and N. clevelandii. The silencing of I2 family members in N. tabacum also compromised the recognition of PcAvr3a1. These results indicate that in this case, non-host resistance is mediated by the same components normally associated with race-specific resistance.

  10. Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket.

    PubMed

    Acosta-Maspons, Alexis; Sepúlveda-García, Edgar; Sánchez-Baldoquín, Laura; Marrero-Gutiérrez, Junier; Pons, Tirso; Rocha-Sosa, Mario; González, Lien

    2014-01-01

    Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

  11. The Nicotiana benthamiana mitogen-activated protein kinase cascade and WRKY transcription factor participate in Nep1(Mo)-triggered plant responses.

    PubMed

    Zhang, Huajian; Li, Deqing; Wang, Meifang; Liu, Jiewen; Teng, Wenjun; Cheng, Baoping; Huang, Qian; Wang, Min; Song, Wenwen; Dong, Suomeng; Zheng, Xiaobo; Zhang, Zhengguang

    2012-12-01

    Many bacterial, fungal, and oomycete species secrete necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) that trigger programmed cell death (PCD) and innate immune responses in dicotyledonous plants. However, how NLP induce such immune responses is not understood. Here, we show that silencing of the MAPKKKα-MEK2-WIPK mitogen-activated protein kinase (MAPK) cascade through virus-induced gene silencing compromises hydrogen peroxide accumulation and PCD induced by Nep1(Mo) from Magnaporthe oryzae. WIPK interacts with NbWRKY2, a transcription factor in Nicotiana benthamiana, in vitro and in vivo, suggesting an effector pathway that mediates Nep1(Mo)-induced cell death. Unexpectedly, salicylic acid-induced protein kinase (SIPK)- and NbWRKY2-silenced plants showed impaired Nep1(Mo)-induced stomatal closure, decreased Nep1(Mo)-promoted nitric oxide (NO) production in guard cells, and a reduction in Nep1(Mo)-induced resistance against Phytophthora nicotianae. Expression studies by real-time polymerase chain reaction suggested that the MEK2-WIPK-NbWRKY2 pathway regulated Nep1(Mo)triggered NO accumulation could be partly dependent on nitrate reductase, which was implicated in NO synthesis. Taken together, these studies demonstrate that the MAPK cascade is involved in Nep1(Mo)-triggered plant responses and MAPK signaling associated with PCD exhibits shared and distinct components with that for stomatal closure.

  12. The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianae encodes a putative fungal transcription factor.

    PubMed

    Chung, Kuang-Ren; Daub, Margaret E; Kuchler, Karl; Schüller, Christoph

    2003-03-07

    The Cercospora nicotianae CRG1 gene is involved in cellular resistance to the perylenequinone toxin, cercosporin, that generates highly toxic singlet oxygen upon exposure to light. The entire open reading frame (ORF) of CRG1 was isolated and sequenced. The gene contains an ORF of 1950bp including a 65-bp intron. The predicted 650 amino acid CRG1 protein contains a Cys(6)Zn(2) binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins, indicating that CRG1 may act functionally as a transcription activator. Targeted gene disruption of CRG1 resulted in mutants that are partially sensitive to cercosporin and reduced in cercosporin production. Genetic complementation revealed that CRG1 fully restored cercosporin resistance, but only slightly restored cercosporin production in a UV-derived mutant (CS10) containing a single nucleotide substitution in crg1. Complementation of a crg1-null mutant, however, yielded strains that are similar to the wild-type in both phenotypes. These results indicate that the transcription regulator CRG1 is involved in the activation of genes associated with cercosporin resistance and production in the fungus Cercospora nicotianae.

  13. Non-host Plant Resistance against Phytophthora capsici Is Mediated in Part by Members of the I2 R Gene Family in Nicotiana spp.

    PubMed Central

    Vega-Arreguín, Julio C.; Shimada-Beltrán, Harumi; Sevillano-Serrano, Jacobo; Moffett, Peter

    2017-01-01

    The identification of host genes associated with resistance to Phytophthora capsici is crucial to developing strategies of control against this oomycete pathogen. Since there are few sources of resistance to P. capsici in crop plants, non-host plants represent a promising source of resistance genes as well as excellent models to study P. capsici – plant interactions. We have previously shown that non-host resistance to P. capsici in Nicotiana spp. is mediated by the recognition of a specific P. capsici effector protein, PcAvr3a1 in a manner that suggests the involvement of a cognate disease resistance (R) genes. Here, we have used virus-induced gene silencing (VIGS) and transgenic tobacco plants expressing dsRNA in Nicotiana spp. to identify candidate R genes that mediate non-host resistance to P. capsici. Silencing of members of the I2 multigene family in the partially resistant plant N. edwardsonii and in the resistant N. tabacum resulted in compromised resistance to P. capsici. VIGS of two other components required for R gene-mediated resistance, EDS1 and SGT1, also enhanced susceptibility to P. capsici in N. edwardsonii, as well as in the susceptible plants N. benthamiana and N. clevelandii. The silencing of I2 family members in N. tabacum also compromised the recognition of PcAvr3a1. These results indicate that in this case, non-host resistance is mediated by the same components normally associated with race-specific resistance. PMID:28261255

  14. Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro).

    PubMed

    Wu, Liming; Wu, Huijun; Chen, Lina; Zhang, Hongyue; Gao, Xuewen

    2017-01-01

    Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up-regulated the expression of the plant defence gene PR-1a and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defence pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.

  15. Attention Alters Perceived Attractiveness.

    PubMed

    Störmer, Viola S; Alvarez, George A

    2016-04-01

    Can attention alter the impression of a face? Previous studies showed that attention modulates the appearance of lower-level visual features. For instance, attention can make a simple stimulus appear to have higher contrast than it actually does. We tested whether attention can also alter the perception of a higher-order property-namely, facial attractiveness. We asked participants to judge the relative attractiveness of two faces after summoning their attention to one of the faces using a briefly presented visual cue. Across trials, participants judged the attended face to be more attractive than the same face when it was unattended. This effect was not due to decision or response biases, but rather was due to changes in perceptual processing of the faces. These results show that attention alters perceived facial attractiveness, and broadly demonstrate that attention can influence higher-level perception and may affect people's initial impressions of one another.

  16. Trichomes as dangerous lollipops: do lizards also use caterpillar body and frass odor to optimize their foraging?

    PubMed

    Stork, William F J; Weinhold, Alexander; Baldwin, Ian T

    2011-12-01

    When attacked by herbivores, plants produce toxic secondary metabolites that function as direct defenses, as well as indirect defenses that attract and reward predators of the offending herbivores. These indirect defenses include both nutritive rewards such as extra floral nectar, as well as informational rewards, such as the production and release of volatile compounds that betray the location of feeding herbivores to predators. Herbivory of Nicotiana attenuata by the tobacco hornworm (Manduca larvae) alters the volatile profiles of both the plant and larval headspace. Herbivory-elicited specific changes in the volatile profiles are detected by arthropod predators of Manduca larvae. The known predators that perceive volatile cues induced by Manduca herbivory of N. attenuata are insects that target Manduca at early developmental stages, when the larvae are still small; large, late-instar larvae may have outgrown these predation risks. However, here we offer evidence that branched chain aliphatic acids derived from the digestion of plant O-acyl sugars from trichomes may betray Manduca larvae to lizard predators during late developmental stages as well.

  17. Trichomes as dangerous lollipops

    PubMed Central

    Stork, William F. J.; Weinhold, Alexander; Baldwin, Ian T.

    2011-01-01

    When attacked by herbivores, plants produce toxic secondary metabolites that function as direct defenses, as well as indirect defenses that attract and reward predators of the offending herbivores. These indirect defenses include both nutritive rewards such as extra floral nectar, as well as informational rewards, such as the production and release of volatile compounds that betray the location of feeding herbivores to predators. Herbivory of Nicotiana attenuata by the tobacco hornworm (Manduca larvae) alters the volatile profiles of both the plant and larval headspace. Herbivory-elicited specific changes in the volatile profiles are detected by arthropod predators of Manduca larvae. The known predators that perceive volatile cues induced by Manduca herbivory of N. attenuata are insects that target Manduca at early developmental stages, when the larvae are still small; large, late-instar larvae may have outgrown these predation risks. However, here we offer evidence that branched chain aliphatic acids derived from the digestion of plant O-acyl sugars from trichomes may betray Manduca larvae to lizard predators during late developmental stages as well. PMID:22095147

  18. Analysis of plant-bacteria interactions in their native habitat: bacterial communities associated with wild tobacco are independent of endogenous jasmonic acid levels and developmental stages.

    PubMed

    Santhanam, Rakesh; Groten, Karin; Meldau, Dorothea G; Baldwin, Ian T

    2014-01-01

    Jasmonic acid (JA) mediates defense responses against herbivores and necrotrophic pathogens but does it influence the recruitment of bacterial communities in the field? We conducted field and laboratory experiments with transformed Nicotiana attenuata plants deficient in jasmonate biosynthesis (irAOC) and empty vector controls (EV) to answer this question. Using both culture-dependent and independent techniques, we characterized root and leaf-associated bacterial communities over five developmental stages, from rosette through flowering of plants grown in their natural habitat. Based on the pyrosequencing results, alpha and beta diversity did not differ among EV and irAOC plants or over ontogeny, but some genera were more abundant in one of the genotypes. Furthermore, bacterial communities were significantly different among leaves and roots. Taxa isolated only from one or both plant genotypes and hence classified as 'specialists' and 'generalists' were used in laboratory tests to further evaluate the patterns observed from the field. The putative specialist taxa did not preferentially colonize the jasmonate-deficient genotype, or alter the plant's elicited phytohormone signaling. We conclude that in N. attenuata, JA signaling does not have a major effect on structuring the bacterial communities and infer that colonization of plant tissues is mainly shaped by the local soil community in which the plant grows.

  19. Uptake of NO, NO 2 and O 3 by sunflower ( Helianthus annuus L.) and tobacco plants ( Nicotiana tabacum L.): dependence on stomatal conductivity

    NASA Astrophysics Data System (ADS)

    Neubert, A.; Kley, D.; Wildt, J.; Segschneider, H. J.; Förstel, H.

    The uptake of NO, NO 2 and O 3 by sunflowers ( Helianthus annuus L. var. giganteus) and tobacco plants ( Nicotiana tabacum L. var. Bel W3), using concentrations representative for moderately polluted air, has been determined by gas exchange experiments. Conductivities for these trace gases were measured at different light fluxes ranging from 820 μEm -2s -1 to darkness. The conductivities to water vapor and the trace gases are highly correlated. It is concluded that the uptake of NO, NO 2 and O 3 by sunflowers and tobacco plants is linearly dependent on stomatal opening. While the uptake of NO is limited by the mesophyll resistance, the uptake of NO 2 is only by diffusion through the stomata. Loss processes by deposition to the leaf surfaces are more pronounced for O 3 than for NO and NO 2.

  20. The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

    PubMed Central

    Resmi, Thulasi Raveendrannair; Hohn, Thomas; Hohn, Barbara; Veluthambi, Karuppannan

    2015-01-01

    Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases. PMID:26008704

  1. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

  2. Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing.

    PubMed

    van den Boogaart, T; Wen, F; Davies, J W; Lomonossoff, G P

    2001-02-01

    Virus resistance in Nicotiana benthamiana plants containing a translatable Pea early browning virus (PEBV) 54K sequence from the 201K replicase gene has been reported previously. Resistant plants contain multiple transgene copies divided between two loci. Analysis of a genetic series containing the two loci in separate homozygous or heterozygous condition suggest that only one of the loci is necessary to induce the resistance. The resistance observed in R2 and R3 generations of lines containing both transgene loci in homozygous condition became less consistent in R4 and R5 generations. This inversely correlated with steady-state transgene transcript levels of the segregating populations. The use of recombinant Potato virus X vectors carrying PEBV 54K sequences showed that the resistance is based upon posttranscriptional gene silencing, is non-strand specific, and recognizes 3' located sequences within the PEBV 54K sequence.

  3. Involvement of the Leaf-Specific Multidrug and Toxic Compound Extrusion (MATE) Transporter Nt-JAT2 in Vacuolar Sequestration of Nicotine in Nicotiana tabacum

    PubMed Central

    Shitan, Nobukazu; Minami, Shota; Morita, Masahiko; Hayashida, Minaho; Ito, Shingo; Takanashi, Kojiro; Omote, Hiroshi; Moriyama, Yoshinori; Sugiyama, Akifumi; Goossens, Alain; Moriyasu, Masataka; Yazaki, Kazufumi

    2014-01-01

    Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues. PMID:25268729

  4. Cotton GhMKK5 affects disease resistance, induces HR-like cell death, and reduces the tolerance to salt and drought stress in transgenic Nicotiana benthamiana

    PubMed Central

    Zhang, Liang; Li, Yuzhen; Lu, Wenjing; Meng, Fei; Wu, Chang-ai; Guo, Xingqi

    2012-01-01

    Mitogen-activated protein kinase (MAPK) cascades are involved in various processes from plant growth and development to biotic and abiotic stress responses. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), play crucial roles in MAPK cascades to mediate a variety of stress responses in plants. However, few MAPKKs have been functionally characterized in cotton (Gossypium hirsutum). In this study, a novel gene, GhMKK5, from cotton belonging to the group C MAPKKs was isolated and characterized. The expression of GhMKK5 can be induced by pathogen infection, abiotic stresses, and multiple defence-related signal molecules. The overexpression of GhMKK5 in Nicotiana benthamiana enhanced the plants’ resistance to the bacterial pathogen Ralstonia solanacearum by elevating the expression of pathogen resistance (PR) genes, including PR1a, PR2, PR4, PR5, and NPR1, but increased the plants’ sensitivity to the oomycete pathogen Phytophthora parasitica var. nicotianae Tucker. Importantly, GhMKK5-overexpressing plants displayed markedly elevated expression of reactive oxygen species-related and cell death marker genes, such as NtRbohA and NtCDM, and resulted in hypersensitive response (HR)-like cell death characterized by the accumulation of H2O2. Furthermore, it was demonstrated that GhMKK5 overexpression in plants reduced their tolerance to salt and drought stresses, as determined by statistical analysis of seed germination, root length, leaf water loss, and survival rate. Drought obviously accelerated the cell death phenomenon in GhMKK5-overexpressing plants. These results suggest that GhMKK5 may play an important role in pathogen infection and the regulation of the salt and drought stress responses in plants. PMID:22442420

  5. A Glycosyltransferase from Nicotiana alata Pollen Mediates Synthesis of a Linear (1,5)-α-L-Arabinan When Expressed in Arabidopsis1[OPEN

    PubMed Central

    Ho, Yin Ying; Moller, Isabel E.; Koh, Poh-Ling; Bacic, Antony

    2016-01-01

    The walls of Nicotiana alata pollen tubes contain a linear arabinan composed of (1,5)-α-linked arabinofuranose residues. Although generally found as a side chain on the backbone of the pectic polysaccharide rhamnogalacturonan I, the arabinan in N. alata pollen tubes is considered free, as there is no detectable rhamnogalacturonan I in these walls. Carbohydrate-specific antibodies detected arabinan epitopes at the tip and along the shank of N. alata pollen tubes that are predominantly part of the primary layer of the bilayered wall. A sequence related to ARABINAN DEFICIENT1 (AtARAD1), a presumed arabinan arabinosyltransferase from Arabidopsis (Arabidopsis thaliana), was identified by searching an N. alata pollen transcriptome. Transcripts for this ARAD1-like sequence, which we have named N. alata ARABINAN DEFICIENT-LIKE1 (NaARADL1), accumulate in various tissues, most abundantly in the pollen grain and tube, and encode a protein that is a type II membrane protein with its catalytic carboxyl terminus located in the Golgi lumen. The NaARADL1 protein can form homodimers when transiently expressed in Nicotiana benthamiana leaves and heterodimers when coexpressed with AtARAD1. The expression of NaARADL1 in Arabidopsis led to plants with more arabinan in their walls and that also exuded a guttation fluid rich in arabinan. Chemical and enzymatic characterization of the guttation fluid showed that a soluble, linear α-(1,5)-arabinan was the most abundant polymer present. These results are consistent with NaARADL1 having an arabinan (1,5)-α-arabinosyltransferase activity. PMID:26850276

  6. Expression of the cercosporin toxin resistance gene ( CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren; Daub, Margaret E; Ehrenshaft, Marilyn

    2003-09-01

    The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene ( PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1- PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.

  7. Na⁺/H⁺ exchanger 1 participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system.

    PubMed

    Chen, Xianyang; Bao, Hexigeduleng; Guo, Jie; Jia, Weitao; Tai, Fang; Nie, Lingling; Jiang, Ping; Feng, Juanjuan; Lv, Sulian; Li, Yinxin

    2014-11-01

    Despite the importance of NHX1 (Na(+)/H(+) exchanger 1) in plant salt tolerance, little is known about its other functions. In this study, intriguingly, it was found that NHX1 participated in plant disease defence against Phytophthora parasitica var. nicotianae (Ppn) in Nicotiana benthamiana. NbNHX1 was originally isolated from N. benthamiana, and characterized. The subcellular localization of NbNHX1 with its C-terminus fused with green fluorescent protein indicated that NbNHX1 localized primarily to the tonoplast. Tobacco rattle virus-induced NbNHX1 silencing led to reduced H(+) efflux from the vacuole to cytoplasts, and decreased Ppn resistance in N. benthamiana. After attack by Ppn, NbNHX1-silenced plants exhibited impaired ability to scavenge reactive oxidative species (ROS) induced by the pathogen. Pea early browning virus-mediated ectopic expression of SeNHX1 (from Salicornia europaea) or AtNHX1 (from Arabidopsis thaliana) both conferred enhanced Ppn resistance to N. benthamiana, with a lower H2O2 concentration after Ppn inoculation. Further investigation of the role of NHX1 demonstrated that transient overexpression of NbNHX1 improved the vacuolar pH and cellular ROS level in N. benthamiana, which was coupled with an enlarged NAD(P) (H) pool and higher expression of ROS-responsive genes. In contrast, NbNHX1 silencing led to a lower pH in the vacuole and a lower cellular ROS level in N. benthamiana, which was coupled with a decreased NAD(P) (H) pool and decreased expression of ROS-responsive genes. These results suggest that NHX1 is involved in plant disease defence; and regulation of vacuolar pH by NHX1, affecting the cellular oxidation state, primes the antioxidative system which is associated with Ppn resistance in tobacco.

  8. Genetically Altered Plant Species

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Researchers in Robert Ferl's lab at the University of Florida in Gainesville, genetically altered this Arabdopsis Thaliana (a brassica species) plant to learn how extreme environments, such as the low atmospheric pressure on Mars, affect plant genes. They inserted green fluorescent protein (GFP) near the on/off switches for anoxia and drought genes. When those genes were turned on after exposure to reduced atmospheric pressure, GFP was turned on as well, causing cells expressing those genes to glow green under a blue light. The natural fluorescence of chlorophyll accounts for the red glow.

  9. [Unusual myopic fundus alteration].

    PubMed

    Münzenberg, C; Paulsen, F; Kalinski, T; Dmitriew, A; Duncker, G I W; Sel, S

    2009-07-01

    A 44-year-old female patient reported a "black dot" which had been in front of the right eye for more than 4 days and which moved together with eye movements. The optical coherence tomography (OCT) image of the right macula showed large cystic cavities and thickening within the retinal pigment epithelium (RPE) near the fovea centralis as well as small bore cystic alterations, which indicated an event in the region of the choroid. Fluorescein angiography and indocyanine green angiography excluded choroidal neovascularization (CNV). The diagnosis revealed a broad superficial choroidal blood vessel mimicking a subretinal hemorrhage.

  10. Molecular heterogeneity of photosystem I. psaD, psaE, psaF, psaH, and psaL are all present in isoforms in Nicotiana spp.

    PubMed

    Obokata, J; Mikami, K; Hayashida, N; Nakamura, M; Sugiura, M

    1993-08-01

    The protein composition of photosystem I (PSI) was examined in Nicotiana spp. by high-resolution polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and immunoblot analysis. Five PSI proteins show polymorphism in an amphidiploid species, Nicotiana tabacum, but not in its ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana spp. appear to have at least 18 PSI proteins per genome that range in molecular mass from 3 to 20 kD. They include the products of nuclear genes psaD, psaE, psaF, psaG, psaH, psaK, and psaL, the product of chloroplast gene psaC, N-terminally blocked proteins of 4.5 and 3.0 kD, and an unidentified protein of 12.5 kD. The psaD, psaF, psaH, and psaL products have two isoforms each that are distinguished by different mobilities in polyacrylamide gel electrophoresis, and the psaE product has four isoforms. The two isoforms of the psaD product have distinct amino acid sequences, indicating that they are encoded by different genes within the genome. Four isoforms of the psaE products can be classified into two groups by N-terminal amino acid sequence, indicating that at least two psaE genes are present in the genome. To examine whether the polymorphic nature of PSI is peculiar to Nicotiana spp., we carried out immunoblot analysis of the psaD and psaE products in isogenic lines of tomato (Lycopersicon esculentum), Arabidopsis thaliana, red bean (Vigna angularis), and corn (Zea mays). Two electrophoretically distinct isoforms were found for the psaD products of tomato, A. thaliana, and corn, and two isoforms of psaE products were detected in tomato, A. thaliana, and red bean. These results suggest that the nuclear-encoded subunits of PSI, except for the psaG and psaK products, generally have two isoforms.

  11. Altered fingerprints: analysis and detection.

    PubMed

    Yoon, Soweon; Feng, Jianjiang; Jain, Anil K

    2012-03-01

    The widespread deployment of Automated Fingerprint Identification Systems (AFIS) in law enforcement and border control applications has heightened the need for ensuring that these systems are not compromised. While several issues related to fingerprint system security have been investigated, including the use of fake fingerprints for masquerading identity, the problem of fingerprint alteration or obfuscation has received very little attention. Fingerprint obfuscation refers to the deliberate alteration of the fingerprint pattern by an individual for the purpose of masking his identity. Several cases of fingerprint obfuscation have been reported in the press. Fingerprint image quality assessment software (e.g., NFIQ) cannot always detect altered fingerprints since the implicit image quality due to alteration may not change significantly. The main contributions of this paper are: 1) compiling case studies of incidents where individuals were found to have altered their fingerprints for circumventing AFIS, 2) investigating the impact of fingerprint alteration on the accuracy of a commercial fingerprint matcher, 3) classifying the alterations into three major categories and suggesting possible countermeasures, 4) developing a technique to automatically detect altered fingerprints based on analyzing orientation field and minutiae distribution, and 5) evaluating the proposed technique and the NFIQ algorithm on a large database of altered fingerprints provided by a law enforcement agency. Experimental results show the feasibility of the proposed approach in detecting altered fingerprints and highlight the need to further pursue this problem.

  12. Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana

    PubMed Central

    Garcia, J.P.; Beingesser, J.; Bohorov, O.; Bohorova, N.; Goodman, C.; Kim, D.; Pauly, M.; Velasco, J.; Whaley, K.; Zeitlin, L.; Roy, C.J.; Uzal, F.A.

    2014-01-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 =1.6 μg/kg) and resulted in a mean time from inoculation to death of 18 and 180 minutes when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 minutes post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication. PMID:24950050

  13. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.

    PubMed

    Ritala, Anneli; Dong, Lemeng; Imseng, Nicole; Seppänen-Laakso, Tuulikki; Vasilev, Nikolay; van der Krol, Sander; Rischer, Heiko; Maaheimo, Hannu; Virkki, Arho; Brändli, Johanna; Schillberg, Stefan; Eibl, Regine; Bouwmeester, Harro; Oksman-Caldentey, Kirsi-Marja

    2014-04-20

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 μg/g dry weight (DW) and a maximum of 31.3 μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag.

  14. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

    PubMed

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

  15. Biological and chemical induction of resistance to the Globodera tabacum solanacearum in oriental and flue-cured tobacco (Nicotiana tabacum L.).

    PubMed

    Parkunan, Venkatesan; Johnson, Charles S; Eisenback, Jon D

    2009-09-01

    The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field.

  16. Effect of Potato Virus Y on the NADP-Malic Enzyme from Nicotiana tabacum L.: mRNA, Expressed Protein and Activity

    PubMed Central

    Doubnerová, Veronika; Müller, Karel; Čeřovská, Noemi; Synková, Helena; Spoustová, Petra; Ryšlavá, Helena

    2009-01-01

    The effect of biotic stress induced by viral infection (Potato virus Y, strain NTN and O) on NADP-malic enzyme (EC 1.1.1.40) in tobacco plants (Nicotiana tabacum L., cv. Petit Havana, SR1) was tested at the transcriptional, translational and activity level. The increase of enzyme activity in infected leaves was correlated with the increased amount of expressed protein and with mRNA of cytosolic NADP-ME isoform. Transcription of the chloroplastic enzyme was not influenced by viral infection. The increase of the enzyme activity was also detected in stems and roots of infected plants. The effect of viral infection induced by Potato virus Y, NTN strain, causing more severe symptoms, was compared with the effect induced by milder strain PVYO. The observed increase in NADP-malic enzyme activity in all parts of the studied plants was higher in the case of PVYNTN strain than in the case of strain PVYO. The relevance of NADP-malic enzyme in plants under stress conditions was discussed. PMID:20111689

  17. Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

    PubMed

    Scalabrin, Elisa; Radaelli, Marta; Capodaglio, Gabriele

    2016-06-01

    The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes.

  18. Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

    SciTech Connect

    Altabella, T.; Chrispeels, M.J. )

    1990-06-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{sub r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

  19. An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

    PubMed

    Jutras, Philippe V; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Michaud, Dominique; Donini, Marcello

    2016-01-01

    The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

  20. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    PubMed Central

    Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Yuan, Zheng; Zhang, Dabing

    2016-01-01

    Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise. PMID:27635142

  1. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases

    PubMed Central

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host. PMID:26648951

  2. Multivariate optimization of a headspace solid-phase microextraction method followed by gas chromatography with mass spectrometry for the determination of terpenes in Nicotiana langsdorffii.

    PubMed

    Ardini, Francisco; Carro, Marina Di; Abelmoschi, Maria Luisa; Grotti, Marco; Magi, Emanuele

    2014-07-01

    A simple and sensitive procedure based on headspace solid-phase microextraction and gas chromatography with mass spectrometry was developed for the determination of five terpenes (α-pinene, limonene, linalool, α-terpineol, and geraniol) in the leaves of Nicotiana langsdorffii. The microextraction conditions (extraction temperature, equilibration time, and extraction time) were optimized by means of a Doehlert design. The experimental design showed that, for α-pinene and limonene, a low temperature and a long extraction time were needed for optimal extraction, while linalool, α-terpineol, and geraniol required a high temperature and a long extraction time. The chosen compromise conditions were temperature 60°C, equilibration time 15 min and extraction time 50 min. The main analytical figures of the optimized method were evaluated; LODs ranged from 0.07 ng/g (α-pinene) to 8.0 ng/g (geraniol), while intraday and interday repeatability were in the range 10-17% and 9-13%, respectively. Finally, the procedure was applied to in vitro wild-type and transgenic specimens of N. langsdorffii subjected to abiotic stresses (chemical and heat stress). With the exception of geraniol (75-374 ng/g), low concentration levels of terpenes were measured (ng/g level or lower); some interesting variations in terpene concentration induced by abiotic stress were observed.

  3. Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana

    PubMed Central

    Yan, Yan; Jia, Haihong; Wang, Fang; Wang, Chen; Liu, Shuchang; Guo, Xingqi

    2015-01-01

    WRKY proteins constitute transcriptional regulators involved in various biological processes, especially in coping with diverse biotic and abiotic stresses. However, in contrast to other well-characterized WRKY groups, the functions of group III WRKY transcription factors are poorly understood in the economically important crop cotton (Gossypium hirsutum). In this study, a group III WRKY gene from cotton, GhWRKY27a, was isolated and characterized. Our data indicated that GhWRKY27a localized to the nucleus and that GhWRKY27a expression could be strongly induced by abiotic stresses, pathogen infection, and multiple defense-related signaling molecules. Virus-induced gene silencing (VIGS) of GhWRKY27a enhanced tolerance to drought stress in cotton. In contrast, GhWRKY27a overexpression in Nicotiana benthamiana markedly reduced plant tolerance to drought stress, as determined through physiological analyses of leaf water loss, survival rates, and the stomatal aperture. This susceptibility was coupled with reduced stomatal closure in response to abscisic acid and decreased expression of stress-related genes. In addition, GhWRKY27a-overexpressing plants exhibited reduced resistance to Rhizoctonia solani infection, mainly demonstrated by the transgenic lines exhibiting more severe disease symptoms, accompanied by attenuated expression of defense-related genes in N. benthamiana. Taken together, these findings indicated that GhWRKY27a functions in negative responses to drought tolerance and in resistance to R. solani infection. PMID:26483697

  4. Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    PubMed Central

    Nemchinov, Lev G.; Boutanaev, Alexander M.; Postnikova, Olga A.

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  5. Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L.

    PubMed

    Chen, Dan; Deng, Yingtian; Zhao, Jie

    2012-01-15

    Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.

  6. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

    PubMed Central

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  7. Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.

    PubMed Central

    Marin, E; Nussaume, L; Quesada, A; Gonneau, M; Sotta, B; Hugueney, P; Frey, A; Marion-Poll, A

    1996-01-01

    Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location. Images PMID:8665840

  8. An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

    PubMed Central

    Jutras, Philippe V.; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Donini, Marcello

    2016-01-01

    The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories. PMID:27893815

  9. Synergistic infection of BrYV and PEMV 2 increases the accumulations of both BrYV and BrYV-derived siRNAs in Nicotiana benthamiana

    PubMed Central

    Zhou, Cui-Ji; Zhang, Xiao-Yan; Liu, Song-Yu; Wang, Ying; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2017-01-01

    Viral synergism is caused by co-infection of two unrelated viruses, leading to more severe symptoms or increased titres of one or both viruses. Synergistic infection of phloem-restricted poleroviruses and umbraviruses has destructive effects on crop plants. The mechanism underlying this synergy remains elusive. In our study, synergism was observed in co-infections of a polerovirus Brassica yellows virus (BrYV) and an umbravirus Pea enation mosaic virus 2 (PEMV 2) on Nicotiana benthamiana, which led to (1) increased titres of BrYV, (2) appearance of severe symptoms, (3) gain of mechanical transmission capacity of BrYV, (4) broader distribution of BrYV to non-vascular tissues. Besides, profiles of virus-derived small interfering RNAs (vsiRNAs) from BrYV and PEMV 2 in singly and doubly infected plants were obtained by small RNA deep sequencing. Our results showed that accumulation of BrYV vsiRNAs increased tremendously and ratio of positive to negative strand BrYV vsiRNAs differed between singly infected and co-infected plants. Positions to which the BrYV vsiRNAs mapped to the viral genome varied considerably during synergistic infection. Moreover, target genes of vsiRNAs were predicted and annotated. Our results revealed the synergistic characteristics during co-infection of BrYV and PEMV 2, and implied possible effects of synergism have on vsiRNAs. PMID:28345652

  10. A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

    PubMed Central

    Li, Fangfang; Zhao, Nan; Xu, Xiongbiao; Wang, Yaqin; Yang, Xiuling; Liu, Shu-Sheng; Wang, Aiming; Zhou, Xueping

    2017-01-01

    A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway. PMID:28212430

  11. NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense1

    PubMed Central

    Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

    2005-01-01

    Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family. PMID:16126865

  12. Targeting of a Nicotiana plumbaginifolia H+-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

    PubMed Central

    Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

    2004-01-01

    The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+-ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane–span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting. PMID:15208389

  13. Overexpression of cotton GhMKK4 enhances disease susceptibility and affects abscisic acid, gibberellin and hydrogen peroxide signalling in transgenic Nicotiana benthamiana.

    PubMed

    Li, Yuzhen; Zhang, Liang; Lu, Wenjing; Wang, Xiuling; Wu, Chang-Ai; Guo, Xingqi

    2014-01-01

    Mitogen-activated protein kinase (MAPK) cascades are involved in plant development, stress responses and hormonal signal transduction. MAPK kinases (MAPKKs), as the key nodes in these cascades, link MAPKs and MAPKK kinases (MAPKKKs). In this study, GhMKK4, a novel group C MAPKK gene from cotton (Gossypium hirsutum), was isolated and identified. Its expression can be induced by various stresses and signalling molecules. The overexpression of GhMKK4 in Nicotiana benthamiana enhanced its susceptibility to bacterial and fungal pathogens, but had no significant effects on salt or drought tolerance. Notably, the overexpressing plants showed increased sensitivity to abscisic acid (ABA) and gibberellin A3 (GA3), and ABA and gibberellin (GA) signalling were affected on infection with Ralstonia solanacearum bacteria. Furthermore, the overexpressing plants showed more reactive oxygen species (ROS) accumulation and stronger inhibition of catalase (CAT), a ROS-scavenging enzyme, than control plants after salicylic acid (SA) treatment. Interestingly, two genes encoding ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), the key enzymes in polyamine synthesis, exhibited reduced R. solanacearum-induced expression in overexpressing plants. These findings broaden our knowledge about the functions of MAPKKs in diverse signalling pathways and the negative regulation of disease resistance in the cotton crop.

  14. N-Glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential

    PubMed Central

    Hamorsky, Krystal Teasley; Kouokam, J. Calvin; Jurkiewicz, Jessica M.; Nelson, Bailey; Moore, Lauren J.; Husk, Adam S.; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-01

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants. PMID:25614217

  15. N-glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential.

    PubMed

    Hamorsky, Krystal Teasley; Kouokam, J Calvin; Jurkiewicz, Jessica M; Nelson, Bailey; Moore, Lauren J; Husk, Adam S; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-23

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

  16. Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies

    PubMed Central

    Petre, Benjamin; Saunders, Diane G. O.; Sklenar, Jan; Lorrain, Cécile; Krasileva, Ksenia V.; Win, Joe; Duplessis, Sébastien; Kamoun, Sophien

    2016-01-01

    Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors. PMID:26863009

  17. Exclusion of a Proton ATPase from the Apical Membrane Is Associated with Cell Polarity and Tip Growth in Nicotiana tabacum Pollen Tubes[W

    PubMed Central

    Certal, Ana C.; Almeida, Ricardo B.; Carvalho, Lara M.; Wong, Eric; Moreno, Nuno; Michard, Erwan; Carneiro, Jorge; Rodriguéz-Léon, Joaquín; Wu, Hen-Ming; Cheung, Alice Y.; Feijó, José A.

    2008-01-01

    Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca2+, H+, K+, and Cl− -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H+-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H+ effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H+ flux profiles. Furthermore, there is no net flux of H+ in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes. PMID:18364468

  18. Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees.

    PubMed

    Helsper, J P F G; Ruyter-Spira, C P; Kwakman, P H S; Bleeker, W K; Keizer, L C P; Bade, J B; Te Velde, A A; Zaat, S A J; Verbeek, M; Creemers-Molenaar, J

    2011-09-01

    Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⁻¹. There is a significant linear relationship (P<0.001) between hEGF concentration and induction of hEGF-receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.

  19. Nicotiana glauca engineered for the production of ketocarotenoids in flowers and leaves by expressing the cyanobacterial crtO ketolase gene.

    PubMed

    Zhu, Changfu; Gerjets, Tanja; Sandmann, Gerhard

    2007-12-01

    Nicotiana glauca is a tobacco species that forms flowers with carotenoid-pigmented petals, sepals, pistil, ovary and nectary tissue. The carotenoids produced are lutein, ss-carotene as well as some violaxanthin and antheraxanthin. This tobacco species was genetically modified for ketocarotenoid biosynthesis by transformation with a cyanobacterial crtO ketolase gene under the 35S CaMV promoter. In the transformants, ketocarotenoids were detected in both leaves and flowers. Although astaxanthin was not detected other ketocarotenoids such as 4'-ketolutein, echinenone, 3'-hydroxyechinenone and 4-ketozeaxanthin were present. Accumulation of ketocarotenoids in leaves decreased their photosynthetic efficiency moderately. Under the green house conditions used no impairment of growth and development compared to the wild type was observed. In the crtO-transformants, an unexpected up-regulation of total carotenoid biosynthesis in leaves and especially in flower petals was observed. This led to a total ketocarotenoid concentration in leaves of 136.6 (young) or 156.1 (older) mug/g dry weight and in petals of 165 mug/g dry weight. In our engineered plants, the ketocarotenoid pathway is one step short of astaxanthin. Strategies are discussed to improve N. glauca flowers as a biological system for astaxanthin.

  20. Ectopic Expression of Capsicum-Specific Cell Wall Protein Capsicum annuum Senescence-Delaying 1 (CaSD1) Delays Senescence and Induces Trichome Formation in Nicotiana benthamiana

    PubMed Central

    Seo, Eunyoung; Yeom, Seon-In; Jo, SungHwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

    2012-01-01

    Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation. PMID:22441673

  1. Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves

    PubMed Central

    Outchkourov, Nikolay S.; Carollo, Carlos A.; Gomez-Roldan, Victoria; de Vos, Ric C. H.; Bosch, Dirk; Hall, Robert D.; Beekwilder, Jules

    2014-01-01

    Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species. PMID:25339964

  2. Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1 enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana.

    PubMed

    Li, Xinlong; Zhang, Yali; Yin, Ling; Lu, Jiang

    2017-03-01

    The NBS-LRR proteins encoded by the majority of R genes represent important intracellular receptors that directly or indirectly recognize pathogen effector proteins, which subsequently activate plant defense responses. In this study, a novel Plasmopara viticola-induced TIR-NBS-LRR gene, named VaRGA1, was cloned from leaf tissues of a highly downy mildew-resistant Vitis amurensis "Shuanghong" grapevine. The fluorescence signal of the VaRGA1-GFP fusion protein was clearly partitioned to the cytoplasm and nucleus. The expression of the VaRGA1 gene was strongly induced during early stages of infection by P. viticola, and was also significantly upregulated after drought and salt treatments. Accordingly, grapevine leaves transiently expressing the VaRGA1 gene manifested increased resistance to P. viticola, and the overexpression of the VaRGA1 gene in Nicotiana benthamiana conferred enhanced resistance to Phytophthora parasitica through the activation of salicylic acid (SA) signaling and phenylpropanoid pathways and could also increase tolerance to drought and salt stresses at the germination and vegetable growth stages. These findings indicate that the grapevine VaRGA1 gene may function as the immune and non-immune receptors against biotic and abiotic stresses and that there may be signaling overlap between biotic and abiotic responses.

  3. Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

    PubMed

    Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

    2012-04-01

    Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

  4. A conserved region in the Closterovirus 1a polyprotein drives extensive remodeling of endoplasmic reticulum membranes and induces motile globules in Nicotiana benthamiana cells.

    PubMed

    Gushchin, V A; Karlin, D G; Makhotenko, A V; Khromov, A V; Erokhina, T N; Solovyev, A G; Morozov, S Yu; Agranovsky, A A

    2017-02-01

    In infected plant cells, closterovirus replicative polyproteins 1a and 1ab drive membrane remodeling and formation of multivesicular replication platforms. Polyprotein 1a contains a variable Central Region (CR) between the methyltransferase and helicase domains. In a previous study, we have found that transient expression of the Beet yellows virus CR-2 segment (aa 1305-1494) in Nicotiana benthamiana induces the formation of ~1µm mobile globules originating from the ER membranes. In the present study, sequence analysis has shown that a part of the CR named the "Zemlya region" (overlapping the CR-2), is conserved in all members of the Closterovirus genus and contains a predicted amphipathic helix (aa 1368-1385). By deletion analysis, the CR-2 region responsible for the induction of 1-μm globules has been mapped to aa 1368-1432. We suggest that the conserved membrane-modifying region of the BYV 1a may be involved in the biogenesis of closterovirus replication platforms.

  5. Spatial differences in (Z)-3-hexen-1-ol production preferentially reduces Spodoptera litura larva attack on the young leaves of Nicotiana benthamiana.

    PubMed

    Zhang, Yuqian; Fu, Xiumin; Wang, Feiyan; Yang, Ziyin

    2016-11-01

    Plants synthesize specialized metabolites which possess extremely important ecological functions including direct defense, indirect defense, and signaling. The optimal defense theory (ODT) proposes that defensive metabolites are preferentially allocated to the tissues with high fitness value or in locations that are easily injured. In our present study, using the model plant Nicotiana benthamiana, we found that direct defense of N. benthamiana against Spodoptera litura (Fabricius) larvae showed spatial differences in the sites producing defensive chemicals. The upper leaves possessed significantly stronger direct defense ability than the middle and lower leaves. Interestingly, the strong defense ability of the upper leaves was not due to occurrences of well-known defensive metabolites such as nicotine and chlorogenic acid. After damage, the middle and lower leaves emitted higher amounts of (Z)-3-hexen-1-ol than the upper leaves, which could both attract larvae and significantly increase the amount of middle and lower leaf eaten by the larvae. The spatial difference in (Z)-3-hexen-1-ol emission may be due to spatial differences in expression of lipoxygenase (NbLOX2), which is responsible for the formation and emission of (Z)-3-hexen-1-ol. This study provided new insight into ODT, showing that plants effectively protect easily injured tissues through reduction in concentration of herbivore-feeding stimulant in the tissues.

  6. Biological and Chemical Induction of Resistance to the Globodera tabacum solanacearum in Oriental and Flue-Cured Tobacco (Nicotiana tabacum L.)

    PubMed Central

    Johnson, Charles S.; Eisenback, Jon D.

    2009-01-01

    The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field. PMID:22736815

  7. Difference of physiological characters in dark green islands and yellow leaf tissue of Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves.

    PubMed

    Shang, Jing; Xi, De-Hui; Yuan, Shu; Xu, Fei; Xu, Mo-Yun; Qi, Hai-Long; Wang, Shao-Dong; Huang, Qing-Rong; Wen, Lin; Lin, Hong-Hui

    2010-01-01

    Dark green islands (DGIs) are a common symptom of plants systemically infected with the mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow leaf tissue that is full of virus particles. In Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves, the respiration and photosynthesis capabilities of DGIs and yellow leaf tissues were measured. The results showed that the cyanide-resistant respiration was enhanced in yellow leaf tissue and the photosynthesis was declined, while in DGIs they were less affected. The activities of the oxygen-scavenging enzymes catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in infected leaves were significantly higher than those in the healthy leaves, and the enzyme activities in DGIs were always lower than in the yellow leaf tissues. Reactive oxygen species (ROS) staining showed that the hydrogen peroxide content in yellow leaf tissues was apparently higher than that in DGIs, while the superoxide content was on the contrary. Formation of DGIs may be a strategy of the host plants resistance to the CMV infection.

  8. Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

    PubMed

    Saeed, Muhammad; Krczal, Gabi; Wassenegger, Michael

    2015-04-01

    Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and βC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or βC1 resulted in re-expression of GFP. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. These results indicated that Rep, TrAP and βC1 proteins of CLCuKoV-CLCuMuB can re-activate the expression of a transcriptionally silenced GFP transgene in N. benthamiana. Although Rep, TrAP, or βC1 proteins have, for other begomoviruses or begomoviruses-betasatellites, been previously shown to have TGS suppressor activity, this is the first report demonstrating that a single begomovirus-betasatellite complex encodes three suppressors of TGS.

  9. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

    PubMed

    Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

    2016-01-15

    Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress.

  10. Salinity-induced accumulation of endogenous H2S and NO is associated with modulation of the antioxidant and redox defense systems in Nicotiana tabacum L. cv. Havana.

    PubMed

    da Silva, Cristiane Jovelina; Batista Fontes, Elizabeth Pacheco; Modolo, Luzia Valentina

    2017-03-01

    Salinity is one of the abiotic factors that most affect crop growth and production. This study focused on the effect of high salinity on the endogenous levels of the signaling molecules hydrogen sulfite (H2S) and nitric oxide (NO) in Nicotiana tabacum leaves and the extent of these for the biochemically-driven plant tolerance to such abiotic stress. The NaCl treatment for 10days led to an expressive augment of H2S and NO levels. This increase was correlated with the raise of l-Cys and l-Arg and the induction of l-cysteine desulfhydrase, cyanoalanine synthase, cysteine synthase, nitrate reductase and arginase, enzymes known to be involved in the biosynthesis of H2S or NO. The enzymatic antioxidant system (superoxide dismutase and catalase activity) was boosted and the non-enzymatic antioxidant glutathione was intensively oxidized in leaves upon stress allowing plants to cope with oxidative stress. Lower stomatal conductance was observed in stressed plants in comparison with control ones. Moreover, the high activity of antioxidant enzymes and high rate of glutathione oxidation following salt stress were considerably decreased upon NO or H2S scavenging. Thus, increment in NO and H2S levels and their interplay, along with metabolic and physiological changes, contributed to tobacco survival to extreme salinity conditions.

  11. Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22.

    PubMed

    Mhlongo, Msizi I; Piater, Lizelle A; Madala, Ntakadzeni E; Steenkamp, Paul A; Dubery, Ian A

    2016-01-01

    Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role.

  12. Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

    PubMed

    Bates, H J; Farjah, M; Osman, T A; Buck, K W

    1995-06-01

    A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

  13. Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22

    PubMed Central

    Mhlongo, Msizi I.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Steenkamp, Paul A.; Dubery, Ian A.

    2016-01-01

    Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role. PMID:26978774

  14. The fungal-specific β-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants.

    PubMed

    Wawra, Stephan; Fesel, Philipp; Widmer, Heidi; Timm, Malte; Seibel, Jürgen; Leson, Lisa; Kesseler, Leona; Nostadt, Robin; Hilbert, Magdalena; Langen, Gregor; Zuccaro, Alga

    2016-10-27

    β-glucans are well-known modulators of the immune system in mammals but little is known about β-glucan triggered immunity in planta. Here we show by isothermal titration calorimetry, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy that the FGB1 gene from the root endophyte Piriformospora indica encodes for a secreted fungal-specific β-glucan-binding lectin with dual function. This lectin has the potential to both alter fungal cell wall composition and properties, and to efficiently suppress β-glucan-triggered immunity in different plant hosts, such as Arabidopsis, barley and Nicotiana benthamiana. Our results hint at the existence of fungal effectors that deregulate innate sensing of β-glucan in plants.

  15. The fungal-specific β-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants

    PubMed Central

    Wawra, Stephan; Fesel, Philipp; Widmer, Heidi; Timm, Malte; Seibel, Jürgen; Leson, Lisa; Kesseler, Leona; Nostadt, Robin; Hilbert, Magdalena; Langen, Gregor; Zuccaro, Alga

    2016-01-01

    β-glucans are well-known modulators of the immune system in mammals but little is known about β-glucan triggered immunity in planta. Here we show by isothermal titration calorimetry, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy that the FGB1 gene from the root endophyte Piriformospora indica encodes for a secreted fungal-specific β-glucan-binding lectin with dual function. This lectin has the potential to both alter fungal cell wall composition and properties, and to efficiently suppress β-glucan-triggered immunity in different plant hosts, such as Arabidopsis, barley and Nicotiana benthamiana. Our results hint at the existence of fungal effectors that deregulate innate sensing of β-glucan in plants. PMID:27786272

  16. Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat, drought and their combination in Nicotiana tabacum plants.

    PubMed

    Lubovská, Zuzana; Dobrá, Jana; Storchová, Helena; Wilhelmová, Naďa; Vanková, Radomíra

    2014-11-01

    Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.

  17. The N-terminal fragment of the tomato torrado virus RNA1-encoded polyprotein induces a hypersensitive response (HR)-like reaction in Nicotiana benthamiana.

    PubMed

    Wieczorek, Przemysław; Obrępalska-Stęplowska, Aleksandra

    2016-07-01

    The hypersensitive response (HR) is a defence reaction observed during incompatible plant-pathogen interactions in plants infected with a wide range of fungi, bacteria and viruses. Here, we show that an N-terminal polyprotein fragment encoded by tomato torrado virus RNA1, located between the first ATG codon and the protease cofactor (ProCo) motif, induces an HR-like reaction in Nicotiana benthamiana. Agrobacterium tumefaciens-mediated transient expression of the first 105 amino acids (the calculated molecular weight of the fragment was ca. 11.33 kDa, hereafter refered to as the 11K domain) from ToTV RNA1 induced an HR-like phenotype in infiltrated leaves. To investigate whether the 11K domain could influence the virulence and pathogenicity of a recombinant virus, we created a potato virus X (PVX) with the 11K coding sequence inserted under a duplicated coat protein promoter. We found that 11K substantially increased the virulence of the recombinant virus. Disease phenotype induced in N. benthamiana by PVX-11K was characterized by strong local and systemic necrosis. This was not observed when the 11K domain was expressed from PVX in an antisense orientation. Further analyses revealed that the 11K domain could not suppress posttranscriptional gene silencing (PTGS) of green fluorescent protein (GFP) in the N. benthamiana 16c line. In silico analysis of the predicted secondary structure of the 11K domain indicated the presence of two putative helices that are highly conserved in tomato-infecting representatives of the genus Torradovirus.

  18. DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana Contributes to Potyvirus Movement and Transports to Plasmodesmata via the Early Secretory Pathway and the Actomyosin System1[OPEN

    PubMed Central

    Geng, Chao; Cong, Qian-Qian; Li, Xiang-Dong; Mou, An-Li; Gao, Rui; Liu, Jin-Liang; Tian, Yan-Ping

    2015-01-01

    The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5′-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement. PMID:25540331

  19. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

    PubMed Central

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

    2016-01-01

    Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1–0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746

  20. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

    PubMed Central

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  1. DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana contributes to potyvirus movement and transports to plasmodesmata via the early secretory pathway and the actomyosin system.

    PubMed

    Geng, Chao; Cong, Qian-Qian; Li, Xiang-Dong; Mou, An-Li; Gao, Rui; Liu, Jin-Liang; Tian, Yan-Ping

    2015-02-01

    The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5'-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.

  2. Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation.

    PubMed

    Lombardi, Raffaele; Donini, Marcello; Villani, Maria Elena; Brunetti, Patrizia; Fujiyama, Kazuhito; Kajiura, Hiroyuki; Paul, Matthew; Ma, Julian K-C; Benvenuto, Eugenio

    2012-10-01

    We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

  3. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

    PubMed

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora.

  4. Tolerance to clomazone herbicide is linked to the state of LHC, PQ-pool and ROS detoxification in tobacco (Nicotiana tabacum L.).

    PubMed

    Darwish, Majd; Vidal, Véronique; Lopez-Lauri, Félicie; Alnaser, Osama; Junglee, Sanders; El Maataoui, Mohamed; Sallanon, Huguette

    2015-03-01

    In this study, plantlets of two tobacco (Nicotiana tabacum L.) varieties that are clomazone-tolerant (cv. Xanthi) and clomazone-sensitive (cv. Virginie vk51) were subjected to low concentration of clomazone herbicide. The oxygen-evolving rate of isolated chloroplasts, chlorophyll a fluorescence transients, JIP-test responses, hydrogen peroxide contents, antioxidant enzyme activities, cytohistological results and photosynthetic pigment contents were recorded. The results indicated that the carotenoid content was 2-fold higher in Virginie, which had greater clomazone sensitivity than Xanthi. Virginie exhibited noticeable decreases in the LHC content (Chl a/b ratio), the maximum photochemical quantum efficiency of PSII (Fv/Fm), the performance index on the absorption basis (PIabs), and the electron flux beyond the first PSII QA evaluated as (1-VJ) with VJ=(FJ-F0)/(Fm-F0) as well as increases in the rate of photon absorption (ABS/RC) and the energy dissipation as heat (DI0/RC). These results suggest that PSII photoinhibition occurred as a consequence of more reduced PQ-pool and accumulated QA(-). The oxygen evolution measurements indicate that PSI electron transport activity was not affected by clomazone. The more significant accumulation of H2O2 in Virginie compared to Xanthi was due to the absence of ROS-scavenging enzymes, and presumably induced programmed cell death (PCD). The symptoms of PCD were observed by cytohistological analysis, which also indicated that the leaf tissues of clomazone-treated Virginie exhibited significant starch accumulation compared to Xanthi. Taken together, these results indicate that the variable tolerance to clomazone observed between Virginie and Xanthi is independent of the carotenoid content and could be related to the state of the LHC, the redox state of the PQ-pool, and the activity of detoxification enzymes.

  5. Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2014-01-01

    Introduction: In order to employ Nicotiana tabacum cells as a profitable natural bioreactor for production of bio-functional "Soluble human TRAIL" (ShTRAIL), endoplasmic reticulum (ER) targeted expression and innovative extraction procedures were exploited. Methods: At first, the ShTRAIL encoding gene was sub-cloned into designed H2 helper vector to equip it with potent TMV omega leader sequences, ER sorting signal peptide, poly-histidine tag and ER retention signal peptide (KDEL). Then, the ER targeted ShTRAIL cassette was sequentially sub-cloned into "CaMV-35S" helper and "pGreen-0179" final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum . Next, the ShTRAIL protein was extracted through both phosphate and innovative ascorbate extraction buffers. Subsequently, oligomerization state of the ShTRAIL was evaluated through cross-linking assay and western blot analysis. Then, semi-quantitative western blot analysis was performed to estimate the ShTRAIL production. Finally, biological activity of the ShTRAIL was evaluated through MTT assay. Results: The phosphate buffer extracted ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy increased the ShTRAIL’s production level up to about 20 μg/g of fresh weight of N. tabacum . MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line. Conclusion: Endoplasmic reticulum expression and reductive ascorbate buffer extraction procedure can be employed to enhance the stability and overall production level of bio-functional recombinant ShTRAIL from transgenic N. tabacum cells. PMID:25337465

  6. Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

    PubMed Central

    Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

    2008-01-01

    After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. PMID:18689443

  7. RNAi based simultaneous silencing of all forms of light-dependent NADPH:protochlorophyllide oxidoreductase genes result in the accumulation of protochlorophyllide in tobacco (Nicotiana tabacum).

    PubMed

    Talaat, Neveen B

    2013-10-01

    Conversion of protochlorophyllide (Pchlide) into chlorophyllide (Chlide), a key step in chlorophyll biosynthesis, is mediated by a light-dependent NADPH:protochlorophyllide oxidoreductase (POR). POR exists in multiple isoforms that share high degree of homology. RNAi-mediated gene silencing approach was used to suppress the expression of POR genes in order to study its role in the Chls biosynthesis in tobacco (Nicotiana tabacum L.). The transgenic plants were devoid of chlorophyll pigments and resembled albino plants. Northern blot analysis confirmed the degradation of POR transcripts into 21-23 bp fragments. Pigment analysis showed the accumulation of various intermediate compounds of Chl biosynthesis pathway including Pchlide. However, no trace of chlorophyll was observed. As compared to wild type, POR-silenced plants accumulated larger (60%) amounts of Pchlide from its endogenous substrate. When leaf discs of WT and POR-silenced plants were treated with exogenous ALA both WT and POR-silenced plants accumulated large amounts of tetrapyrrolic intermediates demonstrating that Pchlide biosynthesis potential was not suppressed in POR-silenced plants. Upon illumination, WT plants photo-transformed large amounts of Pchlide to Chlide. However, POR-silenced plants almost completely failed to do so. Results demonstrate that the antisense approaches to drop expression of individual POR isoforms have provided valuable insights into the role of distinct PORs during greening. Moreover, data illustrate that the POR is the only enzyme that can convert the Pchlide to Chlide and there is no alternate enzyme that can substitute the POR in higher plants. Thus, this investigation describes ideal mechanism for the silencing of POR isozymes in tobacco.

  8. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

    SciTech Connect

    Lim, Hyoun-Sub; Nam, Jiryun; Seo, Eun-Young; Nam, Moon; Vaira, Anna Maria; Bae, Hanhong; Jang, Chan-Yong; Lee, Cheol Ho; Kim, Hong Gi; Roh, Mark; Hammond, John

    2014-03-15

    Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP{sub SP}) with that from AltMV-Po (CP{sub Po}) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP{sub Po} [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP{sub SP} but not CP{sub Po} interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP{sub SP} than CP{sub Po} in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein.

  9. Cell physiology of mortality and immortality in a Nicotiana interspecific F1 hybrid complies with the quantitative balance between reactive oxygen and nitric oxide.

    PubMed

    Yamamoto, Takumi; Shomura, Sachiko; Mino, Masanobu

    2017-03-01

    The cultured cell line, GTH4, of an interspecific F1 hybrid between Nicotiana gossei Domin and N. tabacum L. died after a shift in temperature from 37°C to 26°C. Fluctuations in the cellular amounts of reactive oxygen species (ROS) and nitric oxide (NO) were detected in GTH4 after the temperature shift, but not in the mutant, GTH4S, which did not die at 26°C presumably due to the lack of genetic factors involved in cell death. The removal of ROS or NO suppressed cell death in GTH4, suggesting that ROS and NO both acted as mediators of cell death. However, excess amounts of the superoxide anion (O2(-)) or NO alleviated cell death. A series of experiments using generators and scavengers of ROS and NO showed that O2(-) affected the cellular levels of NO, and vice versa, indicating that a quantitative balance between O2(-) and NO was important for hybrid cell death. The combination of NO and hydrogen peroxide (H2O2) was necessary and sufficient to initiate cell death in GTH4 and GTH4S. Hypoxia, which suppressed cell death in GTH4 at 26°C, reduced the generation of H2O2 and NO, but allowed for the production of O2(-), which acted as a suppressor and/or modulator of cell death. The activation of MAPK was involved in the generation of H2O2 in GTG4 cells under normoxic conditions, but promoted O2(-) generation under hypoxic conditions. More protective cellular conditions against ROS, as estimated by the expression levels of genes for ROS-scavenging enzymes, may be involved in the mechanisms responsible for the low cell death rate of GTH4 under hypoxic conditions.

  10. Fertilization Independent Endosperm genes repress NbGH3.6 and regulate the auxin level during shoot development in Nicotiana benthamiana

    PubMed Central

    Zeng, Jun; Ding, Qi; Fukuda, Hiroo; He, Xin-Qiang

    2016-01-01

    The Fertilization Independent Endosperm (FIE) gene is required to restrict endosperm development without fertilization, and it represses flowering during embryo and seedling development in Arabidopsis thaliana. However, the regulatory mechanism of the FIE gene in postembryonic shoot development is not well understood. Silencing of Nicotiana benthamiana homologues of the FIE gene, NbFIE1 and NbFIE2, resulted in the enhanced outgrowth of axillary buds and the impairment of secondary xylem differentiation. RNA sequencing analysis found that one of the auxin-responsive GRETCHEN HAGEN 3 (GH3) family genes, NbGH3.6, was upregulated and maintained a high expression during the time course of silencing NbFIE genes. Chromatin immunoprecipiation (ChIP)-PCR results showed a lack of H3K27me3 marks on NbGH3.6 chromatin in NbFIE-silenced plants compared with negative control plants, indicating that NbGH3.6 was a direct target of NbFIE genes during postembryonic shoot development. Moreover, the free IAA content was reduced significantly in NbFIE-silenced plants, which might cause the enhanced outgrowth of axillary buds as well as impaired secondary xylem differentiation. These results clearly indicated that NbGH3.6 was a primary target of NbFIE genes during postembryonic shoot development, and NbFIE genes regulated axillary bud growth and secondary xylem formation through tuning endogenous auxin homeostasis, possibly by regulating the expression of the NbGH3.6 gene. PMID:26873977

  11. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

  12. Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals.

    PubMed

    Barretto, Sherwin S; Michoux, Franck; Hellgardt, Klaus; Nixon, Peter J

    2017-01-15

    Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotiana tabacum shoots from callus tissue in RITA(®) temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4 min aeration every 8 h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium). A critical aeration rate of 440 ml min(-1) was identified, corresponding to a shear rate of 96.7 s(-1), pneumatic power input of 8.8 mW kg(-1) and initial 20-day pneumatic energy dissipation of 127 J kg(-1), at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production.

  13. Nicotiana benthamiana calreticulin 3a is required for the ethylene-mediated production of phytoalexins and disease resistance against oomycete pathogen Phytophthora infestans.

    PubMed

    Matsukawa, Mizuki; Shibata, Yusuke; Ohtsu, Mina; Mizutani, Aki; Mori, Hitoshi; Wang, Ping; Ojika, Makoto; Kawakita, Kazuhito; Takemoto, Daigo

    2013-08-01

    Mature Nicotiana benthamiana shows strong resistance to the potato late blight pathogen Phytophthora infestans. By screening using virus-induced random gene silencing, we isolated a gene for plant-specific calreticulin NbCRT3a as a required gene for resistance of N. benthamiana against P. infestans. NbCRT3a encodes an endoplasmic reticulum quality-control (ERQC) chaperone for the maturation of glycoproteins, including glycosylated cell-surface receptors. NbCRT3a-silenced plants showed no detectable growth defects but resistance to P. infestans was significantly compromised. Defense responses induced by the treatment with INF1 (a secretory protein of P. infestans), such as production of reactive oxygen species and accumulation of phytoalexins, were suppressed in NbCRT3a-silenced N. benthamiana. Expression of an ethylene-regulated gene for phytoalexin biosynthesis, NbEAS, was reduced in NbCRT3a-silenced plants, whereas the expression of salicylic acid-regulated NbPR-1a was not affected. Consistently, induction of ethylene production by INF1 was suppressed in NbCRT3a-silenced plants. Resistance reactions induced by a hyphal wall components elicitor prepared from P. infestans were also impaired in NbCRT3a-silenced plants. However, cell death induced by active mitogen-activated protein kinase kinase (NbMEK2(DD)) was not affected by the silencing of NbCRT3a. Thus, NbCRT3a is required for the initiation of resistance reactions of N. benthamiana in response to elicitor molecules derived from P. infestans.

  14. PeBL1, a novel protein elicitor from Brevibacillus laterosporus strain A60, activates defense responses and systemic resistance in Nicotiana benthamiana.

    PubMed

    Wang, Haoqian; Yang, Xiufen; Guo, Lihua; Zeng, Hongmei; Qiu, Dewen

    2015-04-01

    We report the identification, characterization, and gene cloning of a novel protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated a protein that was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI-Q-TOF-MS-MS). The 351-bp PeBL1 gene produces a 12,833-Da protein with 116 amino acids that contains a 30-residue signal peptide. The PeBL1 protein was expressed in Escherichia coli. The recombinant protein can induce a typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana, like the endogenous protein. PeBL1-treated N. benthamiana exhibited strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control N. benthamiana. In addition, PeBL1 triggered a cascade of events that resulted in defense responses in plants, including reactive oxygen species (ROS) production, extracellular-medium alkalization, phenolic-compound deposition, and expression of several defense-related genes. Real-time quantitative-PCR analysis indicated that the known defense-related genes PR-1, PR-5, PDF1.2, NPR1, and PAL were upregulated to varying degrees by PeBL1. This research not only provides insights into the mechanism by which beneficial bacteria activate plant systemic resistance, but also sheds new light on a novel strategy for biocontrol using strain A60.

  15. PeBL1, a Novel Protein Elicitor from Brevibacillus laterosporus Strain A60, Activates Defense Responses and Systemic Resistance in Nicotiana benthamiana

    PubMed Central

    Wang, Haoqian; Yang, Xiufen; Guo, Lihua; Zeng, Hongmei

    2015-01-01

    We report the identification, characterization, and gene cloning of a novel protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated a protein that was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI–Q-TOF–MS-MS). The 351-bp PeBL1 gene produces a 12,833-Da protein with 116 amino acids that contains a 30-residue signal peptide. The PeBL1 protein was expressed in Escherichia coli. The recombinant protein can induce a typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana, like the endogenous protein. PeBL1-treated N. benthamiana exhibited strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control N. benthamiana. In addition, PeBL1 triggered a cascade of events that resulted in defense responses in plants, including reactive oxygen species (ROS) production, extracellular-medium alkalization, phenolic-compound deposition, and expression of several defense-related genes. Real-time quantitative-PCR analysis indicated that the known defense-related genes PR-1, PR-5, PDF1.2, NPR1, and PAL were upregulated to varying degrees by PeBL1. This research not only provides insights into the mechanism by which beneficial bacteria activate plant systemic resistance, but also sheds new light on a novel strategy for biocontrol using strain A60. PMID:25662975

  16. Species origin of genomic factors in Nicotiana nudicaulis Watson controlling hybrid lethality in interspecific hybrids between N. nudicaulis Watson and N. tabacum L.

    PubMed

    Liu, Hongshuo; Marubashi, Wataru

    2014-01-01

    Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis × N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris × N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris × N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla × N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla × N. tabacum and N. sylvestris × N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis.

  17. Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus (CLRDV) results in systemic infection in cotton and the model plant Nicotiana benthamiana.

    PubMed

    Delfosse, Verónica C; Casse, María F; Agrofoglio, Yamila C; Kresic, Iván Bonacic; Hopp, Horacio E; Ziegler-Graff, Véronique; Distéfano, Ana J

    2013-07-01

    Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse

  18. Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum.

    PubMed

    Palomares, R; Herrmann, R G; Oelmüller, R

    1991-11-01

    We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b-binding protein of photosystem II (CP24), the Rieske iron-sulphur protein of the cytochrome b6/f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum. The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue-UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

  19. Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits.

    PubMed

    Boes, Alexander; Spiegel, Holger; Edgue, Gueven; Kapelski, Stephanie; Scheuermayer, Matthias; Fendel, Rolf; Remarque, Edmond; Altmann, Friedrich; Maresch, Daniel; Reimann, Andreas; Pradel, Gabriele; Schillberg, Stefan; Fischer, Rainer

    2015-02-01

    One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P. falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2 × 10(6) were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC₅₀ values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.

  20. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2013-10-01

    mice and mice transfused with Syk inhibitor-treated platelets . Platelet lodging was remarkably decreased in lungs of mice transfused with Syk...AD_________________ Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet ...30September2012–29September2013 4. TITLE AND SUBTITLE Complement Activation Alters Platelet Function 5a. CONTRACT NUMBER W81XWH-12-1-0523 5b. GRANT NUMBER

  1. Hypothesis tests for hydrologic alteration

    NASA Astrophysics Data System (ADS)

    Kroll, Charles N.; Croteau, Kelly E.; Vogel, Richard M.

    2015-11-01

    Hydrologic systems can be altered by anthropogenic and climatic influences. While there are a number of statistical frameworks for describing and evaluating the extent of hydrologic alteration, here we present a new framework for assessing whether statistically significant hydrologic alteration has occurred, or whether the shift in the hydrologic regime is consistent with the natural variability of the system. Four hypothesis tests based on shifts of flow duration curves (FDCs) are developed and tested using three different experimental designs based on different strategies for resampling of annual FDCs. The four hypothesis tests examined are the Kolmogorov-Smirnov (KS), Kuiper (K), confidence interval (CI), and ecosurplus and ecodeficit (Eco). Here 117 streamflow sites that have potentially undergone hydrologic alteration due to reservoir construction are examined. 20 years of pre-reservoir record is used to develop the critical value of the test statistic for type I errors of 5% and 10%, while 10 years of post-alteration record is used to examine the power of each test. The best experimental design, based on calculating the mean annual FDC from an exhaustive jackknife resampling regime, provided a larger number of unique values of each test statistic and properly reproduced type I errors. Of the four tests, the CI test consistently had the highest power, while the K test had the second highest power; KS and Eco always had the lowest power. The power of the CI test appeared related to the storage ratio of the reservoir, a rough measure of the hydrologic alteration of the system.

  2. Narrow-Leafed Lupin (Lupinus angustifolius) β1- and β6-Conglutin Proteins Exhibit Antifungal Activity, Protecting Plants against Necrotrophic Pathogen Induced Damage from Sclerotinia sclerotiorum and Phytophthora nicotianae

    PubMed Central

    Jimenez-Lopez, Jose C.; Melser, Su; DeBoer, Kathleen; Thatcher, Louise F.; Kamphuis, Lars G.; Foley, Rhonda C.; Singh, Karam B.

    2016-01-01

    Vicilins (7S globulins) are seed storage proteins and constitute the main protein family in legume seeds, particularly in narrow-leafed lupin (Lupinus angustifolius L.; NLL), where seven vicilin genes, called β1- to β7-conglutin have been identified. Vicilins are involved in germination processes supplying amino acids for seedling growth and plant development, as well as in some cases roles in plant defense and protection against pathogens. The roles of NLL β-conglutins in plant defense are unknown. Here the potential role of five NLL β-conglutin family members in protection against necrotrophic fungal pathogens was investigated and it was demonstrated that recombinant purified 6xHis-tagged β1- and β6-conglutin proteins exhibited the strongest in vitro growth inhibitory activity against a range of necrotrophic fungal pathogens compared to β2, β3, and β4 conglutins. To examine activity in vivo, two representative necrotrophic pathogens, the fungus Sclerotinia sclerotiorum and oomycete Phytophthora nicotianae were used. Transient expression of β1- and β6-conglutin proteins in Nicotiana benthamiana leaves demonstrated in vivo growth suppression of both of these pathogens, resulting in low percentages of hyphal growth and elongation in comparison to control treated leaves. Cellular studies using β1- and β6-GFP fusion proteins showed these conglutins localized to the cell surface including plasmodesmata. Analysis of cellular death following S. sclerotiorum or P. nicotianae revealed both β1- and β6-conglutins suppressed pathogen induced cell death in planta and prevented pathogen induced suppression of the plant oxidative burst as determined by protein oxidation in infected compared to mock-inoculated leaves. PMID:28018392

  3. The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants

    NASA Technical Reports Server (NTRS)

    Persson, S.; Wyatt, S. E.; Love, J.; Thompson, W. F.; Robertson, D.; Boss, W. F.; Brown, C. S. (Principal Investigator)

    2001-01-01

    To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

  4. [Neurocutaneous syndrome with hair alterations].

    PubMed

    Camacho-Martínez, F

    1997-09-01

    There are multiple neurocutaneous syndromes that may show hair alterations such as the interglabellar peak or 'widow's peak', which is an alteration of the hair implantation, in addition to the genohypotrichosis, hypertrichosis and hair shaft dysplasias. In this chapter we will focus on the latter. Out of the unspecific hair shaft dysplasias the only ones showing neurological alterations are trichorrhexis invaginata, observed in the syndrome of Netherton. Among the specific dysplasias we would like to point out monilethrix, and very especially the moniliform hair syndrome, the trichorrhexis nodosa, the pili torti and trichotiodystrophy. The latter is actually a group of syndromes which associates a series of diverse symptoms that have in common hair brittleness, fertility problems and physical and mental retardation, and they constitute the basic syndrome know as 'BIDS syndrome.

  5. THE ALTERATION OF INTRACELLULAR ENZYMES

    PubMed Central

    Kaplan, J. Gordin

    1954-01-01

    1. The ability of homologous series of alcohols, ketones, and aldehydes to cause alteration of intracellular catalase increases approximately threefold for each methylene group added, thus following Traube's rule. Equiactive concentrations of alcohols (methanol to octanol) varied over a 4,000-fold range, yet the average corresponding surface tension was 42 ± 2 dynes/cm., that for ketones 43 ± 2, and for aldehydes (above C1) 41 ± 3. 2. Above C8 the altering activity of alcohols ceased to follow Traube's rule, and at C18 was nil. Yet the surface activities of alcohols from nonanol to dodecanol did follow Traube's rule. These two facts show that the interface which is being affected by these agents is not the cell surface, for if it were, altering activity should not fall off between C9 and C12 where surface activity is undiminished; they show also that micelle formation by short range association of hydrocarbon "tails," usually invoked to explain decrease in biological activity of compounds above C8, is not responsible for this effect in these experiments, in which permeability of the cell membrane probably is involved. 3. The most soluble alcohols and aldehydes (alcohols C1 to C8; aldehydes C1, C2), but not ketones, cause, above optimal concentration, an irreversible inhibition of yeast catalase. 4. The critical concentration of altering agent (i.e., that concentration just sufficient to cause doubling of the catalase activity of the yeast suspension) was independent of the concentration of the yeast cells. 5. Viability studies show that the number of yeast cells killed by the altering agents was not related to the degree of activation of the catalase produced. While all the cells were invariably killed by concentrations of altering agent which produced complete activation, all the cells had been killed by concentrations which were insufficient to cause more than 50 per cent maximal activation. Further, the evidence suggested that the catalase may be partially

  6. The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

    PubMed

    Pavli, Ourania I; Kelaidi, Georgia I; Tampakaki, Anastasia P; Skaracis, George N

    2011-03-04

    To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms

  7. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    PubMed Central

    2009-01-01

    Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant

  8. Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level

    PubMed Central

    Yu, Shi; Pilot, Guillaume

    2014-01-01

    Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants. PMID:25477887

  9. Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

    NASA Technical Reports Server (NTRS)

    Staehelin, L. A.; Giddings, T. H. Jr; Kiss, J. Z.; Sack, F. D.

    1990-01-01

    The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to "young" and "old", polysaccharide-slime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips of Arabidopsis and Nicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types. Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (approximately 30 nm) and are the least stained. Medial cisternae are narrower (approximately 20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4-6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network. Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans

  10. Changes in Air CO2 Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves

    PubMed Central

    Secchi, Francesca; Schubert, Andrea; Lovisolo, Claudio

    2016-01-01

    The aquaporin specific control on water versus carbon pathways in leaves is pivotal in controlling gas exchange and leaf hydraulics. We investigated whether Nicotiana tabacum aquaporin 1 (NtAQP1) and Nicotiana tabacum plasma membrane intrinsic protein 2;1 (NtPIP2;1) gene expression varies in tobacco leaves subjected to treatments with different CO2 concentrations (ranging from 0 to 800 ppm), inducing changes in photosynthesis, stomatal regulation and water evaporation from the leaf. Changes in air CO2 concentration ([CO2]) affected net photosynthesis (Pn) and leaf substomatal [CO2] (Ci). Pn was slightly negative at 0 ppm air CO2; it was one-third that of ambient controls at 200 ppm, and not different from controls at 800 ppm. Leaves fed with 800 ppm [CO2] showed one-third reduced stomatal conductance (gs) and transpiration (E), and their gs was in turn slightly lower than in 200 ppm– and in 0 ppm–treated leaves. The 800 ppm air [CO2] strongly impaired both NtAQP1 and NtPIP2;1 gene expression, whereas 0 ppm air [CO2], a concentration below any in vivo possible conditions and specifically chosen to maximize the gene expression alteration, increased only the NtAQP1 transcript level. We propose that NtAQP1 expression, an aquaporin devoted to CO2 transport, positively responds to CO2 scarcity in the air in the whole range 0–800 ppm. On the contrary, expression of NtPIP2;1, an aquaporin not devoted to CO2 transport, is related to water balance in the leaf, and changes in parallel with gs. These observations fit in a model where upregulation of leaf aquaporins is activated at low Ci, while downregulation occurs when high Ci saturates photosynthesis and causes stomatal closure. PMID:27089333

  11. A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity.

    PubMed

    Kiba, Akinori; Nakano, Masahito; Vincent-Pope, Patrick; Takahashi, Hirotaka; Sawasaki, Tatsuya; Endo, Yaeta; Ohnishi, Kouhei; Yoshioka, Hirofumi; Hikichi, Yasufumi

    2012-07-01

    To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses.

  12. Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophyll a-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, in Nicotiana sp.*

    PubMed Central

    Pinnola, Alberta; Ghin, Leonardo; Gecchele, Elisa; Merlin, Matilde; Alboresi, Alessandro; Avesani, Linda; Pezzotti, Mario; Capaldi, Stefano; Cazzaniga, Stefano; Bassi, Roberto

    2015-01-01

    Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies. PMID:26260788

  13. Altered Vision Near the Hands

    ERIC Educational Resources Information Center

    Abrams, Richard A.; Davoli, Christopher C.; Du, Feng; Knapp, William H., III; Paull, Daniel

    2008-01-01

    The present study explored the manner in which hand position may affect visual processing. We studied three classic visual attention tasks (visual search, inhibition of return, and attentional blink) during which the participants held their hands either near the stimulus display, or far from the display. Remarkably, the hands altered visual…

  14. Art as Alterity in Education

    ERIC Educational Resources Information Center

    Zhao, Guoping

    2014-01-01

    In education, art has often been perceived as entertainment and decoration and is the first subject to go when there are budget cuts or test-score pressures. Drawing on Emmanuel Lévinas's idea of the primacy of radical alterity that breaks the totality of our being, enables self-transformation and ethics, and ensures community as a totality…

  15. Modelling glass alteration in an altered argillaceous environment

    NASA Astrophysics Data System (ADS)

    Bildstein, O.; Trotignon, L.; Pozo, C.; Jullien, M.

    2007-05-01

    The long term behaviour of materials such as glass, steel and clay has been investigated in the context of deep geological disposal of radioactive wastes. The interactions between vitrified wastes, canister corrosion products (CPs) and clay are studied using a modified version of the reaction-transport code Crunch, especially looking at pH changes and possible cementation at the interface with the clayey materials. These perturbations may indeed affect the lifetime of glass matrix in deep repositories, e.g., high pH enhances the rate of glass alteration. This work focuses on the argillite of Bure. The calculations were performed at 323 K with a glass alteration rate switching from a high initial rate to a residual rate according to the sorption capacity of CPs. The time at which this sorption capacity is saturated is crucial to the system in terms of wastes package lifetime. The results show that the glass alteration imposes a high pH value at the interface with CPs and clay: up to a value of 9.2, compared to 7.3 which is the initial pH value in the argillite. Experimental data show that the rate of glass alteration is much higher in such pH conditions. For a R7T7-type glass, the rate is about five times higher at pH 9 than at pH 7. This pH perturbation migrates through the clayey domain as a result of the migration of mobile elements such as boron and sodium, and despite the existence of strong pH buffers in the argillite. The cementation of porosity at the interface between glass and clay is predicted by the model due to the massive precipitation of iron corrosion products and glass alteration products. At this point of the evolution of the system, the pH starts to decrease and the alteration rate of the glass could be significantly reduced. This porosity clogging effect is difficult to confirm by experiments especially since existing data on short term experiments tend to show a pervasive precipitation of silica in the domain instead of a localized precipitation

  16. Genetic alterations and epigenetic alterations of cancer-associated fibroblasts

    PubMed Central

    Du, Heng; Che, Guowei

    2017-01-01

    Cancer-associated fibroblasts (CAFs) are one major type of component identified in the tumor microenvironment. Studies have focused on the genetic and epigenetic status of CAFs, since they are critical in tumor progression and differ phenotypically and functionally from normal fibroblasts. The present review summarizes the recent achievements in understanding the gene profiles of CAFs and pays special attention to their possible epigenetic alterations. A total of 7 possible genetic alterations and epigenetic changes in CAFs are discussed, including gene differential expression, karyotype analysis, gene copy number variation, loss of heterozygosis, allelic imbalance, microsatellite instability, post-transcriptional control and DNA methylation. These genetic and epigenetic characteristics are hypothesized to provide a deep understanding of CAFs and a perspective on their clinical significance. PMID:28123515

  17. 28 CFR 36.402 - Alterations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ACCOMMODATIONS AND IN COMMERCIAL FACILITIES New Construction and Alterations § 36.402 Alterations. (a) General. (1) Any alteration to a place of public accommodation or a commercial facility, after January 26... alteration is a change to a place of public accommodation or a commercial facility that affects or...

  18. Chemosensory alterations and cancer therapies

    SciTech Connect

    Bartoshuk, L.M. )

    1990-01-01

    Taste and olfaction provide sensory information and sensory pleasure. Cancer therapies affect both. Chemotherapy has not been shown to produce dramatic losses of taste or smell, but systematic studies on various chemotherapeutic agents and types of cancer are lacking. Radiation therapy does produce clear losses of both taste and smell. Both chemotherapy and radiation therapy alter the pleasure produced by taste and smell through the formation of conditioned aversions. That is, foods consumed in proximity with the nausea of therapy come to be unpleasant. The impact of conditioned aversions can be diminished by providing a scapegoat food just before therapy. Alterations in foods may be beneficial to the cancer patient. Increasing the concentrations of flavor ingredients can compensate for sensory losses, and providing pureed foods that retain the cognitive integrity of a meal can benefit the patient who has chewing or swallowing problems.

  19. Buccal alterations in diabetes mellitus

    PubMed Central

    2010-01-01

    Long standing hyperglycaemia besides damaging the kidneys, eyes, nerves, blood vessels, heart, can also impair the function of the salivary glands leading to a reduction in the salivary flow. When salivary flow decreases, as a consequence of an acute hyperglycaemia, many buccal or oral alterations can occur such as: a) increased concentration of mucin and glucose; b) impaired production and/or action of many antimicrobial factors; c) absence of a metalloprotein called gustin, that contains zinc and is responsible for the constant maturation of taste papillae; d) bad taste; e) oral candidiasis f) increased cells exfoliation after contact, because of poor lubrication; g) increased proliferation of pathogenic microorganisms; h) coated tongue; i) halitosis; and many others may occur as a consequence of chronic hyperglycaemia: a) tongue alterations, generally a burning mouth; b) periodontal disease; c) white spots due to demineralization in the teeth; d) caries; e) delayed healing of wounds; f) greater tendency to infections; g) lichen planus; h) mucosa ulcerations. Buccal alterations found in diabetic patients, although not specific of this disease, have its incidence and progression increased when an inadequate glycaemic control is present. PMID:20180965

  20. Altered stomatal dynamics in ascorbate oxidase over-expressing tobacco plants suggest a role for dehydroascorbate signalling.

    PubMed

    Fotopoulos, Vasileios; De Tullio, Mario C; Barnes, Jeremy; Kanellis, Angelos K

    2008-01-01

    Control of stomatal aperture is of paramount importance for plant adaptation to the surrounding environment. Here, we report on several parameters related to stomatal dynamics and performance in transgenic tobacco plants (Nicotiana tabacum L., cv. Xanthi) over-expressing cucumber ascorbate oxidase (AO), a cell wall-localized enzyme of uncertain biological function that oxidizes ascorbic acid (AA) to monodehydroascorbic acid which dismutates yielding AA and dehydroascorbic acid (DHA). In comparison to WT plants, leaves of AO over-expressing plants exhibited reduced stomatal conductance (due to partial stomatal closure), higher water content, and reduced rates of water loss on detachment. Transgenic plants also exhibited elevated levels of hydrogen peroxide and a decline in hydrogen peroxide-scavenging enzyme activity. Leaf ABA content was also higher in AO over-expressing plants. Treatment of epidermal strips with either 1 mM DHA or 100 microM hydrogen peroxide resulted in rapid stomatal closure in WT plants, but not in AO-over-expressing plants. This suggests that signal perception and/or transduction associated with stomatal closure is altered by AO over-expression. These data support a specific role for cell wall-localized AA in the perception of environmental cues, and suggest that DHA acts as a regulator of stomatal dynamics.

  1. The Alteration of Plant Morphology by Small Peptides Released from the Proteolytic Processing of the Bacterial Peptide TENGU1[W

    PubMed Central

    Sugawara, Kyoko; Honma, Youhei; Komatsu, Ken; Himeno, Misako; Oshima, Kenro; Namba, Shigetou

    2013-01-01

    Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches’ broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function. PMID:23784461

  2. Altering prolactin concentrations in sows.

    PubMed

    Farmer, C

    2016-07-01

    Prolactin has a multiplicity of actions, but it is of particular importance in gestating and lactating animals. In sows, it is involved in the control of mammary development and also holds essential roles in the lactogenic and galactopoietic processes. Furthermore, low circulating concentrations of prolactin are associated with the agalactia syndrome. The crucial role of prolactin makes it important to understand the various factors that can alter its secretion. Regulation of prolactin secretion is largely under the negative control of dopamine, and dopamine agonists consistently decrease prolactin concentrations in sows. On the other hand, injections of dopamine antagonists can enhance circulating prolactin concentrations. Besides pharmacologic agents, many other factors can also alter prolactin concentrations in sows. The use of Chinese-derived breeds, for instance, leads to increased prolactin concentrations in lactating sows compared with standard European white breeds. Numerous husbandry and feeding practices also have a potential impact on prolactin concentrations in sows. Factors, such as provision of nest-building material prepartum, housing at farrowing, high ambient temperature, stress, transient weaning, exogenous thyrotropin-releasing factor, exogenous growth hormone-releasing factor, nursing frequency, prolonged photoperiod, fasting, increased protein and/or energy intake, altered energy sources, feeding high-fiber diets, sorghum ergot or plant extracts, were all studied with respect to their prolactinemic properties. Although some of these practices do indeed affect circulating prolactin concentrations, none leads to changes as drastic as those brought about by dopamine agonists or antagonists. It appears that the numerous factors regulating prolactin concentrations in sows are still not fully elucidated, and that studies to develop novel applicable ways of increasing prolactin concentrations in sows are warranted.

  3. Altered cell function in microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, Millie

    1991-01-01

    The paper overviews published results from investigations of changes in basic biological parameters taking place as a result of spaceflight exposure. These include changes in the rates of the DNA, mRNA, and protein biosyntheses; changes in the growth rate of an organism; and alterations in the cytoskeleton structure, differentiation, hormone accumulation, and collagen matrix secretion. These results, obtained both in complex biological organisms and on cultured cells, suggest that a basic cellular function is influenced and changed by microgravity. Many of the above mentioned changes are also found to take place in aging cells.

  4. Acid Sulfate Alteration on Mars

    NASA Technical Reports Server (NTRS)

    Ming, D. W.; Morris, R. V.

    2016-01-01

    A variety of mineralogical and geochemical indicators for aqueous alteration on Mars have been identified by a combination of surface and orbital robotic missions, telescopic observations, characterization of Martian meteorites, and laboratory and terrestrial analog studies. Acid sulfate alteration has been identified at all three landing sites visited by NASA rover missions (Spirit, Opportunity, and Curiosity). Spirit landed in Gusev crater in 2004 and discovered Fe-sulfates and materials that have been extensively leached by acid sulfate solutions. Opportunity landing on the plains of Meridiani Planum also in 2004 where the rover encountered large abundances of jarosite and hematite in sedimentary rocks. Curiosity landed in Gale crater in 2012 and has characterized fluvial, deltaic, and lacustrine sediments. Jarosite and hematite were discovered in some of the lacustrine sediments. The high elemental abundance of sulfur in surface materials is obvious evidence that sulfate has played a major role in aqueous processes at all landing sites on Mars. The sulfate-rich outcrop at Meridiani Planum has an SO3 content of up to 25 wt.%. The interiors of rocks and outcrops on the Columbia Hills within Gusev crater have up to 8 wt.% SO3. Soils at both sites generally have between 5 to 14 wt.% SO3, and several soils in Gusev crater contain around 30 wt.% SO3. After normalization of major element compositions to a SO3-free basis, the bulk compositions of these materials are basaltic, with a few exceptions in Gusev crater and in lacustrine mudstones in Gale crater. These observations suggest that materials encountered by the rovers were derived from basaltic precursors by acid sulfate alteration under nearly isochemical conditions (i.e., minimal leaching). There are several cases, however, where acid sulfate alteration minerals (jarosite and hematite) formed in open hydrologic systems, e.g., in Gale crater lacustrine mudstones. Several hypotheses have been suggested for the

  5. Aqueous Alteration of Enstatite Chondrites

    NASA Technical Reports Server (NTRS)

    Zolensky, M. E.; Ziegler, K.; Weisberg, M. K.; Gounelle, M.; Berger, E. L.; Le, L.; Ivanov, A.

    2014-01-01

    The Kaidun meteorite is different from all other meteorites [1], consisting largely of a mixture of “incompatible” types of meteoritic material – carbonaceous and enstatite chondrites, i.e. corre-sponding to the most oxidized and the most reduced samples of meteorite materials, including CI1, CM1-2, CV3, EH3-5, and EL3. In addition to these, minor amounts of ordinary and R chondrites are present. In addition, approximately half of the Kaidun lithologies are new materials not known as separate meteorites. Among these are aqueously altered enstatite chondrites [1], which are of considerable interest because they testify that not all reduced asteroids escaped late-stage oxidation, and hydrolysis, and also because hydrated poorly crystalline Si-Fe phase, which in turn is re-placed by serpentine (Figs 3-5). In the end the only indication of the original presence of metal is the re-sidual carbides. In other enstatite chondrite lithogies (of uncertain type) original silicates and metal have been thoroughly replaced by an assemblage of authi-genic plagioclase laths, calcite boxwork, and occasion-al residual grains of silica, Cr-rich troilite, ilmenite, and rare sulfides including heideite (Fig. 6). Fe and S have been largely leached from the rock (Fig. 4). Again the accessory phases are the first clue to the original character of the rock, which can be verified by O isotopes. It is fortunate that Kaidun displays every step of the alteration process.

  6. Circadian Disorganization Alters Intestinal Microbiota

    PubMed Central

    Voigt, Robin M.; Forsyth, Christopher B.; Green, Stefan J.; Mutlu, Ece; Engen, Phillip; Vitaterna, Martha H.; Turek, Fred W.; Keshavarzian, Ali

    2014-01-01

    Intestinal dysbiosis and circadian rhythm disruption are associated with similar diseases including obesity, metabolic syndrome, and inflammatory bowel disease. Despite the overlap, the potential relationship between circadian disorganization and dysbiosis is unknown; thus, in the present study, a model of chronic circadian disruption was used to determine the impact on the intestinal microbiome. Male C57BL/6J mice underwent once weekly phase reversals of the light:dark cycle (i.e., circadian rhythm disrupted mice) to determine the impact of circadian rhythm disruption on the intestinal microbiome and were fed either standard chow or a high-fat, high-sugar diet to determine how diet influences circadian disruption-induced effects on the microbiome. Weekly phase reversals of the light:dark (LD) cycle did not alter the microbiome in mice fed standard chow; however, mice fed a high-fat, high-sugar diet in conjunction with phase shifts in the light:dark cycle had significantly altered microbiota. While it is yet to be established if some of the adverse effects associated with circadian disorganization in humans (e.g., shift workers, travelers moving across time zones, and in individuals with social jet lag) are mediated by dysbiosis, the current study demonstrates that circadian disorganization can impact the intestinal microbiota which may have implications for inflammatory diseases. PMID:24848969

  7. Occluded motion alters event perception.

    PubMed

    Kawachi, Yousuke; Gyoba, Jiro

    2013-04-01

    We employed audiovisual stream/bounce displays, in which two moving objects with crossing trajectories are more likely to be perceived as bouncing off, rather than streaming through, each other when a brief sound is presented at the coincidence of the two objects. However, Kawachi and Gyoba (Perception 35:1289-1294, 2006b) reported that the presence of an additional moving object near the two objects altered the perception of a bouncing event to that of a streaming event. In this study, we extended this finding and examined whether alteration of the event perception could be induced by the visual context, such as by occluded object motion near the stream/bounce display. The results demonstrated that even when the sound was presented, the continuous occluded motion strongly biased observers' percepts toward the streaming percept during a short occlusion interval (approximately 100 ms). In contrast, when the continuous occluded motion was disrupted by introducing a spatiotemporal gap in the motion trajectory or by removing occlusion cues such as deletion/accretion, the bias toward the streaming percept declined. Thus, we suggest that a representation of object motion generated under a limited occlusion interval interferes with audiovisual event perception.

  8. CADDIS Volume 2. Sources, Stressors and Responses: Flow Alteration

    EPA Pesticide Factsheets

    Introduction to the flow alteration module, when to list flow alteration as a candidate cause, ways to measure flow alteration, simple and detailed conceptual model diagrams for flow alteration, flow alteration module references and literature reviews.

  9. Window to 'Clovis's' Altered Past

    NASA Technical Reports Server (NTRS)

    2004-01-01

    This image taken by the Mars Exploration Rover Spirit shows a rock outcrop dubbed 'Clovis.' The rock was discovered to be softer than other rocks studied so far at Gusev Crater after the rover easily ground a hole (center) into it with its rock abrasion tool. An analysis of the interior of the hole with the rover's alpha particle X-ray spectrometer found higher concentrations of sulfur, bromine and chlorine compared to basaltic, or volcanic, rocks at Gusev. This might indicate that Clovis was chemically altered, and that fluids once flowed through the rock depositing these elements. Spirit's solar panels can be seen in the foreground. This image was taken by the rover's navigation camera on sol 205 (July 31, 2004).

  10. Epigenetic alterations underlying autoimmune diseases.

    PubMed

    Aslani, Saeed; Mahmoudi, Mahdi; Karami, Jafar; Jamshidi, Ahmad Reza; Malekshahi, Zahra; Nicknam, Mohammad Hossein

    2016-01-01

    Recent breakthroughs in genetic explorations have extended our understanding through discovery of genetic patterns subjected to autoimmune diseases (AID). Genetics, on the contrary, has not answered all the conundrums to describe a comprehensive explanation of causal mechanisms of disease etiopathology with regard to the function of environment, sex, or aging. The other side of the coin, epigenetics which is defined by gene manifestation modification without DNA sequence alteration, reportedly has come in to provide new insights towards disease apprehension through bridging the genetics and environmental factors. New investigations in genetic and environmental contributing factors for autoimmunity provide new explanation whereby the interactions between genetic elements and epigenetic modifications signed by environmental agents may be responsible for autoimmune disease initiation and perpetuation. It is aimed through this article to review recent progress attempting to reveal how epigenetics associates with the pathogenesis of autoimmune diseases.

  11. 14 CFR 26.45 - Holders of type certificates-Alterations and repairs to alterations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... category airplanes subject to § 26.43. (b) Fatigue critical alteration structure. For existing and future... alteration data and identify all <