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1

Agrobacterium tumefaciens -mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.)  

Microsoft Academic Search

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and #-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and

A. J. E. Bettany; S. Dalton; E. Timms; B. Manderyck; M. Dhanoa; P. Morris

2003-01-01

2

Agrobacterium tumefaciens -Mediated Transformation of Rosa hybrida using the Green Fluorescent Protein (GFP) Gene  

Microsoft Academic Search

Embryogenic calluses of Rosa hybrida cultivar Tineke were transformed with Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBIN m-gfp5-ER into which the virE\\/virG genes had been inserted. Visualization of GFP-expressing cells enabled visual selection of dividing, embryogenic cell clusters that were transgenic. When the Agrobacterium strain with the bifunctional fusion marker containing additional virE\\/virG genes was used, the number

C. K. Kim; J. D. Chung; S. H. Park; A. M. Burrell; K. K. Kamo; D. H. Byrne

2004-01-01

3

Developing an Agrobacterium tumefaciens -mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus  

Microsoft Academic Search

Agrobacterium\\u000a tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The\\u000a optimal concentration of kanamycin that could effectively inhibit cell growth and division in

Diane E. Darlington; Chiu-Yueh Hung; Jiahua Xie

2009-01-01

4

Identification of antibiotics that are effective 2 in eliminating Agrobacterium tumefaciens  

Microsoft Academic Search

An assay was performed to identify the antibiotics that are most effective againstAgrobacterium tumefaciens strains EHA101 and LBA4404, and to determine if these antibiotics inhibited tobacco callus and shoot formation. We tested\\u000a ten antibiotics: cefotaxime, carbenecillin, erythromycin, spectinomycin, polymixin B, chloramphenicol, methicillin, Augmentin\\u000a 500, Augmentin 250, and moxalactam. The effectiveness of each antibiotic against the two strains was determined by

Natha J. Shackelfordand; Caryl A. Chlan

1996-01-01

5

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber ( Cucumis sativus L. cv. Poinsett76) via Agrobacterium tumefaciens  

Microsoft Academic Search

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis\\u000a from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and

N. Selvaraj; S. Kasthurirengan; A. Vasudevan; M. Manickavasagam; C. W. Choi; A. Ganapathi

2010-01-01

6

The impact of carbenicillin, cefotaxime and vancomycin on chrysanthemum and tobacco TCL morphogenesis and Agrobacterium growth  

Microsoft Academic Search

Agrobacterium-mediated plant genetic transformation requires a two-step process for its success: selection and regeneration of transformed tissues, and the elimination of the transformation vector, Agrobacterium. This study uses carbenicillin (CA), cefotaxime (CF) and vancomycin (VA) singly, or in combination, to eliminate Agrobacterium tumefaciens LBA4404 and AGLO growing on Agrobacterium-favouring (LB) and plant-favouring (MS) media, at transgenic plant selection levels (10

S. Fukai

7

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid  

Microsoft Academic Search

Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral FantasyPhalaenopsis (Baby HatAnn Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for ?-glucuronidase (GUS) and hygromycin resistance.\\u000a The efficiency of transformation was markedly increased by 10?h cocultivation of cell clumps with A. tumefaciens that had been induced with 200??m

M. M. Belarmino; M. Mii

2000-01-01

8

Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species).  

PubMed

Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20-30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 ?M of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 ?M of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future. PMID:24993358

Prasad, Binod; Vadakedath, Nithya; Jeong, Hyun-Jeong; General, Thiyam; Cho, Man-Gi; Lein, Wolfgang

2014-10-01

9

High-efficiency Agrobacterium -mediated transformation of chickpea ( Cicer arietinum L.) and regeneration of insect-resistant transgenic plants  

Microsoft Academic Search

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium\\u000a tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding ?-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 0.3 with 48 h

Meenakshi Mehrotra; Indraneel Sanyal; D. V. Amla

10

Agrobacterium mediated genetic transformation of mint with E.   coli glutathione synthetase gene  

Microsoft Academic Search

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of\\u000a leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), ?-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and

Akhilesh Kumar; Amrita Chakraborty; Srijani Ghanta; Sharmila Chattopadhyay

2009-01-01

11

Evaluation of 12 ?-lactam antibiotics for Agrobacterium -mediated transformation through in planta antibacterial activities and phytotoxicities  

Microsoft Academic Search

The antibacterial activities of 12 ß-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 living in tobacco (Nicotiana tabacum L.) leaf tissues, and their phytotoxicities to tobacco leaf tissues were evaluated. All ß-lactams at minimum bactericidal concentration (MBC) or higher showed weak bactericidal activities against agrobacteria persisting in tobacco leaf tissues. The ß-lactams evaluated were classified into two major groups

Yoichi Ogawa; Masahiro Mii

2005-01-01

12

Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium  

Microsoft Academic Search

Derivatives of potato (Solanum tuberosum cv.'s ‘Maris Bard’ and ‘Desiree’) transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixedcultures of shoot-inducing A. tumefaciens strains T37 and either Agrobacterium strain LBA1834(pRAL1834), (Hille et al. 1983) or LBA4404(pBIN6; pRAL4404), (Bevan 1984). Two small-scale feasibility

G. Ooms; M. M. Burrell; A. Karp; M. Bevan; J. Hille

1987-01-01

13

Carbohydrate Metabolism in Agrobacterium tumefaciens  

PubMed Central

The activity of pentose cycling (PC) reactions in Agrobacterium tumefaciens is much greater than that normally found in bacteria, and in this regard the organism represents a unique category. Equations specifically derived from radiorespirometric data for bacteria with high PC activity in the presence of an alternate pathway are presented. A. tumefaciens utilizes d-glucose by strictly aerobic mechanisms involving the Entner-Doudoroff (ED) and PC pathways; relative participation by the ED pathway is 55% and by the PC cycle, 44%. The 3-ketoglycose-synthesizing system in the bacterium does not affect the relative participation of these two pathways. Radiorespirometric and enzymatic analyses clearly demonstrate that the Embden-Meyerhof-Parnas pathway does not function. Studies on the oxidation of pyruvic, acetic, succinic, and glutamic acids show that terminal respiration includes both the tricarboxylic acid and glyoxylic acid cycles. PMID:4745418

Arthur, Larry O.; Bulla, Lee A.; Julian, Grant St.; Nakamura, Lawrence K.

1973-01-01

14

Transformation of rice mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient

Yukoh Hiei; Toshihiko Komari; Tomoaki Kubo

1997-01-01

15

Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.  

PubMed

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns. PMID:14615907

Luo, H; Hu, Q; Nelson, K; Longo, C; Kausch, A P; Chandlee, J M; Wipff, J K; Fricker, C R

2004-04-01

16

Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production  

PubMed Central

Background: Artemisinin, a sesquiterpene lactone endoperoxide isolated from the medicinal plant Artemisia annua L., is a choice and effective drug for malaria treatment. Due to the low yield of artemisinin in plants, there is a need to enhance the production of artemisinin from A. annua and biotechnological technique may be one of the methods that can be used for the purpose. Aim: To study the transformation efficiency of Agrobacterium tumefaciens in A. annua that could be applied to enhance the production of artemisinin by means of transgenic plants. Setting and Designs: The factors influencing Agrobacterium-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain and effect of organosilicone surfactants. Three strains of A. tumefaciens, that is, LBA4404, GV1301, and AGL1 harboring the binary vector pCAMBIA 1303 have been used for transformation. The evaluation was based on transient ?-glucuronidase (GUS). Materials and Methods: Plant cell cultures were inniatiated from the seeds of A. annua using the germination Murashige and Skoog medium. A. tumefaciens harboring pCAMBIA were tranformed into the leaves of A.annua cultures from 2-week-old-seedling and 2-month-old-seedling for 15 min by vacuum infiltration. Transformation efficiency was determinated by measuring of blue area (GUS expression) on the whole leaves explant using ImageJ 1.43 software. Two organosilicon surfactants, that is, Silwet L-77 and Silwet S-408 were used to improve the transformation efficiency. Results: The transformation frequency with AGL1 strain was higher than GV3101 and LBA4404 which were 70.91, 49.25, and 45.45%, respectively. Effect of organosilicone surfactants, that is, Silwet L-77 and Silwet S-408 were tested on A. tumefaciens AGL1 and GV3101 for their level of transient expression, and on A. rhizogenes R1000 for its hairy root induction frequency. For AGL1, Silwet S-408 produced higher level of expression than Silwet L-77, were 2.3- and 1.3-fold, respectively. For GV3101, Silwet L-77 was still higher than Silwet S-408, were 1.5- and 1.4-fold, respectively. However, GV3101 produced higher levels of expression than AGL1. The area of GUS expression spots of AGL1, LBA4404, and GV3101 strains was 53.43%, 41.06%, and 30.51%, respectively. Conclusion: A. tumefaciens AGl1 strain was the most effective to be transformed in to A. annua than GV3101 and LBA4404 strain. Surfactant Silwet S-408 produced the highest efficiency of transformation. PMID:24914301

Elfahmi; Suhandono, Sony; Chahyadi, Agus

2014-01-01

17

Cellulose Synthesis in Agrobacterium tumefaciens  

SciTech Connect

We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants including CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one preliminary experiment of this type and have successfully complemented an A. tumefaciens CelC mutant with the homologous gene (yhjM) from E. coli.

Alan R. White; Ann G. Matthysse

2004-07-31

18

Agrobacterium tumefaciens is a diazotrophic bacterium  

SciTech Connect

This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate {sup 15}N supplied as {sup 15}N{sub 2}. As with most other well-characterized diazotrophic bacteria, the presence of NH{sub 4}{sup +} in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship.

Kanvinde, L.; Sastry, G.R.K. (Univ. of Leeds (England))

1990-07-01

19

Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus  

PubMed Central

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2–3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. PMID:18327592

Spetz, Carl; Haugslien, Sissel; Xing, Shaochen; Dees, Merete W.; Moe, Roar; Blystad, Dag-Ragnar

2008-01-01

20

Agroinfiltration by Cytokinin-Producing Agrobacterium sp. Strain GV3101 Primes Defense Responses in Nicotiana tabacum.  

PubMed

Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies. PMID:25054409

Sheikh, Arsheed Hussain; Raghuram, Badmi; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin; Sinha, Alok Krishna

2014-11-01

21

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens  

Microsoft Academic Search

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which ?-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited

H. I. Kokko; S. O. Kärenlampi

1998-01-01

22

Shape-dependent bactericidal activity of TiO2 for the killing of Gram-negative bacteria Agrobacterium tumefaciens under UV torch irradiation.  

PubMed

This paper demonstrated the relative bactericidal activity of photoirradiated (6W-UV Torch, ??>?340 nm and intensity?=?0.64 mW/cm(2)) P25-TiO2 nanoparticles, nanorods, and nanotubes for the killing of Gram-negative bacterium Agrobacterium tumefaciens LBA4404 for the first time. TiO2 nanorod (anatase) with length of 70-100 nm and diameter of 10-12 nm, and TiO2 nanotube with length of 90-110 nm and diameter of 9-11 nm were prepared from P-25 Degussa TiO2 (size, 30-50 nm) by hydrothermal method and compared their biocidal activity both in aqueous slurry and thin films. The mode of bacterial cell decomposition was analyzed through transmission electron microscopy (TEM), Fourier transform-infrared (FT-IR), and K(+) ion leakage. The antimicrobial activity of photoirradiated TiO2 of different shapes was found to be in the order P25-TiO2?>?nanorod?>?nanotube which is reverse to their specific surface area as 54?

Aminedi, Raghavendra; Wadhwa, Gunveen; Das, Niranjan; Pal, Bonamali

2013-09-01

23

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens  

Microsoft Academic Search

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens–mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation

Kristin J Mayo; Barbara J Gonzales; Hugh S Mason

2006-01-01

24

Agrobacterium tumefaciens responses to plant-derived signaling molecules  

PubMed Central

As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium–plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including ?-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium–plant interactions. PMID:25071805

Subramoni, Sujatha; Nathoo, Naeem; Klimov, Eugene; Yuan, Ze-Chun

2014-01-01

25

Transformation of forage legumes using Agrobacterium tumefaciens  

Microsoft Academic Search

Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce

K. J. Webb

1986-01-01

26

Transformation of Brassica napus with Agrobacterium tumefaciens based vectors  

Microsoft Academic Search

A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6–7 week old plants were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultured for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly

Joyce Fry; Arlene Barnason; Robert B. Horsch

1987-01-01

27

Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens  

Microsoft Academic Search

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100

Stephen L. Sain; Kwabena K. Oduro; Douglas B. Furtek

1994-01-01

28

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens  

Microsoft Academic Search

Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed

Sharon E. Radke; Joann C. Turner; Daniel Facciotti

1992-01-01

29

Transformation of forage legumes using Agrobacterium tumefaciens.  

PubMed

Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines. PMID:24247771

Webb, K J

1986-04-01

30

Transformation of Brassica napus L. using Agrobacterium tumefaciens : developmentally regulated expression of a reintroduced napin gene  

Microsoft Academic Search

Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter

S. E. Radke; B. M. Andrews; M. M. Moloney; M. L. Crouch; J. C. Kridl; V. C. Knauf

1988-01-01

31

Progress of cereal transformation technology mediated by Agrobacterium tumefaciens  

PubMed Central

Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.

Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

2014-01-01

32

Development of Transgenic Papaya through Agrobacterium-Mediated Transformation.  

PubMed

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and ?-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime?+?50?mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi. PMID:24066284

Azad, Md Abul Kalam; Rabbani, Md Golam; Amin, Latifah; Sidik, Nik Marzuki

2013-01-01

33

Development of Transgenic Papaya through Agrobacterium-Mediated Transformation  

PubMed Central

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and ?-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime?+?50?mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi. PMID:24066284

Azad, Md. Abul Kalam; Rabbani, Md. Golam; Amin, Latifah; Sidik, Nik Marzuki

2013-01-01

34

Transformation of white spruce and other conifer species by Agrobacterium tumefaciens  

Microsoft Academic Search

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced

David Ellis; Dane Roberts; Ben Sutton; Wayne Lazaroff; David Webb; Barry Flinn

1989-01-01

35

Mutants of Agrobacterium tumefaciens with elevated vir gene expression  

SciTech Connect

Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.

Pazour, G.J.; Ta, C.N.; Das, A. (University of Minnesota, St. Paul (USA))

1991-08-15

36

The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58  

Microsoft Academic Search

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences

Derek W. Wood; Joao C. Setubal; Rajinder Kaul; Dave E. Monks; Joao P. Kitajima; Vagner K. Okura; Yang Zhou; Lishan Chen; Gwendolyn E. Wood; Nalvo F. Almeida; Lisa Woo; Yuching Chen; Ian T. Paulsen; Peter D. Karp; Donald Bovee Sr; Peter Chapman; James Clendenning; Glenda Deatherage; Will Gillet; Charles Grant; Tatyana Kutyavin; Ruth Levy; Meng-Jin Li; Erin McClelland; Anthony Palmieri; Christopher Raymond; Gregory Rouse; Channakhone Saenphimmachak; Zaining Wu; Pedro Romero; David Gordon; Shiping Zhang; Heayun Yoo; Yumin Tao; Phyllis Biddle; Mark Jung; William Krespan; Michael Perry; Bill Gordon-Kamm; Li Liao; Sun Kim; Carol Hendrick; Zuo-Yu Zhao; Maureen Dolan; Forrest Chumley; Scott V. Tingey; Jean-Francois Tomb; Milton P. Gordon; Maynard V. Olson; Eugene W. Nester

2001-01-01

37

Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans  

Microsoft Academic Search

SUMMARY Agrobacterium tumefaciens is widely used for plant DNA trans- formation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phy- tophthora infestans, the causal agent of potato late blight. Binary T-DNA vectors containing neomycin phosphotransferase (npt ) and ?-glucuronidase (gus) fused to oomycete

Irma Vijn; Francine Govers

2003-01-01

38

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions  

SciTech Connect

The authors isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned, R. meliloti exo loci. They also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-..beta..-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.

Cangelosi, G.A.; Hung, L.; Puvanesarajah, V.; Stacey, G.; Ozga, D.A.; Leigh, J.A.; Nester, E.W.

1987-05-01

39

Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system.  

PubMed

Agrobacterium tumefaciens has been widely used as a tool for transgenesis in plants. The availability of its genome sequence should facilitate the directed engineering of improved properties; however, the current genome engineering options are laborious. Here, we investigated whether the lambda Red operon can be applied for recombineering of the A. tumefaciens genome. First, we built an expression plasmid for A. tumefaciens employing a tetracycline-inducible promoter to regulate the Red operon. This multicopy plasmid was then itself modified in A. tumefaciens to verify that the Red operon was functional. Then, we modified the endogenous A. tumefaciens tumor-inducing plasmid and the linear chromosome. These results extend recombineering technology to a new host and indicate a fast and convenient way to engineer the A. tumefaciens genome for functional genomics and strain improvements. PMID:24297480

Hu, Shengbiao; Fu, Jun; Huang, Fan; Ding, Xuezhi; Stewart, A Francis; Xia, Liqiu; Zhang, Youming

2014-03-01

40

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots by Agrobacterium tumefaciens and Agrobacterium rhizogenes  

Microsoft Academic Search

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls

J. Vlasák; M. Ond?ej

1985-01-01

41

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens -mediated transformation  

Microsoft Academic Search

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg\\/L AgNO3 and 0.5–2 mg\\/L BA in combination

Donna G. Barfield; Eng-Chong Pua

1991-01-01

42

Transformation of suspension cultures of bromegrass ( Bromus inermis ) by Agrobacterium tumefaciens  

Microsoft Academic Search

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of\\u000a cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS)\\u000a and

Toshihide Nakamura; Masaya Ishikawa

2006-01-01

43

Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation  

Microsoft Academic Search

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations\\u000a of 200–500 mg\\/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect

Z.-M. Cheng; J. A. Schnurr; J. A. Kapaun

1998-01-01

44

Developmental Effects of Zeatin, Ribosyl-Zeatin, and Agrobacterium tumefaciens B6 on Certain Mosses  

PubMed Central

Eight species of mosses studied were divided into two groups on the basis of their developmental responses to ribosyl-trans-zeatin and Agro-bacterium tumefaciens B6. All eight produced either gametophores or callus on the protonema in response to 6-(?,?-dimethylallylamino) purine and trans-zeatin. Three which produced normal gametophores with A. tumefaciens yielded callus or abnormal gametophores with ribosyl-trans-zeatin. Ribosyl-trans-zeatin and A. tumefaciens were relatively ineffective on five other mosses. Characteristics of protonemal growth common to each of these two groups are described. PMID:16659608

Spiess, Luretta D.

1976-01-01

45

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum  

Microsoft Academic Search

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight

Khaled M. S. A. Salem; Barry V. Charlwood

1995-01-01

46

An enrichment technique for auxotrophs of Agrobacterium tumefaciens using a combination of carbenicillin and lysozyme.  

PubMed

A procedure to enrich for auxotrophic and fermentation mutants of Agrobacterium tumefaciens is described. The method is based on the amplification of the killing power of carbenicillin by the addition of lysozyme. Isolation frequencies of some types of mutants are presented, with and without the application of the proposed procedure. The yield of mutants is usually enhanced a hundredfold per enrichment treatment. PMID:1104767

Klapwijk, P M; de Jonge, A J; Schilperoort, R A; Rörsch, A

1975-11-01

47

Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis  

PubMed Central

Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930

Aktas, Meriyem; Danne, Linna; Moller, Philip; Narberhaus, Franz

2014-01-01

48

Transformation of radish ( Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus  

Microsoft Academic Search

A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in

Byong-Jin Park; Zaochang Liu; Akira Kanno; Toshiaki Kameya

2005-01-01

49

Agrobacterium-mediated genetic transformation using cotyledons in Japanese pear (Pyrus pyrifolia).  

PubMed

Genetic transformation was successfully established producing both transformed adventitious shoots and calli in Japanese pear (Pyrus pyrifolia Nakai) by using cotyledons as explants. Cotyledons of five cultivars were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying the pBIN19-sgfp, which contained a green fluorescent protein gene and the neomycin phosphotransferase gene. In order to increase transformation efficiency, sonication and ethylenedioxybis (ethylamine)-N,N,N',N'-tetraacetic acid (EGTA) treatments were applied, which could produce physical wounds across the tissue and prevent plant defense reaction, respectively. Green fluorescent protein (GFP) fluorescence was evaluated two weeks and five months after Agrobacterium inoculation as measures of transient and stable transformations, respectively. As a result, sonication significantly increased both transient and stable expression of GFP fluorescence, whereas EGTA treatment did not show a positive effect on either. Out of 18 regenerated plantlets obtained, one plant regenerated from 'Agenosho Shinanashi' showed stable GFP fluorescence. This plant was confirmed as a transformant by PCR and genomic Southern blotting. Three other transformed regenerated shoots by myb gene showed red color, which were derived from 'Imamuraaki' by the same transformation method. Transformation system in this study was shown to be reproducible since plural transformants were obtained. PMID:24273422

Nakajima, Ikuko; Sato, Yoshihiko; Saito, Toshihiro; Moriguchi, Takaya; Yamamoto, Toshiya

2013-09-01

50

Agrobacterium-mediated genetic transformation using cotyledons in Japanese pear (Pyrus pyrifolia)  

PubMed Central

Genetic transformation was successfully established producing both transformed adventitious shoots and calli in Japanese pear (Pyrus pyrifolia Nakai) by using cotyledons as explants. Cotyledons of five cultivars were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying the pBIN19-sgfp, which contained a green fluorescent protein gene and the neomycin phosphotransferase gene. In order to increase transformation efficiency, sonication and ethylenedioxybis (ethylamine)-N,N,N?,N?-tetraacetic acid (EGTA) treatments were applied, which could produce physical wounds across the tissue and prevent plant defense reaction, respectively. Green fluorescent protein (GFP) fluorescence was evaluated two weeks and five months after Agrobacterium inoculation as measures of transient and stable transformations, respectively. As a result, sonication significantly increased both transient and stable expression of GFP fluorescence, whereas EGTA treatment did not show a positive effect on either. Out of 18 regenerated plantlets obtained, one plant regenerated from ‘Agenosho Shinanashi’ showed stable GFP fluorescence. This plant was confirmed as a transformant by PCR and genomic Southern blotting. Three other transformed regenerated shoots by myb gene showed red color, which were derived from ‘Imamuraaki’ by the same transformation method. Transformation system in this study was shown to be reproducible since plural transformants were obtained. PMID:24273422

Nakajima, Ikuko; Sato, Yoshihiko; Saito, Toshihiro; Moriguchi, Takaya; Yamamoto, Toshiya

2013-01-01

51

Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.  

PubMed Central

The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations. Images PMID:8407849

Girbes, T; Barbieri, L; Ferreras, M; Arias, F J; Rojo, M A; Iglesias, R; Alegre, C; Escarmis, C; Stirpe, F

1993-01-01

52

Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall  

Microsoft Academic Search

We compared the binding of Agrobacterium tumefaciens by freshly isolated root cap cells with susceptibility of plants to crown gall tumorigenesis. A high binding reaction was strongly correlated with susceptibility to tumorigenesis in a survey of the binding of strain B6 to cells from 48 species in 17 families. In reciprocal experiments with nine virulent A. tumefaciens strains, tumors developed

M. C. Hawes; S. G. Pueppke

1987-01-01

53

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens  

Microsoft Academic Search

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did

F. A. Krens; L. Molendijk; G. J. Wullems; R. A. Schilperoort

1985-01-01

54

Restricted electron flux increases coenzyme Q 10 production in Agrobacterium tumefaciens ATCC4452  

Microsoft Academic Search

Coenzyme Q10 (CoQ10) is an electron carrier in the respiration chain with antioxidant activity. The effects of aeration and agitation rates, dissolved oxygen levels, an electron flux inhibitor (azide), a proton gradient releaser (2,4-dinitrophenol (DNP)), and an oxidative stressor (hydrogen peroxide (H2O2)) on the intracellular CoQ10 contents in Agrobacterium tumefaciens ATCC4452 were investigated. With a decrease of dissolved oxygen level

Gi-Sub Choi; Yong-Sung Kim; Jin-Ho Seo; Yeon-Woo Ryu

2005-01-01

55

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana  

Microsoft Academic Search

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per

Maria Cec??lia dos Reis; Maria Helena Pelegrinelli Fungaro; Rubens Tadeu Delgado Duarte; Luciana Furlaneto; Marcia Cristina Furlaneto

2004-01-01

56

Agrobacterium tumefaciens -mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii  

Microsoft Academic Search

Conidia of the phytopathogenic fungus Calonectria morganii were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. Agrobacterium tumefaciens-mediated transformation yielded stable hygromycin B-resistant clones (average number 106 per 107 conidia). Putative transformants appeared to be mitotically and meiotically stable. The

Stefan Malonek; Friedhelm Meinhardt

2001-01-01

57

Agrobacterium tumefaciens -mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum  

Microsoft Academic Search

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per\\u000a 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants

Ji-ye Wang; Hong-ye Li

2008-01-01

58

Agrobacterium tumefaciens -mediated transformation of broccoli ( Brassica oleracea var. italica ) and cabbage ( B. oleracea var. capitata )  

Microsoft Academic Search

Transgenic broccoli (Brassica oleracea var. italica) was produced by two Agrobacterium tumefaciens-mediated transformation methods. One used flowering stalk explants from mature plants; the other used hypocotyl and petiole explants from in vitro-grown seedlings. Several hundred transformants containing a Bacillus thuringiensis ?-endotoxin gene (CryIA(c)-type) and the neomycin phosphotransferase gene were recovered. Rooted transformants were obtained in as little as 3 months

Timothy D. Metz; Ram Dixit; Elizabeth D. Earle

1995-01-01

59

Transformation of pollen embryo-derived explants by Agrobacterium tumefaciens in Hyoscyamus niger  

Microsoft Academic Search

Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient ß-glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were

Shanjun Tu; R. S. Sangwan; V. Raghavan; D. P. S. Verma; B. S. Sangwan-Norreel

2005-01-01

60

Agrobacterium tumefaciens -mediated expression of gusA in maize tissues  

Microsoft Academic Search

To develop a system forAgrobacterium-mediated transformation of maize (Zea mays L.), we have investigated histochemically the transient expression of ?-glucuronidase (GUS) activity in maize seedling tissue segments using binary vectors that allow minimal (pKIWI105 and pCNL1) or undetectable (p35S-GUS-INT and pCNL56) levels of GUS activity inA. tumefaciens. Tissue segments from three- to five-day-old sterile seedlings of maize genotype A188 were

Steven W. Ritchie; Chang-Nong Lui; James C. Sellmer; Halina Kononowicz; Thomas K. Hodges; Stanton B. Gelvin

1993-01-01

61

Two chromosomal loci involved in production of exopolysaccharide in Agrobacterium tumefaciens.  

PubMed Central

The chromosomal locus pscA (exoC) of Agrobacterium tumefaciens LBA4301 has been cloned by complementation of the avirulent and exopolysaccharide (EPS)-deficient mutant LBA4301 pscA. We have also identified a new locus, termed psdA (polysaccharide depression) and located 16 kilobases from pscA in the A. tumefaciens chromosome, that negatively affects EPS production when it is present in more than one copy in A. tumefaciens LBA4301. Subcloning, transposon mutagenesis, and transcriptional analysis have been conducted for both loci and indicate that pscA and psdA are transcribed in the same orientation. Acidic-EPS assays showed that psdA depresses succinoglycan production and that its negative effect increases with the copy number of the gene. Virulence tests of psdA transconjugants on Datura stramonium showed no visible alteration in virulence, while LBA4301 pscA was totally avirulent. Images PMID:2921249

Kamoun, S; Cooley, M B; Rogowsky, P M; Kado, C I

1989-01-01

62

Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens  

PubMed Central

The ?-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse ?-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJ homologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ?pleC and ?divK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ?pleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions. PMID:23437210

Fuqua, Clay

2013-01-01

63

Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass.  

PubMed

Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant. PMID:12765291

Chai, Ming-Liang; Wang, Bing-Liang; Kim, Jae-Yeoul; Lee, Jong-Min; Kim, Doo-Hwan

2003-01-01

64

Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens  

PubMed Central

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens. PMID:25330313

Moller, Philip; Overloper, Aaron; Forstner, Konrad U.; Wen, Tuan-Nan; Sharma, Cynthia M.; Lai, Erh-Min; Narberhaus, Franz

2014-01-01

65

Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.  

PubMed

A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed. PMID:6321432

Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

1984-03-01

66

Interaction between Meloidogyne incognita and Agrobacterium tumefaciens or Fusarium oxysporum f. sp. lycopersici on Tomato.  

PubMed

Agrobacterium tumefaciens stimulated and Fusarium oxysporum f. sp. lycopersici inhibited development and reproduction of Meloidogyne incognita when applied to the opposite split root of tomato, Lycopersicon esculentum cv. Tropic, plants. The lowest rate of nematode reproduction occurred after 2,000 juveniles were applied and the fungus was present in the opposite split root. The effects of all three pathogens alone on the growth of roots and shoots of tomato plants were evident, but M. incognita had a greater effect alone than did either of the other pathogens. The length of split roots was reduced by the infection of M. incognita and A. tumefaciens or F. oxysporum f. sp. lycopersici. The number of galls induced by nematodes on roots was higher where the bacterium was applied and lower where the fungus was applied to the opposite split root. PMID:19283119

El-Sherif, A G; Elwakil, M A

1991-04-01

67

Phosphorus Limitation Enhances Biofilm Formation of the Plant Pathogen Agrobacterium tumefaciens through the PhoR-PhoB Regulatory System  

Microsoft Academic Search

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phos- phate-replete conditions, despite slower overall growth under low-phosphate conditions. Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results. The increase in surface interactions under phosphate limitation was observed in both static culture and

Thomas Danhorn; Morten Hentzer; Michael Givskov; Matthew R. Parsek; Clay Fuqua

2004-01-01

68

The ntrA gene of Agrobacterium tumefaciens: identification, cloning, and phenotype of a site-directed mutant.  

PubMed Central

A 3.6-kb EcoRI fragment containing the ntrA gene of Agrobacterium tumefaciens was cloned by using the homologous ntrA gene of Rhizobium meliloti as a probe. Construction of an ntrA mutant of A. tumefaciens by site-directed insertional mutagenesis demonstrated the requirement of the ntrA gene for nitrate utilization and C4-dicarboxylate transport but not for vir gene expression or tumorigenesis. PMID:1556090

Wu, Z L; Charles, T C; Wang, H; Nester, E W

1992-01-01

69

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens.  

PubMed

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution. PMID:11919722

Leloup, L; Lai, E-M; Kado, C I

2002-03-01

70

An efficient Agrobacterium tumefaciens -mediated genetic transformation of “Egusi” melon ( Colocynthis citrullus L.)  

Microsoft Academic Search

Cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harbouring genes coding for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII) or plasmid pBBRacdS harbouring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Six weeks after\\u000a co-cultivation, more than 35% of explants produced shoots

Valentine Otang Ntui; Raham Sher khan; Dong Poh Chin; Ikuo Nakamura; Masahiro Mii

2010-01-01

71

Virus-induced gene silencing and Agrobacterium tumefaciens-mediated transient expression in Nicotiana tabacum.  

PubMed

Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the corresponding Verticillium effector Ave1 through Agrobacterium tumefaciens-mediated transient transformation (agroinfiltration), we established a rapid system for assessing the requirement of candidate plant genes for Ve1-mediated immune signaling. PMID:24643561

Zhang, Zhao; Thomma, Bart P H J

2014-01-01

72

Accumulation of glycolipids and other non-phosphorous lipids in Agrobacterium tumefaciens grown under phosphate deprivation.  

PubMed

Phosphate deficiency is characteristic for many natural habitats, resulting in different physiological responses in plants and bacteria including the replacement of phospholipids by glycolipids and other phosphorous-free lipids. The plant pathogenic bacterium Agrobacterium tumefaciens, which is free of glycolipids under full nutrition, harbors an open reading frame (ORF) coding for a processive glycosyltransferase (named as Pgt). This glycosyltransferase was previously shown to synthesize glucosylgalactosyldiacylglycerol (GGD) and digalactosyldiacylglycerol (DGD) after heterologous expression. The native function of this enzyme and the conditions for its activation remained unknown. We show here that Pgt is active under phosphate deprivation synthesizing GGD and DGD in Agrobacterium. A corresponding deletion mutant (?pgt) is free of these two glycolipids. Glycolipid accumulation is mainly regulated by substrate (diacylglycerol) availability. Diacylglycerol and the total fatty acid pool are characterized by an altered acyl composition in dependence of the phosphate status with a strong decrease of 18:1 and concomitant increase of 19:0 cyclo during phosphate deprivation. Furthermore, Agrobacterium accumulates two additional unknown glycolipids and diacylglycerol trimethylhomoserine (DGTS) during phosphate deprivation. Accumulation of all these lipids is accompanied by a reduction in phospholipids from 75 to 45% in the wild type. A further non-phosphorous lipid, ornithine lipid, was not increased but its degree of hydroxylation was elevated under phosphate deprivation. The lack of GGD and DGD in the ?pgt mutant has no effect on growth and virulence of Agrobacterium, suggesting that these two lipids are functionally replaced by DGTS and the two unknown glycolipids under phosphate deprivation. PMID:22923441

Geske, Thomas; Vom Dorp, Katharina; Dörmann, Peter; Hölzl, Georg

2013-01-01

73

Agrobacterium tumefaciens-mediated transformation of the lichen fungus, Umbilicaria muehlenbergii.  

PubMed

Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT) for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway. PMID:24386304

Park, Sook-Young; Jeong, Min-Hye; Wang, Hai-Ying; Kim, Jung A; Yu, Nan-Hee; Kim, Sungbeom; Cheong, Yong Hwa; Kang, Seogchan; Lee, Yong-Hwan; Hur, Jae-Seoun

2013-01-01

74

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens.  

PubMed

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0-80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved. PMID:24232587

Ellis, D; Roberts, D; Sutton, B; Lazaroff, W; Webb, D; Flinn, B

1989-05-01

75

Agrobacterium tumefaciens-Mediated Transformation of the Lichen Fungus, Umbilicaria muehlenbergii  

PubMed Central

Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT) for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway. PMID:24386304

Wang, Hai-Ying; Kim, Jung A.; Yu, Nan-Hee; Kim, Sungbeom; Cheong, Yong Hwa; Kang, Seogchan; Lee, Yong-Hwan; Hur, Jae-Seoun

2013-01-01

76

Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens.  

PubMed Central

Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells. PMID:8752352

Matthysse, A G; Yarnall, H A; Young, N

1996-01-01

77

Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens  

Microsoft Academic Search

A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein

V. Levée; E. Garin; K. Klimaszewska; A. Séguin

1999-01-01

78

Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends  

SciTech Connect

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)

2012-09-05

79

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)  

NASA Technical Reports Server (NTRS)

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

1998-01-01

80

Influence of Agrobacterium tumefaciens strain on the production of transgenic peas ( Pisum sativum L.).  

PubMed

We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants. KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean. The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids. The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons. Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants. We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures. PMID:12819922

Grant, J E; Thomson, L M J; Pither-Joyce, M D; Dale, T M; Cooper, P A

2003-08-01

81

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens.  

PubMed

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine. PMID:24241754

Mackay, J; Séguin, A; Lalonde, M

1988-06-01

82

Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens.  

PubMed

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome-one on the circular chromosome and six on the linear chromosome-suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species. PMID:21795751

Lassalle, Florent; Campillo, Tony; Vial, Ludovic; Baude, Jessica; Costechareyre, Denis; Chapulliot, David; Shams, Malek; Abrouk, Danis; Lavire, Céline; Oger-Desfeux, Christine; Hommais, Florence; Guéguen, Laurent; Daubin, Vincent; Muller, Daniel; Nesme, Xavier

2011-01-01

83

Identification and Characterization of a Second Quorum-Sensing System in Agrobacterium tumefaciens A6  

PubMed Central

Quorum sensing (QS) is a widespread mechanism of bacterial communication in which individual cells produce and respond to small chemical signals. In Agrobacterium tumefaciens, an acylhomoserine lactone-dependent QS mechanism is known to regulate the replication and conjugation of the tumor-inducing (Ti) plasmid. Most of the QS regulatory proteins are encoded within the Ti plasmid. Among them, TraI is the LuxI-type enzyme synthesizing the QS signal N-3-oxooctanoyl-l-homoserine lactone (3OC8HSL), TraR is the LuxR-type transcriptional factor that recognizes 3OC8HSL, and TraM is an antiactivator that antagonizes TraR. Recently, we identified a TraM homolog encoded by the traM2 gene in the chromosomal background of A. tumefaciens A6. In this study, we further identified additional homologs (TraI2 and TraR2) of TraI and TraR in this strain. We showed that similar to TraI, TraI2 could predominantly synthesize the QS signal 3OC8HSL. We also showed that TraR2 could recognize 3OC8HSL and activate the tra box-containing promoters as efficiently as TraR. Further analysis showed that traM2, traI2, and traR2 are physically linked on a mobile genetic element that is not related to the Ti plasmid. These findings indicate that A. tumefaciens A6 carries a second QS system that may play a redundant role in the regulation of the replication and conjugation of the Ti plasmid. PMID:24464459

Wang, Chao; Yan, Chunlan; Fuqua, Clay

2014-01-01

84

Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens.  

PubMed

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5' leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5' UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5' UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5' leader region upstream of the gene of interest. PMID:25117444

Ahmad, Tauqeer; Venkataraman, Srividhya; Hefferon, Kathleen; AbouHaidar, Mounir G

2014-09-12

85

Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.  

PubMed Central

The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a. Images PMID:457613

Loper, J E; Kado, C I

1979-01-01

86

Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens  

PubMed Central

The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

Kado, Clarence I.

2014-01-01

87

Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants  

PubMed Central

Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report ?-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10?min with 80?W, A. tumefaciens infection of 30?min and co-cultivation of 2 d in 1/2 MS medium containing 100??M acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. Conclusions In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants. PMID:22748182

2012-01-01

88

High-efficiency transformation of Lycium barbarum mediated by Agrobacterium tumefaciens and transgenic plant regeneration via somatic embryogenesis  

Microsoft Academic Search

We have developed a reliable and high-frequency system of transformation and regeneration via somatic embryogenesis (SE) of Lycium barbarum. Leaf segments were co-cultivated with Agrobacterium tumefaciens EHA101 (pIG121Hm) carrying the neomycin phosphotransferase II gene as a selectable marker and an intron-#-glucuronidase (GUS) gene as a reporter marker. On the medium for callus-induction, which contained 50 mg l-1 kanamycin (Km), approximately

Z. Hu; J. Yang; G. Guo; G. Zheng

2002-01-01

89

Regeneration of transgenic Picea glauca, P. Mariana , and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens  

Microsoft Academic Search

Summary  Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58\\/pMP90\\/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (?-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation\\u000a procedure. Of the three

Krystyna Klimaszewska; Denis Lachance; Gervais Pelletier; Marie-Anne Lelu; Armand Séguin

2001-01-01

90

T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives  

Microsoft Academic Search

We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a\\/b binding protein\\/octopine synthase (cab\\/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and

Jonathan D. G. Jones; David E. Gilbert; Karen L. Grady; Richard A. Jorgensen

1987-01-01

91

Cotransformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants  

Microsoft Academic Search

Co-transformation was investigated as a method that would allow the use of a selectable marker during plant regeneration\\u000a followed by recovery of progeny which contain the desired gene(s) but lack a marker gene. Rapeseed (Brassica napus cv `212\\/86') and tobacco (Nicotiana tabacum cv `Xanthi NC') were co-cultivated with a single Agrobacterium tumefaciens strain containing two binary plasmids. Genes from both

M. Daley; V. C. Knauf; K. R. Summerfelt; J. C. Turner

1998-01-01

92

Agrobacterium tumefaciens Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion  

Microsoft Academic Search

This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens. In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth.

Zhiyong Ding; Peter J. Christie

2003-01-01

93

Bud regeneration from Eucalyptus globulus clones and seedlings through hormonal imbalances induced by Agrobacterium tumefaciens strain 82.139  

Microsoft Academic Search

Bud regeneration from seedling hypocotyls and stem cuttings arising from micropropagated plantlets of Eucalyptus globulus was achieved using inoculation with the wild Agrobacterium tumefaciens strain 82.139 which induced shooty tumors. The adventitious buds regenerated on the shooty tumors were untransformed as revealed by the use of a C58pMP90\\/T139GUS-INT strain harbouring the intron inactivated uidA (gus) reporter gene. Subsequent rooting and

A Azmi; W Dewitte; C Drevet; H Van Onckelen; P Landré; A. M Boudet; L Jouanin; D Chriqui

1997-01-01

94

Transgenic Christmas Trees Regenerated from Agrobacterium tumefaciens -mediated Transformation of Zygotic Embryos Using the Green Fluorescence Protein as a Reporter  

Microsoft Academic Search

Mature zygotic embryos of recalcitrant Christmas tree species Fraser fir [Abies fraseri (Pursh) Poir], and Nordmann fir (Abies nordmanniana L.k.), and Virginia pine (Pinus virginiana Mill.) were used as explants for Agrobacterium tumefaciens strain GV3850-mediated transformation using the gfp (green fluorescent protein) gene as a reporter. Factors including media used for inoculation and co-cultivation, concentrations\\u000a of acetosyringone, and antibiotics in

Wei Tang; Ronald J. Newton

2005-01-01

95

Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in Agrobacterium tumefaciens  

PubMed Central

The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed. PMID:24626341

Lin, Jer-Sheng; Wu, Hsin-Hui; Hsu, Pang-Hung; Ma, Lay-Sun; Pang, Yin-Yuin; Tsai, Ming-Daw; Lai, Erh-Min

2014-01-01

96

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens.  

PubMed

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation. PMID:17921337

Louwerse, Jeanine D; van Lier, Miranda C M; van der Steen, Dirk M; de Vlaam, Clementine M T; Hooykaas, Paul J J; Vergunst, Annette C

2007-12-01

97

A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.  

PubMed Central

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity. Images PMID:2001991

Zhang, L H; Kerr, A

1991-01-01

98

[Establishment of high efficiency genetic transformation system of maize mediated by Agrobacterium tumefaciens].  

PubMed

In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25 degrees C for co-culture temperature, 0.7 ODx15 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively. PMID:19933098

WEI, Kai-Fa

2009-11-01

99

The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization*  

PubMed Central

The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of ?-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe147, that are important for SSA association; BlcRF147A existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcRF147A retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcRF147A or BlcRF147A-DNA. As well as in the SSA-binding site, Phe147 is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization. PMID:21467043

Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling

2011-01-01

100

The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization  

SciTech Connect

The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of {gamma}-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe{sup 147}, that are important for SSA association; BlcR{sup F147A} existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcR{sup F147A} retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcR{sup F147A} or BlcR{sup F147A}-DNA. As well as in the SSA-binding site, Phe{sup 147} is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization.

Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling (IMCB-Singapore); (Indiana)

2012-02-08

101

Mutational analysis of the VirG protein, a transcriptional activator of Agrobacterium tumefaciens virulence genes.  

PubMed Central

The VirG protein of Agrobacterium tumefaciens is required in conjunction with the VirA protein for transcriptional activation of the virulence (vir) genes in response to plant phenolic compounds. These proteins are members of a family of two component regulatory systems. vir genes are activated via a cascade of phosphorylation reactions involving a specific aspartic acid residue of the VirG protein. We have conducted a mutational analysis of the VirG protein. By mutating conserved and nonconserved aspartic acid residues in the N-terminal domain, we demonstrated that two of three conserved aspartic acid residues located in two different regions are important for the phosphorylation of VirG by VirA phosphate. A third conserved N-terminal region was also shown to be critical for the biological function of VirG as a transcriptional activator. The identification of phosphorylatable but biologically inactive mutated VirG proteins suggests that not only phosphorylation but also a conformational change is necessary for its activity. We further demonstrated that phosphorylation is not required for sequence-specific binding to a vir gene regulatory sequence (vir box) and that the C-terminal domain is sufficient for DNA binding. The data support the model of a two-domain structure for the VirG protein and demonstrate that the sequence homologies to other two-component regulatory systems reflect both functional and structural homologies. Images PMID:2211523

Roitsch, T; Wang, H; Jin, S G; Nester, E W

1990-01-01

102

Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens.  

PubMed

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037. PMID:6313860

Pankhurst, C E; Broughton, W J; Wieneke, U

1983-08-01

103

Transformation of Solanum integrifolium poir via Agrobacterium tumefaciens: Plant regeneration and progeny analysis.  

PubMed

The wild species Solanum integrifolium represents a source of pest and disease resistance genes for breeding strategies of the cultivated species Solanum melongena. Somatic hybridization via protoplast fusion between the two species may provide a valuable tool for transferring polygenic traits into the cultivated species. The availability of S.integrifolium cells carrying dominant selectable markers would facilitate the heterokaryon rescue. An appropriate methodology for in vitro culture and plant regeneration from leaf explants of S.integrifolium is reported. Efficient leaf-disk transformation via co-cultivation with Agrobacterium tumefaciens led to the regeneration of transformed plants carrying the reporter genes GUS and NPT-II. Transformed individuals were obtained through selection on kanamycin-containing medium. Stable genetic transformation was assessed by histochemical and enzymatic assays for GUS and NPT-II activity, by the ability of leaf disks to initiate callus on Km-containing medium, Southern blot analyses of the regenerated plants, and genetic analysis of their progenies. Selfed-seed progeny of individual transformed plants segregated seedlings capable to root and grow in selective condition, while untransformed progeny did not. Genetic analyses of progeny behaviour showed that the reporter gene NPT-II segregated as single as well as two independent Mendelian factors. In two cases an excess of kanamycin-sensitive seedlings was obtained, not fitting into any genetic hypothesis. PMID:24213029

Rotino, G L; Perrone, D; Ajmone-Marsan, P; Lupotto, E

1992-02-01

104

Transformation of Montmorency sour cherry ( Prunus cerasus L.) and Gisela 6 ( P. cerasus × P. canescens ) cherry rootstock mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus × P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ß-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars

Guo-Qing Song; K. C. Sink

2006-01-01

105

Agrobacterium tumefaciens ?-glucosidase is also an effective ?-xylosidase, and has a high transglycosylation activity in the presence of alcohols  

Microsoft Academic Search

Agrobacterium tumefaciens?-glucosidase, Cbg1 was extensively characterised and found to be a retaining aryl-glucosidase and an aryl-xylosidase. Cbg1s specificity for p-nitrophenyl ?-d-xylopyranoside was 73% that for p-nitrophenyl ?-d-glucopyranoside when measured by the ratio kcat\\/Km. The enzyme also hydrolysed p-nitrophenyl ?-d-fucopyranoside, and p-nitrophenyl ?-d-galactopyranoside with moderate efficiency. The enzyme released only terminal glucose from p-nitrophenyl ?-cellobioside and had a 20?000-fold preference for

Derek K Watt; Hiroshi Ono; Kiyoshi Hayashi

1998-01-01

106

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants  

Microsoft Academic Search

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0 %) with 2,4-D (5.0 ?M) and 2-iP (50.0 ?M) or 2,4-D (50.0 ?M) and TDZ (5.0 ?M) under light\\u000a conditions. These calli were co-cultured

Gulshan Chhabra; Darshna Chaudhary; Manish Sainger; Pawan K. Jaiwal

2011-01-01

107

Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens.  

PubMed

Sequencing of a 7277 bp fragment adjacent to the chvH locus of Agrobacterium tumefaciens revealed four open reading frames (ORFs), designated ameR, ameA, ameB and ameC, respectively. These ORFs exhibit amino acid similarities to components of Resistance-Nodulation-Cell Division (RND) type efflux systems. AmeA and AmeB show high homology to membrane fusion proteins (MFP) and RND-type transporters, whereas AmeC shows similarity to NodT and other members of outer membrane factor families. Mutations of the ameA and ameB genes did not affect the susceptibility profile of the wild-type strain to several detergents and antibiotics. In contrast, mutations of the ameC gene dramatically affected the susceptibility of the strain to these same inhibitory compounds. RT-PCR analysis demonstrated that the ameABC genes form an operon. In addition, ameC gene has its own promoter gene located in the intergenic region between ameB and ameC. Mapping upstream of ameA is ameR, which encodes a protein that shows similarity especially at its N-terminal end to the TetR family of bacterial transcriptional regulators. AmeR negatively regulates expression of the ameABC operon. A mutation in ameR increased the resistance of the cells to several antimicrobial agents. This regulatory locus appears to be in the same operon as a gene located upstream which codes for a product that has high similarity to numerous 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetases. The possible role of the putative efflux pump coded by the ame genes is discussed. PMID:11404022

Peng, W T; Nester, E W

2001-05-30

108

Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea.  

PubMed

Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus. PMID:20952653

Baumgartner, Kendra; Fujiyoshi, Phillip; Foster, Gary D; Bailey, Andy M

2010-12-01

109

Agrobacterium -mediated transformation of the wetland monocot Typha latifolia L. (Broadleaf cattail)  

Microsoft Academic Search

An Agrobacterium-mediated model transformation system was standardized for the wetland monocot Typha latifolia L. to achieve the long-term objective of introducing candidate genes for phytoremediation. Two binary plasmid vectors, pCAMBIA1301\\/EHA105 and pTOK233\\/LBA4404, both containing the gus (ß-glucuronidase) and hptII (hygromycin phosphotransferase II) genes, were used for transformation. Fifty-day-old 5 mg\\/l picloram-derived calli were cocultivated and selected on medium containing 20 mg\\/l or 40 mg\\/l

Rangaraj Nandakumar; Li Chen; Suzanne M. D. Rogers

2005-01-01

110

Lon protease of the alpha-proteobacterium Agrobacterium tumefaciens is required for normal growth, cellular morphology and full virulence.  

PubMed

The ATP-dependent Lon (La) protease is ubiquitous in nature and regulates a diverse set of physiological responses in bacteria. In this paper a lon mutant of the alpha-proteobacterium Agrobacterium tumefaciens C58 has been characterized. Unlike lon mutants of Escherichia coli, the lon mutant of A. tumefaciens grows very slowly, is not filamentous and exhibits normal resistance to UV irradiation. The mutant retains motility and chemotaxis, produces apparently normal amounts of exopolysacchride, but displays severe defects in cell morphology, with 80 % of the mutant cells appearing Y-shaped. Lon protease of A. tumefaciens shares high homology with its counterparts in E. coli and in Sinorhizobium meliloti, and functionally complements an E. coli lon mutant for defects in morphology and RcsA-mediated regulation of capsular polysaccharide production. Mutations at sites of Lon(At) corresponding to the ATP-binding site and the active site serine of the E. coli Lon protease abolish complementation of phenotypes of the A. tumefaciens and E. coli lon mutants. The nucleotide sequence upstream of A. tumefaciens lon contains an element similar to the consensus sigma(32) heat-shock promoter of E. coli. Northern and Western blot analyses indicated that expression of lon is induced by elevated temperature, albeit to a much lower level than that of groEL. The lon mutant is highly attenuated for virulence, suggesting that Lon may be required for the proper expression, assembly or function of the VirB/D4-mediated T-DNA transfer system. PMID:16549682

Su, Shengchang; Stephens, Bonnie B; Alexandre, Gladys; Farrand, Stephen K

2006-04-01

111

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation.  

PubMed

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR. PMID:18471930

Tanaka, Hidenori; Toyama, Jun; Hashiguchi, Masatsugu; Kutsuna, Yasuyo; Tsuruta, Shin-ichi; Akashi, Ryo; Hoffmann, Franz

2008-08-25

112

A stable and efficient Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Digitalis purpurea L.  

PubMed

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) thidiazuron (TDZ) (a cytokine) and 0.1 mg L(-1) 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter ?-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4-6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach. PMID:24272685

Li, Ying; Gao, Zhenrui; Piao, Chunlan; Lu, Kaiwen; Wang, Zhiping; Cui, Min-Long

2014-02-01

113

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants1[C][W][OA  

PubMed Central

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown “filler” sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant’s DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate. PMID:22797657

Singer, Kamy; Shiboleth, Yoel M.; Li, Jianming; Tzfira, Tzvi

2012-01-01

114

Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta.  

PubMed

The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization. PMID:24981331

Ma, Lay-Sun; Hachani, Abderrahman; Lin, Jer-Sheng; Filloux, Alain; Lai, Erh-Min

2014-07-01

115

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.  

PubMed Central

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli. PMID:8824616

Jones, A L; Lai, E M; Shirasu, K; Kado, C I

1996-01-01

116

Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs  

E-print Network

the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided A .tumefaciens can survive in mouse bloodstream and direct expression of its T- DNA within mouse organs. Our data

Citovsky, Vitaly

117

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.  

PubMed Central

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria. Images PMID:2211508

Robinette, D; Matthysse, A G

1990-01-01

118

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.  

PubMed

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria. PMID:2211508

Robinette, D; Matthysse, A G

1990-10-01

119

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens  

Microsoft Academic Search

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca (Moench) Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, con- taining the neomycin phosphotransferase II (nptII) gene providing kanamycin

Julie Belles-Isles; Mathieu Dusabenyagasani; Francine M. Tremblay

2001-01-01

120

Agrobacterium tumefaciens-mediated transformation of the causative agent of Valsa canker of apple tree Valsa mali var. mali.  

PubMed

Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10(6) conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa. PMID:24554343

Hu, Yang; Dai, Qingqing; Liu, Yangyang; Yang, Zhe; Song, Na; Gao, Xiaoning; Voegele, Ralf Thomas; Kang, Zhensheng; Huang, Lili

2014-06-01

121

Expression of Nitrite and Nitric Oxide Reductases in Free-Living and Plant-Associated Agrobacterium tumefaciens C58 Cells†  

PubMed Central

A number of the bacteria that form associations with plants are denitrifiers. To learn more about how the association with plants affects expression of denitrification genes, the regulation of nitrite and nitric oxide reductases was investigated in Agrobacterium tumefaciens. Analysis of free-living cells revealed that expression of the genes encoding nitrite and nitric oxide reductases, nirK and nor, respectively, requires low-oxygen conditions, nitric oxide, and the transcriptional regulator NnrR. Expression of nor was monitored in plant-associated bacteria using nor-gfp fusion expression. In root association experiments, only a small percentage of the attached cells were fluorescent, even when they were incubated under a nitrogen atmosphere. Inactivation of nirK had no significant effect on the ability of A. tumefaciens to bind to plant roots regardless of the oxygen tension, but it did decrease the occurrence of root-associated fluorescent cells. When wild-type cells containing the gfp fusion were infiltrated into leaves, most cells eventually became fluorescent. The same result was obtained when a nirK mutant was used, suggesting that nitric oxide activated nor expression in the endophytic bacteria. Addition of a nitric oxide synthase inhibitor to block nitric oxide generation by the plant prevented gfp expression in infiltrated nitrite reductase mutants, demonstrating that plant-derived nitric oxide can activate nor expression in infiltrated cells. PMID:16085833

Baek, Seung-Hun; Shapleigh, James P.

2005-01-01

122

Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis.  

PubMed

Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction. PMID:24860585

Lin, Yi-Han; Pierce, B Daniel; Fang, Fang; Wise, Arlene; Binns, Andrew N; Lynn, David G

2014-01-01

123

In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens?  

PubMed Central

Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase. PMID:19181804

Aktas, Meriyem; Narberhaus, Franz

2009-01-01

124

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants.  

PubMed

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0 %) with 2,4-D (5.0 ?M) and 2-iP (50.0 ?M) or 2,4-D (50.0 ?M) and TDZ (5.0 ?M) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for ?-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11-13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation. PMID:23573002

Chhabra, Gulshan; Chaudhary, Darshna; Sainger, Manish; Jaiwal, Pawan K

2011-04-01

125

Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis  

PubMed Central

Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction. PMID:24860585

Lin, Yi-Han; Pierce, B. Daniel; Fang, Fang; Wise, Arlene; Binns, Andrew N.; Lynn, David G.

2014-01-01

126

Optimization of the uidA gene transfer into somatic embryos of rose via Agrobacterium tumefaciens  

E-print Network

of chromosome numbers [4,11,20]. The tools of genetic engineering via plant transformation offer a highly into Agrobacerium tumefaciens strain GV3101, and used for transformation of leaf, undifferentiated callus, and primary embryogenic callus. Although all sources of explants showed GUS activity following transformation

Korban, Schuyler S.

127

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens -induced crown galls and in the host stems of Ricinus communis L  

Microsoft Academic Search

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions,

Roni Aloni; Katja S. Pradel; Cornelia I. Ullrich

1995-01-01

128

Differential Expression of Crown Gall Tumor Markers in Transformants Obtained after in vitro Agrobacterium tumefaciens-Induced Transformation of Cell Wall Regenerating Protoplasts Derived from Nicotiana tabacum  

Microsoft Academic Search

To obtain transformation of plant cells, we incubated 3-day-old cell wall-regenerating protoplasts from tobacco with Agrobacterium tumefaciens harboring tumor-inducing plasmids. Putative transformed tobacco cells were selected by phytohormone autotrophic growth and were shown to be transformed by the detection of the tumor cell specific enzymes lysopine dehydrogenase or nopaline dehydrogenase. This was substantiated by the detection, in transformed tumor tissues,

George J. Wullems; Lucy Molendijk; Gert Ooms; Robbert A. Schilperoort

1981-01-01

129

An efficient method for the production of transgenic plants of peanut ( Arachis hypogaea L .) through Agrobacterium tumefaciens-mediated genetic transformation  

Microsoft Academic Search

Cotyledon explants from mature peanut seeds (Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%). Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II (nptII) and ß-glucuronidase (GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to

Kiran K Sharma; Vanamala Anjaiah

2000-01-01

130

Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority and Rule 23a, Note 1 as applied to the corresponding specific epithets. Opinion 94.: Judicial Commission of the International Committee on Systematics of Prokaryotes.  

PubMed

The Judicial Commission affirms that, according to the Rules of the International Code of Nomenclature of Bacteria (including changes made to the wording), the COMBINATION: Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has PRIORITY: over the COMBINATION: Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority as applied to the corresponding SPECIFIC EPITHETS: . The TYPE SPECIES: of the genus is Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942, even if treated as a LATER HETEROTYPIC SYNONYM: of Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942. Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 is typified by the strain defined on the Approved Lists of Bacterial Names and by strains known to be derived from the NOMENCLATURAL TYPE: . PMID:25288664

Tindall, B J

2014-10-01

131

6-Hydroxy-3-Succinoylpyridine Hydroxylase Catalyzes a Central Step of Nicotine Degradation in Agrobacterium tumefaciens S33  

PubMed Central

Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8±1.85 µmol min?1 mg protein?1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP. PMID:25054198

Huang, Haiyan; Wang, Shuning

2014-01-01

132

Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C4 dicotyledon that is closely related to Arabidopsis thaliana  

PubMed Central

In leaves of most C4 plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C3 plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C4 pathway, and there are few genes that have been shown to be important for the cell biology and development of C4 leaves. To facilitate functional analysis of C4 photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C4 species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C4 species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of ?-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C4 plant and should significantly accelerate the analysis of mechanisms underlying C4 photosynthesis. PMID:20150516

Newell, Christine A.; Brown, Naomi J.; Liu, Zheng; Pflug, Alexander; Gowik, Udo; Westhoff, Peter; Hibberd, Julian M.

2010-01-01

133

6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.  

PubMed

Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8 ± 1.85 µmol min-1 mg protein-1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53 ± 0.03 mM 2,5-DHP was produced from 0.76 ± 0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP. PMID:25054198

Li, Huili; Xie, Kebo; Huang, Haiyan; Wang, Shuning

2014-01-01

134

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens  

PubMed Central

The plant pathogen Agrobacterium tumefaciens encodes predicted iron-responsive regulators, Irr and RirA, that function in several other bacteria to control the response to environmental iron levels. Deletion mutations of irr and rirA, alone and in combination, were evaluated for their impact on cellular iron response. Growth was severely diminished in the ?irr mutant under iron-limiting conditions, but reversed to wild-type levels in an ?irr ?rirA mutant. The level of uncomplexed iron in the ?irr mutant was decreased, whereas the ?rirA mutant exhibited elevated iron levels. Sensitivity of the ?irr and ?rirA mutants to iron-activated antimicrobial compounds generally reflected their uncomplexed-iron levels. Expression of genes that encode iron uptake systems was decreased in the ?irr mutant, whereas that of iron utilization genes was increased. Irr function required a trihistidine repeat likely to mediate interactions with heme. Iron uptake genes were derepressed in the ?rirA mutant. In the ?irr ?rirA mutant, iron uptake and utilization genes were derepressed, roughly combining the phenotypes of the single mutants. Siderophore production was elevated in the rirA mutant, but most strongly regulated by an RirA-controlled sigma factor. Expression of rirA itself was regulated by Irr, RirA, and iron availability, in contrast to irr expression, which was relatively stable in the different mutants. These studies suggest that in A. tumefaciens, the Irr protein is most active under low-iron conditions, inhibiting iron utilization and activating iron acquisition, while the RirA protein is active under high-iron conditions, repressing iron uptake. PMID:21602352

Hibbing, Michael E.; Fuqua, Clay

2011-01-01

135

Agrobacterium tumefaciens pTAR parA promoter region involved in autoregulation, incompatibility and plasmid partitioning.  

PubMed

The locus responsible for directing proper plasmid partitioning of Agrobacterium tumefaciens pTAR is contained within a 1259 base-pair region. Insertions or deletions within this locus can result in the loss of the plasmid's ability to partition properly. One protein product (parA), approximately 25,000 Mr, is expressed from the par locus in Escherichia coli and A. tumefaciens protein analysis systems in vitro. DNA sequence analysis of the locus revealed a single 23,500 Mr open reading frame, confirming the protein data. A 248 base-pair region immediately upstream from the 23,500 Mr open reading frame, containing an array of 12 seven-base-pair palindromic repeats each of which are separated by exactly ten base-pairs of A + T-rich (75%) sequence, not only serves to provide the promoter but is also involved in parA autoregulation. In addition, this region containing a set of 12 seven-base-pair palindromic repeats, is responsible for plasmid-associated incompatibility within Inc Ag-1 and also functions as the cis-acting recognition site at which parA interacts to bring about partitioning. Transcriptional analysis indicated that only the DNA strand responsible for parA is actively transcribed, and that active transcription of the opposite strand of par can inhibit the production of parA, resulting in plasmid destabilization. The presence of the par locus in a plasmid results in stable inheritance within a wide range of members of Rhizobiaceae. Segregation rates of par-defective derivatives can be influenced by the host. PMID:3586028

Gallie, D R; Kado, C I

1987-02-01

136

Factors affecting Agrobacterium tumefaciens -mediated genetic transformation of Lycium barbarum L  

Microsoft Academic Search

Summary  Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation,\\u000a including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included

Zhong Hu; Yi-Rui Wu; Wei Li; Huan-Huan Gao

2006-01-01

137

Presence of Unintended Agrobacterium tumefaciens Cloning Vector Sequences in Genetically Modified Plants  

Microsoft Academic Search

Agrobacterium transformation was used in the production of genetically modified plants from oilseed rape (Brassica napus) and tobacco (Nicotiana tabacum). After inoculation stop with the antibiotic timentin, a subsequent one-week treatment eliminated the vector bacterium from\\u000a the oilseed rape plate explant cultures. From the tobacco, however, we recorded vector-derived signals one week after potting\\u000a the regenerants in the greenhouse and

Katarina Björklöf; Michael Färdig; Kirsten S. Jørgensen

2006-01-01

138

Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58.  

PubMed

Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit. PMID:22684062

Mase, Tomoko; Yabuki, Hideya; Okai, Masahiko; Ohtsuka, Jun; Imai, Fabiana Lica; Nagata, Yuji; Tanokura, Masaru

2012-06-01

139

Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis.  

PubMed

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose. PMID:21873475

Choi, Jin-Geun; Ju, Yo-Han; Yeom, Soo-Jin; Oh, Deok-Kun

2011-10-01

140

Improvement in the Thermostability of d-Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis ?  

PubMed Central

The S213C, I33L, and I33L S213C variants of d-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of d-psicose. PMID:21873475

Choi, Jin-Geun; Ju, Yo-Han; Yeom, Soo-Jin; Oh, Deok-Kun

2011-01-01

141

Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose.  

PubMed

The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50 degrees C and pH 8.0 with Mn2+. The turnover number (k(cat)) and catalytic efficiency (k(cat)/Km) of the enzyme for D-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30 degrees C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50 degrees C after 100 min, corresponding to a conversion yield of 32.9%. PMID:16461638

Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

2006-02-01

142

Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose  

PubMed Central

The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%. PMID:16461638

Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

2006-01-01

143

Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity  

PubMed Central

Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants. PMID:25389429

Ishibashi, Nanako; Kitakura, Saeko; Terakura, Shinji; Machida, Chiyoko; Machida, Yasunori

2014-01-01

144

Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains.  

PubMed

Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain. PMID:25107375

Zhang, Yan-Jun; Zhao, Jin-Jin; Xie, Ming; Peng, De-Liang

2014-10-01

145

Improvement of Arabidopsis thaliana seed transformation efficiency  

Microsoft Academic Search

Agrobacterium tumefaciens induced transgenosis by treatment of germinatingArabidopsis thaliana seed embryos has been achieved with differentAgrobacterium strains including the strain LBA4404, which was ineffective in seed transformation experiments of the other authors. The\\u000a frequency of transgenosis was increased several times by application of acetosyringone to the growingA. tumefaciens suspension cultures. The DNA demethylating agent 5-azacytidine partly restored the distorted Mendelian

D. PAVINGEROVJk; M. Ond?ej

1995-01-01

146

Agrobacterium tumefaciens-Mediated Transformation of Valsa mali: An Efficient Tool for Random Insertion Mutagenesis  

PubMed Central

Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT), with the optimal transformation conditions as follows: 106/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200??M acetosyringone (AS) in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 106 recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels. PMID:24381526

Wang, Caixia; Guan, Xiangnan; Wang, Hanyan; Li, Guifang; Dong, Xiangli; Wang, Guoping

2013-01-01

147

Optimization of culture conditions and scale-up to pilot and plant scales for coenzyme Q 10 production by Agrobacterium tumefaciens  

Microsoft Academic Search

This report describes the optimization of culture conditions for coenzyme Q10 (CoQ10) production by Agrobacterium\\u000a tumefaciens KCCM 10413, an identified high-CoQ10-producing strain (Kim et al., Korean patent. 10-0458818, 2002b). Among the conditions tested, the pH and the dissolved oxygen (DO) levels were the key factors affecting CoQ10 production. When the pH and DO levels were controlled at 7.0 and 0–10%,

Suk-Jin Ha; Sang-Yong Kim; Jin-Ho Seo; Deok-Kun Oh; Jung-Kul Lee

2007-01-01

148

Mutational analysis of the active site residues of a D: -psicose 3-epimerase from Agrobacterium tumefaciens.  

PubMed

D-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of D: -fructose to D-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn(2+) are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also in substrate binding. PMID:19859667

Kim, Hye-Jung; Yeom, Soo-Jin; Kim, Kwangsoo; Rhee, Sangkee; Kim, Dooil; Oh, Deok-Kun

2010-02-01

149

Genome sequence and mutational analysis of plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a zinc-lead mine tailing.  

PubMed

The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006

Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J; Rensing, Christopher; Wei, Gehong

2012-08-01

150

Genome Sequence and Mutational Analysis of Plant-Growth-Promoting Bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a Zinc-Lead Mine Tailing  

PubMed Central

The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006

Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J.

2012-01-01

151

Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT.  

PubMed

The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens. PMID:25227896

Bhubhanil, Sakkarin; Sittipo, Panida; Chaoprasid, Paweena; Nookabkaew, Sumontha; Sukchawalit, Rojana; Mongkolsuk, Skorn

2014-11-01

152

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains.  

PubMed

An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which--although indispensable--was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta. PMID:15876681

Pniewski, Tomasz; Kapusta, Józef

2005-01-01

153

Expression of two insect-resistant genes cryIA (b&c)\\/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids  

Microsoft Academic Search

The synthesizedBacillus thuringiensis insecticidal protein gene cryIA(b&c) and the synthesized geneGNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes ofNicotiana tabacum var. K326 have been transformed withAgrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern\\u000a blot analyses show

Zhibin Wang; Sandui Guo

1999-01-01

154

Roles of Ile66 and Ala107 of D-psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its substrate, D-fructose.  

PubMed

Using site-directed mutagenesis, we investigated the roles of Ile66 and Ala107 of D: -psicose 3-epimerase from Agrobacterium tumefaciens in binding O6 of its true substrate, D: -fructose. When Ile66 was substituted with alanine, glycine, cysteine, leucine, phenylalanine, tryptophan, tyrosine or valine, all the mutants dramatically increased the K (m) for D: -tagatose but slightly decreased the K (m) for D: -fructose, indicating that Ile66 is involved in substrate recognition. When Ala107 was substituted by either isoleucine or valine, the substituted mutants had lower thermostability than the wild-type enzyme whereas the proline-substituted mutant had higher thermostability. Thus, Ala107 is involved in enzyme stability. PMID:19728106

Kim, Hye-Jung; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Dooil; Oh, Deok-Kun

2010-01-01

155

Quantitative Image Analysis and Modeling Indicate the Agrobacterium tumefaciens Type IV Secretion System Is Organized in a Periodic Pattern of Foci  

PubMed Central

The Gram negative plant pathogen Agrobacterium tumefaciens is uniquely capable of genetically transforming eukaryotic host cells during the infection process. DNA and protein substrates are transferred into plant cells via a type IV secretion system (T4SS), which forms large cell-envelope spanning complexes at multiple sites around the bacterial circumference. To gain a detailed understanding of T4SS positioning, the spatial distribution of fluorescently labeled T4SS components was quantitatively assessed to distinguish between random and structured localization processes. Through deconvolution microscopy followed by Fourier analysis and modeling, T4SS foci were found to localize in a non-random periodic pattern. These results indicate that T4SS complexes are dependent on an underlying scaffold or assembly process to obtain an organized distribution suitable for effective delivery of substrates into host cells. PMID:22860087

Cameron, Todd A.; Roper, Marcus; Zambryski, Patricia C.

2012-01-01

156

Characterization of a novel Agrobacterium tumefaciens Galactarolactone Cycloisomerase Enzyme for Direct Conversion of d-Galactarolactone to 3-Deoxy-2-keto-l-threo-hexarate*  

PubMed Central

Microorganisms use different pathways for d-galacturonate catabolism. In the known microbial oxidative pathway, d-galacturonate is oxidized to d-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and ?-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes d-galacturonic acid to d-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the d-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on d-galactarolactone and d-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from d-galactaro-1,4-lactone and d-glucaro-1,4-lactone conversion is in both cases the l-threo form of 3-deoxy-2-keto-hexarate. PMID:22493433

Andberg, Martina; Maaheimo, Hannu; Boer, Harry; Penttila, Merja; Koivula, Anu; Richard, Peter

2012-01-01

157

The Ctp type IVb pilus locus of Agrobacterium tumefaciens directs formation of the common pili and contributes to reversible surface attachment.  

PubMed

Agrobacterium tumefaciens can adhere to plant tissues and abiotic surfaces and forms biofilms. Cell surface appendages called pili play an important role in adhesion and biofilm formation in diverse bacterial systems. The A. tumefaciens C58 genome sequence revealed the presence of the ctpABCDEFGHI genes (cluster of type IV pili; Atu0216 to Atu0224), homologous to tad-type pilus systems from several bacteria, including Aggregatibacter actinomycetemcomitans and Caulobacter crescentus. These systems fall into the type IVb pilus group, which can function in bacterial adhesion. Transmission electron microscopy of A. tumefaciens revealed the presence of filaments, significantly thinner than flagella and often bundled, associated with cell surfaces and shed into the external milieu. In-frame deletion mutations of all of the ctp genes, with the exception of ctpF, resulted in nonpiliated derivatives. Mutations in ctpA (a pilin homologue), ctpB, and ctpG decreased early attachment and biofilm formation. The adherence of the ctpA mutant could be restored by ectopic expression of the paralogous pilA gene. The ?ctpA ?pilA double pilin mutant displayed a diminished biovolume and lower biofilm height than the wild type under flowing conditions. Surprisingly, however, the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants formed normal biofilms and showed enhanced reversible attachment. In-frame deletion of the ctpA pilin gene in the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants caused the same attachment-deficient phenotype as the ctpA single mutant. Collectively, these findings indicate that the ctp locus is involved in pilus assembly and that nonpiliated mutants, which retain the CtpA pilin, are proficient in attachment and adherence. PMID:24914181

Wang, Yi; Haitjema, Charles H; Fuqua, Clay

2014-08-15

158

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry ( Prunus cerasus L.) cultivar Montmorency  

Microsoft Academic Search

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24h

Guo-Qing Song; Kenneth C. Sink

2005-01-01

159

Genetic transformation of flax ( Linum usitatissimum ) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase  

Microsoft Academic Search

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Kmr callus developing on hypocotyl sections. The totipotency of the Kmr callus was dependent upon its origin. T-DNA was visualised

Nazir Basiran; Philip Armitage; Roderick John Scott; John Draper

1987-01-01

160

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.  

PubMed

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro. PMID:16369768

Song, Guo-Qing; Sink, K C

2006-03-01

161

Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells  

PubMed Central

ABSTRACT Type IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain of Agrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in the A. tumefaciens octopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression following vir induction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles. vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiple vir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates. PMID:22027007

Aguilar, Julieta; Cameron, Todd A.; Zupan, John; Zambryski, Patricia

2011-01-01

162

Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants  

PubMed Central

Background Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. Results In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. Conclusion We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues. PMID:20696066

2010-01-01

163

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.  

PubMed

KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying. PMID:23160638

Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui

2013-03-01

164

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices  

Microsoft Academic Search

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted

Alix Pigeaire; Deborah Abernethy; Penelope M. Smith; Kaylene Simpson; Natalie Fletcher; Chin-Yi Lu; Craig A. Atkins; Edwina Cornish

1997-01-01

165

Genetic transformation and regeneration of transgenic plants in grapevine ( Vitis rupestris S.)  

Microsoft Academic Search

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and theß-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl-1) was

L. Martinelli; G. Mandolino

1994-01-01

166

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci  

Microsoft Academic Search

Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to

R. Batchvarova; V. Nikolaeva; S. Slavov; S. Bossolova; V. Valkov; S. Atanassova; S. Guelemerov; A. Atanassov; H. Anzai

1998-01-01

167

A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans.  

PubMed

We evaluated the usefulness and robustness of Agrobacterium tumefaciens-mediated transformation (ATMT) as a high-throughput transformation tool for pathogenicity gene discovery in the filamentous phytopathogen Leptosphaeria maculans. Thermal asymmetric interlaced polymerase chain reaction allowed us to amplify the left border (LB) flanking sequence in 135 of 400 transformants analysed, and indicated a high level of preservation of the T-DNA LB. In addition, T-DNA preferentially integrated as a single copy in gene-rich regions of the fungal genome, with a probable bias towards intergenic and/or regulatory regions. A total of 53 transformants out of 1388 (3.8%) showed reproducible pathogenicity defects when inoculated on cotyledons of Brassica napus, with diverse altered phenotypes. Co-segregation of the altered phenotype with the T-DNA integration was observed for 6 of 12 transformants crossed. If extrapolated to the whole collection, this indicates that 1.9% of the collection actually corresponds to tagged pathogenicity mutants. The preferential insertion into gene-rich regions along with the high ratio of tagged mutants renders ATMT a tool of choice for large-scale gene discovery in L. maculans. PMID:16979359

Blaise, Françoise; Rémy, Estelle; Meyer, Michel; Zhou, Ligang; Narcy, Jean-Paul; Roux, Jacqueline; Balesdent, Marie-Hélène; Rouxel, Thierry

2007-02-01

168

Development of inhibitors against TraR quorum-sensing system in Agrobacterium tumefaciens by molecular modeling of the ligand-receptor interaction.  

PubMed

The quorum sensing (QS) inhibitors that antagonize TraR, a receptor protein for N-3-oxo-octanoyl-L-homoserine lactones (3-oxo-C8-HSL), a QS signal of Agrobacterium tumefaciens were developed. The structural analogues of 3-oxo-C8-HSL were designed by in silico molecular modeling using SYBYL packages, and synthesized by the solid phase organic synthesis (SPOS) method, where the carboxamide bond of 3-oxo-C8-HSL was replaced with a nicotinamide or a sulfonamide bond to make derivatives of N-nicotinyl-L-homoserine lactones or N-sulfonyl-L-homoserine lactones. The in vivo inhibitory activities of these compounds against QS signaling were assayed using reporter systems and compared with the estimated binding energies from the modeling study. This comparison showed fairly good correlation, suggesting that the in silico interpretation of ligand-receptor structures can be a valuable tool for the pre-design of better competitive inhibitors. In addition, these inhibitors also showed anti-biofilm activities against Pseudomonas aeruginosa. PMID:19855933

Kim, Cheoljin; Kim, Jaeeun; Park, Hyung-Yeon; Park, Hee-Jin; Kim, Chan Kyung; Yoon, Jeyong; Lee, Joon-Hee

2009-11-30

169

Synthesis of Methylerythritol Phosphate Analogues and Their Evaluation as Alternate Substrates for IspDF and IspE from Agrobacterium tumefaciens.  

PubMed

The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts d-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. d-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five d-MEP analogues-d-erythritol-4-phosphate (EP), d-3-methylthrietol-4-phosphate (MTP), d-3-ethylerythritol-4-phosphate (EEP), d-1-amino-3-methylerythritol-4-phosphate (NMEP), and d-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes. PMID:25184438

Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E; Lherbet, Christian; Coates, Robert M; Poulter, C Dale

2014-10-01

170

Hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA  

SciTech Connect

A binary-vectory strategy was used to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. Plasmids were constructed (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs were tested in trans with complementary each of regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results the authors suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

Hood, E.E.; Helmer, G.L.; Fraley, R.T.; Chilton, M.D.

1986-12-01

171

Establishment of Agrobacterium tumefaciens-Mediated Transformation of an Oleaginous Fungus, Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding?  

PubMed Central

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5? strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ?3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ?3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain. PMID:19581481

Ando, Akinori; Sumida, Yosuke; Negoro, Hiroaki; Suroto, Dian Anggraini; Ogawa, Jun; Sakuradani, Eiji; Shimizu, Sakayu

2009-01-01

172

Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding.  

PubMed

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5(-) strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for omega3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/10(8) spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal omega3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain. PMID:19581481

Ando, Akinori; Sumida, Yosuke; Negoro, Hiroaki; Suroto, Dian Anggraini; Ogawa, Jun; Sakuradani, Eiji; Shimizu, Sakayu

2009-09-01

173

Evidence for Host Genome Involvement in Cytokinin Metabolism by Male and Female Cells of Mercurialis annua Transformed by Strain 15,955 of Agrobacterium tumefaciens  

PubMed Central

When male and female individuals of a dioecious species Mercurialis annua L. were inoculated with the same strain of Agrobacterium tumefaciens (15,955), the corresponding tumor tissues of each sex clearly differed in their endogenous cytokinin content; only the male tumors had a morphogenetic feminizing effect on male flowers. In male tumor tissues, zeatin (Z) in higher quantity than ribosyl-zeatin (RZ) became the major metabolite in contrast with the general situation for crown-galls; the female tumor tissues were characterized by an increase of total endogenous cytokinins and by the appearance of some specific metabolites such as a methyl-thio-Z and several glycosylated Z derivatives that had not been detected in healthy apices. In both male and female tumor tissues, the cis form of RZ, present in healthy apices as 30% of trans-RZ form, was no longer detectable. Quantitative and qualitative differences characterize male and female tumor tissues (host genes expression) but since differences also appeared between healthy male and female apices and their corresponding tumor tissues (TDNA gene expression), it can be tentatively concluded that a complex interaction between host cytokinin genes and those of TDNA control the endogenous metabolism of tumor tissues. Images Fig. 1 PMID:16663368

Guerin, B.; Kahlem, G.; Teller, G.; Durand, Bernard

1984-01-01

174

Evidence for Host Genome Involvement in Cytokinin Metabolism by Male and Female Cells of Mercurialis annua Transformed by Strain 15,955 of Agrobacterium tumefaciens.  

PubMed

When male and female individuals of a dioecious species Mercurialis annua L. were inoculated with the same strain of Agrobacterium tumefaciens (15,955), the corresponding tumor tissues of each sex clearly differed in their endogenous cytokinin content; only the male tumors had a morphogenetic feminizing effect on male flowers.In male tumor tissues, zeatin (Z) in higher quantity than ribosyl-zeatin (RZ) became the major metabolite in contrast with the general situation for crown-galls; the female tumor tissues were characterized by an increase of total endogenous cytokinins and by the appearance of some specific metabolites such as a methyl-thio-Z and several glycosylated Z derivatives that had not been detected in healthy apices.In both male and female tumor tissues, the cis form of RZ, present in healthy apices as 30% of trans-RZ form, was no longer detectable.Quantitative and qualitative differences characterize male and female tumor tissues (host genes expression) but since differences also appeared between healthy male and female apices and their corresponding tumor tissues (TDNA gene expression), it can be tentatively concluded that a complex interaction between host cytokinin genes and those of TDNA control the endogenous metabolism of tumor tissues. PMID:16663368

Guerin, B; Kahlem, G; Teller, G; Durand, B

1984-01-01

175

Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.  

PubMed

Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS?+?1 mg/l BAP?+?1 mg/l NAA, while indirect regeneration from callus was obtained on MS?+?1 mg/l BAP?+?2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling. PMID:24154495

Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju

2014-05-01

176

The thuEFGKAB Operon of Rhizobia and Agrobacterium tumefaciens Codes for Transport of Trehalose, Maltitol, and Isomers of Sucrose and Their Assimilation through the Formation of Their 3-Keto Derivatives  

PubMed Central

The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism. PMID:23772075

Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Bhuvaneswari, T. V.

2013-01-01

177

The thuEFGKAB operon of rhizobia and agrobacterium tumefaciens codes for transport of trehalose, maltitol, and isomers of sucrose and their assimilation through the formation of their 3-keto derivatives.  

PubMed

The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism. PMID:23772075

Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Jensen, John Beck; Bhuvaneswari, T V

2013-09-01

178

[Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].  

PubMed

The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement. PMID:16755923

Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

2006-05-01

179

The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain.  

PubMed Central

Products of the approximately 9.5-kb virB operon are proposed to direct the export of T-DNA/protein complexes across the Agrobacterium tumefaciens envelope en route to plant cells. The presence of conserved nucleoside triphosphate (NTP)-binding domains in VirB4 and VirB11 suggests that one or both proteins couple energy, via NTP hydrolysis, to T-complex transport. To assess the importance of VirB4 for virulence, a nonpolar virB4 null mutation was introduced into the pTiA6NC plasmid of strain A348. The 2.37-kb virB4 coding sequence was deleted precisely by oligonucleotide-directed mutagenesis in vitro. The resulting delta virB4 mutation was exchanged for the wild-type allele by two sequential recombination events with the counterselectable Bacillus subtilis sacB gene. Two derivatives, A348 delta B4.4 and A348 delta B4.5, sustained a nonpolar deletion of the wild-type virB4 allele, as judged by Southern blot hybridization and immunoblot analyses with antibodies specific for VirB4, VirB5, VirB10, and VirB11. Transcription of wild-type virB4 from the lac promoter restored virulence to the nonpolar null mutants on a variety of dicotyledonous species, establishing virB4 as an essential virulence gene. A substitution of glutamine for Lys-439 and a deletion of Gly-438, Lys-439, and Thr-440 within the glycine-rich NTP-binding domain (Gly-Pro-Iso-Gly-Arg-Gly-Lys-Thr) abolished complementation of A348 delta B4.4 or A348 delta B4.5, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Merodiploids expressing both the mutant and wild-type virB4 alleles exhibited lower virulence than A348, suggesting that VirB4, a cytoplasmic membrane protein, may contribute as a homo- or heteromultimer to A. tumefaciens virulence. Images PMID:8449880

Berger, B R; Christie, P J

1993-01-01

180

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system  

PubMed Central

Background The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ?FaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. Conclusion The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this. PMID:25048842

2014-01-01

181

Agrobacterium-mediated genetic transformation of Prunus salicina .  

E-print Network

??We report Agrobacterium tumefaciens-mediated transformation of two Prunus salicina varieties, ‘Angeleno’ and ‘Larry Anne’, using a modification of the hypocotyl slice technique previously described for… (more)

Urtubia, Carolina

2008-01-01

182

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders.  

PubMed Central

Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids. PMID:1400174

Cook, D M; Farrand, S K

1992-01-01

183

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.  

PubMed Central

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity. Images PMID:2203743

Ward, J E; Dale, E M; Christie, P J; Nester, E W; Binns, A N

1990-01-01

184

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica.  

PubMed

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25+/-2 degrees C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained. PMID:16470412

Wei, Xiaoping; Gou, Xiaoping; Yuan, Tong; Russell, Scott D

2006-06-01

185

ENHANCING THE FREQUENCY OF SOMATIC EMBRYOGENESIS FOLLOWING AGROBACTERIUM-MEDIATED TRANSFORMATION OF  

E-print Network

ENHANCING THE FREQUENCY OF SOMATIC EMBRYOGENESIS FOLLOWING AGROBACTERIUM-MEDIATED TRANSFORMATION embryogenic explants along with the location of embryogenesis- and transformation-competent cells embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection

Korban, Schuyler S.

186

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes.  

PubMed

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position. PMID:16876192

Kim, Kwangsoo; Kim, Hye-Jung; Oh, Deok-Kun; Cha, Sun-Shin; Rhee, Sangkee

2006-09-01

187

Agrobacterium -mediated genetic transformation of a Dendrobium orchid  

Microsoft Academic Search

A protocol was developed to obtain stable transgenic orchids (Dendrobium nobile) via Agrobacterium-mediated transformation of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strains AGL1 and EHA105 were used, with each containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing ß-glucuronidase gene (gus-int) as a reporter gene. PLBs were co-cultivated with A. tumefaciens,

Shuzhen Men; Xiaotian Ming; Rongwei Liu; Chunhong Wei; Yi Li

2003-01-01

188

Agrobacterium -Mediated Transformation of Common Bermudagrass ( Cynodon dactylon )  

Microsoft Academic Search

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. We have been able to transform the species using Agrobacterium-mediated approach. In seven experiments reported here, a total of 67 plates of calluses and suspensions were infected with Agrobacterium tumefaciens strains, and nine hygromycin B resistant calluses were

L. Li; R. Li; S. Fei; R. Qu

2005-01-01

189

Status Perkembangan Perbaikan Sifat Genetik Padi Menggunakan Transformasi Agrobacterium  

Microsoft Academic Search

Development Status of Rice Genetic Improvement using Agrobacterium Transformation. Syamsidah Rahmawati. Genetic transformation of rice becomes an important re- search area in recent years. Rice is staple food for almost half of world population and has been extensively used as a plant model system for monocotyledonous plant. Compare to direct DNA transfer techniques (PEG, electroporation, and DNA bombardment), Agrobacterium tumefaciens-mediated

Syamsidah Rahmawati

190

Tumor DNA Structure in Plant Cells Transformed by A. tumefaciens  

Microsoft Academic Search

Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA)

Patricia Zambryski; Marcelle Holsters; Kelly Kruger; Ann Depicker; Josef Schell; Marc van Montagu; Howard M. Goodman

1980-01-01

191

Susceptibility of dry bean (Phaseolus vulgaris L.) to Agrobacterium infection: Transformation of cotyledonary and hypocotyl tissues  

Microsoft Academic Search

A germinating-seed assay was developed to determine the susceptibility of dry bean (Phaseolus vulgaris L.) to infection by Agrobacterium tumefaciens. Seedlings infected one to three days after germination were more susceptible to A. tumefaciens infection than seedlings germinated for five to seven days and the galls that formed on the one to three day seedlings were significantly larger. Nineteen genotypes

Phillip McClean; Paula Chee; Bruce Held I; Jorge Simental; Roger F. Drong; Jerry Slightom

1991-01-01

192

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA.  

PubMed

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined. PMID:3341112

Donson, J; Gunn, H V; Woolston, C J; Pinner, M S; Boulton, M I; Mullineaux, P M; Davies, J W

1988-01-01

193

Augmentin ® as an alternative antibiotic for growth suppression of Agrobacterium for tomato ( Lycopersicon esculentum ) transformation  

Microsoft Academic Search

Augmentin® was used as an alternative antibiotic for suppressing Agrobacterium tumefaciens during tomato (Lycopersicon esculentum) transformation. The efficiency of Augmentin compared with Timentin® to suppress the growth of Agrobacterium was at concentrations of 300 and 100 mg l-1, respectively. In addition, the high concentration up to 500 mg l-1 of both antibiotics showed no significant toxicity to shoot regeneration. The

S. Ieamkhang; O. Chatchawankanphanich

2005-01-01

194

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method  

E-print Network

transformation is a process of genetic manipulation by which foreign genes are introduced into plant cellsAgrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method Xiuren of various methods for Agrobacterium tumefaciens­mediated transformation of Arabidopsis thaliana. Among these

Franks, Robert

195

Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F. v. semperflorens  

Microsoft Academic Search

By identifying antibiotics that eliminated Agrobacterium tumefaciens with the least phytotoxic effects, we were able to select appropriate A. tumefaciens strains for a more efficient transformation of seasonal Fragaria vesca and everbearing F. v. semperflorens. An efficient and reproducible method of shoot regeneration from leaf discs was developed with optimal shoot regeneration obtained on medium supplemented with 0.25 mg l-1

M. Alsheikh; H.-P. Suso; M. Robson; N. Battey; A. Wetten

2002-01-01

196

Mechanism of the 3-ketolactose test for Agrobacterium  

Microsoft Academic Search

All of Kern's (1966) claims concerning the mechanism and the conditions of the 3-ketolactose test for the specific detection of Agrobacterium tumefaciens and A. radiobacter, were experimentally refuted. Kern's unknown compound, which he holds to be responsible for the test, was identified by us as lactulose, an impurity arising from lactose by heatsterilization; it is not responsible for a positive

M. Bernaerts; J. Ley

1967-01-01

197

Agrobacterium -mediated transformation of Solanum tuberosum L. cv. ‘Russet Burbank’  

Microsoft Academic Search

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. ‘Russet Burbank’. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100

C. A. Newell; R. Rozman; M. A. Hinchee; E. C. Lawson; L. Haley; P. Sanders; W. Kaniewski; N. E. Tumer; R. B. Horsch; R. T. Fraley

1991-01-01

198

A new Agrobacterium strain isolated from aerial tumors on Ficus benjamina L.  

PubMed

Crown gall tumors, collected from branches of 1-year-old weeping fig (Ficus benjamina L.) trees, yielded both tumorigenic and nonpathogenic agrobacteria. On the basis of classical diagnostic tests, the nonpathogenic strains were identified as Agrobacterium tumefaciens, whereas the tumorigenic strains could not be assigned to any of the known terrestrial Agrobacterium spp. The tumorigenic strains also differed from other members of the genus by producing more acid from mannitol. According to cluster analysis of carbon substrate oxidation (GN Microplate; Biolog, Inc.) and fatty acid content, the tumorigenic fig strains were distinct from strains of A. tumefaciens, Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi. Furthermore, they had unusual opine metabolism, inducing tumors that synthesized nopaline and three recently discovered opines: chrysopine (d-lactone of N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-5-oxo-L-proline. The nonpathogenic A. tumefaciens strains present in the same tumors were unable to degrade any of the opines tested. The phylogenetic position of the tumorigenic fig strain AF3.10 was inferred from comparing its rrs (i.e., 16S rRNA gene) sequence with those from the type strains of Agrobacterium and Rhizobium species. The analysis showed that strain AF3.10 clustered with A. tumefaciens and A. rubi but not with A. vitis and was far removed from A. rhizogenes. However, the sequence was significantly different from those of A. tumefaciens and A. rubi to suggest that the tumorigenic fig strain may be a new Agrobacterium species that is as different from A. tumefaciens and A. rubi as these two species are from one another. PMID:7887626

Bouzar, H; Chilton, W S; Nesme, X; Dessaux, Y; Vaudequin, V; Petit, A; Jones, J B; Hodge, N C

1995-01-01

199

Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium -mediated plant transformation  

Microsoft Academic Search

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to

G. Vancanneyt; R. Schmidt; A. O'Connor-Sanchez; L. Willmitzer; M. Rocha-Sosa

1990-01-01

200

Agrobacterium -mediated transformation of cereals — from technique development to its application  

Microsoft Academic Search

Agrobacterium tumefaciens is a very useful vector to transfer foreign genes into dicotyledonous cells. Monocotyledonous, especially cereals, were considered\\u000a outside the host range of the bacteria. The main, alternative technique of transformation developed for them was delivery\\u000a of naked DNA (e.g. microprojectile bombardment, electroporation of protoplasts). The results of Agrobacterium-mediated transformation of cereals accumulated during the last few years confirmed

Anna Nadolska-Orczyk; Wac?aw Orczyk; Anna Przetakiewicz

2000-01-01

201

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium  

Microsoft Academic Search

We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant\\u000a calli. The results of transient GUS (?-glucuronidase) assays suggest that some of these mutants are blocked at an early step\\u000a in the Agrobacterium-mediated transformation process, whereas others are

J. Nam; K. S. Mysore; C. Zheng; M. K. Knue; A. G. Matthysse; S. B. Gelvin

1999-01-01

202

Odyssey of agrobacterium T-DNA.  

PubMed

Agrobacterium tumefaciens, a plant pathogen, is characterized by the unique feature of interkingdom DNA transfer. This soil bacterium is able to transfer a fragment of its DNA, called T-DNA (transferred DNA), to the plant cell where T-DNA is integrated into the plant genome leading to "genetic colonization" of the host. The fate of T-DNA, its processing, transfer and integration, resembles the journey of Odysseus, although our hero returns from its long trip in a slightly modified form. PMID:11833771

Ziemienowicz, A

2001-01-01

203

Shoot regeneration and Agrobacterium -mediated transformation of Fragaria vesca L  

Microsoft Academic Search

An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg\\/l) and indole-3-butyric acid (0.25 mg\\/l) induced the maximum percentage of shoot regeneration (98%) and the highest

Iman Mansouri; José A. Mercado; Victoriano Valpuesta; José M. López-Aranda; Fernando Pliego-Alfaro; Miguel A. Quesada

1996-01-01

204

Agrobacterium -mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration  

Microsoft Academic Search

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and ?-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were\\u000a transformed in the presence of 100 ?M acetosyringone using 90 s sonication plus 10 min

Ningxia Du; Paula M. Pijut

2009-01-01

205

Efficient Agrobacterium-mediated Transformation of Prunus serotina via Sonication and Vacuum-Infiltration  

E-print Network

. tumefaciens. This transformation system will be useful for future genetic modification and overEfficient Agrobacterium-mediated Transformation of Prunus serotina via Sonication and Vacuum-mediated transformation of an elite mature genotype (#3) of black cherry using an RNAi construct for the AGAMOUS (AG) gene

206

Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration  

Microsoft Academic Search

Transgenic pakchoi (Brassica rapa L. ssp. chinensis) plants were obtained in the progeny of plants infiltrated by an Agrobacterium tumefaciens strain carrying a gene for resistance to the herbicide phosphinotricin (Basta). Genetic analysis demonstrates the transmission of the herbicide resistant trait to the progeny. Molecular analyses show that the transgene was inserted in the plant genome and expressed. This work

Cao Ming Qing; Liu Fan; Yao Lei; David Bouchez; Colette Tourneur; Li Yan; Christophe Robaglia

2000-01-01

207

L-glutamine and L-glutamic acid facilitate successful agrobacterium infection of recalcitrant tea cultivars.  

PubMed

The first step in Agrobacterium tumefaciens infection of plants is the establishment of cell-cell contact between the two partners. However, failure to establish such contact makes many plants and explants recalcitrant to A. tumefaciens infection. Tea is one such example where even the popular inducer, acetosyringone failed to facilitate A. tumefaciens infection due to the presence of high amounts of bactericidal/bacteriostatic polyphenols. Quinones are formed as a result of polyphenols oxidation. They cause tissue browning and necrosis during the process of transformation, and in turn prevent A. tumefaciens infection. Compounds such as polyphenol adsorbents, i.e., polyvinylpyrrolidone and charcoal, and antioxidants like cysteine and ascorbic acid were screened to overcome tissue browning. Although these compounds enhanced the growth of A. tumefaciens, these failed to facilitate the infection of the leaves of either Kangra Jat, Tocklai Variety-1, UPASI-9, UPASI-10, and Stock-449 cultivars of tea. On the contrary, the presence of filter sterilized L-glutamine and L-glutamic acid in the co-cultivation medium facilitated successful A. tumefaciens infection of recalcitrant tea leaves. L-Glutamine and glutamic acid form harmless adducts by binding to quinones. Therefore, their presence in the co-cultivation medium allowed the tea leaves to remain living and appealing to the infecting A. tumefaciens. Successful A. tumefaciens infection of tea leaves was confirmed by positive signals in GUS assay, PCR, and Dot blot. PMID:23712792

Kumar, Nitish; Gulati, Ashu; Bhattacharya, Amita

2013-08-01

208

High-frequency transformation of Arabidopsis thaliana by Agrobacterium tumefaciens  

Microsoft Academic Search

Incorporation of 5 mg\\/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation\\u000a protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants\\u000a ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density

Michael C. Clarke; Wenbin Wei; Keith Lindsey

1992-01-01

209

Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens.  

PubMed Central

Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become established only infrequently in recipients with a nopaline Ti plasmid and, vice versa, nopaline Ti plasmids were only rarely established in recipients with an octopine Ti plasmid. Rare clones in which the incoming octopine (nopaline) Ti plasmid had been established despite the presence of a nopaline (octopine) Ti plasmid appeared to harbor cointegrates consisting of the entire incoming Ti plasmid and the entire resident Ti plasmid. The integration event invariably had occurred in a region of the plasmids that is highly conserved in evolution and that is essential for oncogenicity. These results show that octopine and nopaline Ti plasmids cannot be maintained as separate replicons by one and the same cell. Therefore, be definition, these plasmids belong to the same incompatibility group, which has been names inc Rh-1. Agrobacterial non-Ti octopine and nopaline plasmids were found to belong to another incompatibility group. The tumorigenic properties of strains harboring two different Ti plasmids, in a cointegrate structure, were indicative of the virulence genes of both of them being expressed. The agrobacterial non-Ti octopine and nopaline plasmids did not influence the virulence properties encoded by the Ti plasmid. Images PMID:7410319

Hooykaas, P J; den Dulk-Ras, H; Ooms, G; Schilperoort, R A

1980-01-01

210

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

A rapid Agrobacferium fumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. lhe explants were inoculated with a disarmed A. tumefa- ciens strain C58 (ABI) harboring the binary vector pMON18365 containing the p-glucuronidase gene with an intron, and a select- able marker, the neomycin phosphotransferase II gene. Various factors

Ming Cheng; Joyce E. Fry; Shengzhi Pang; Huaping Zhou; Catherine M. Hironaka; David R. Duncan; Timothy W. Conner; Yuechun Wan

1997-01-01

211

Occurrence of Enzymes Involved in Biosynthesis of Indole3Acetic Acid from Indole3Acetonitrile in Plant-Associated Bacteria, Agrobacterium and Rhizobium  

Microsoft Academic Search

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular

Michihiko Kobayashi; Takahisa Suzuki; Takayuki Fujita; Masatoshi Masuda; Sakayu Shimizu

1995-01-01

212

Agrobacterium?-mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB)  

Microsoft Academic Search

A protocol was developed for establishing embryogenic suspension cultures from in vitro-grown, thin shoot-tip sections of\\u000a the banana cultivar Rasthali. The best medium for callus induction was an MS-based medium supplemented with 2?mg\\/l 2,4-D and\\u000a 0.2?mg\\/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were\\u000a subsequently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens

T. R. Ganapathi; N. S. Higgs; P. J. Balint-Kurti; C. J. Arntzen; G. D. May; J. M. Van Eck

2001-01-01

213

Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.  

PubMed

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.). PMID:24243208

Sparks, Caroline A; Doherty, Angela; Jones, Huw D

2014-01-01

214

Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression  

Microsoft Academic Search

Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed

Markus Gorfer; Sylvia Klaubauf; Dragana Bandian; Joseph Strauss

2007-01-01

215

Expression of virB2 protein-containing structures on Agrobacterium in mating cultures.  

PubMed

Exocellular structures containing VirB2 proteins were, for the first time, localized on the surface of Agrobacterium by transmission electron microscopy. Using colloidal gold (CG)-labeled VirB2-specific antibodies, it was shown that VirB2 proteins enter into the composition of short surface pili, which emerge at the poles of acetosyringone (AS)-inducedAgrobacterium cells. However, cells of the Ti plasmidless A. tumefaciens strain UBAPF-2 and cells not induced with AS were incapable of pilus synthesis. In suspension, mating Agrobacterium cells were connected together by short thick bridges. It was found that these bridges did not include as part of their structure CG-labeled VirB1 and VirB2 proteins. We did not find the tetracycline-resistant transconjugants after mating of A. tumefaciens donor cells harboring binary systems with plasmid-free A. tumefaciens GM-I9023 in vir-induced and vir-uninduced conditions. However, the same strains can transfer pSUP106 plasmid via a vir-dependent way. We found that activated vir genes slightly stimulate pTd33 plasmid transfer via a tra-dependent pathway to plasmid-free strain UBAPF-2. It seems, that vir-induced T-DNA/plasmid DNA transfer machinery is not essential for the conjugation process between agrobacterial cells but may participate in this process. PMID:11816972

Kurbanova, I V; Velikov, V A; Chumakov, M I

2001-09-01

216

Genotypic variability of soybean response to agrobacterium strains harboring the ti or ri plasmids.  

PubMed

Twenty four diverse cultivars of soybean (Glycine max [L.] Merrill) and three lines of its annual wild progenitor Glycine soja Sieb and Zucc. were tested for their response to Agrobacterium strains harboring either the Ti (tumor-inducing) plasmid (pTi) from Agrobacterium tumefaciens or the Ri (root-inducing) plasmid (pRi) from Agrobacterium rhizogenes following uniform wounding and inoculation. Based upon gall weight at 8 weeks postinfection, three G. max cultivars (Biloxi, Jupiter, and Peking) and one G. soja line, Plant Introduction (PI) 398.693B, were judged highly susceptible to A. tumefaciens strain A348 (pTiA6), ten genotypes moderately susceptible, 11 weakly susceptible, and two nonsusceptible. Of 26 genotypes inoculated with strain R1000 (pRiA4b), only seven responded in a clearly susceptible fashion by forming small, fleshy roots at internodal infection sites. Cotyledons excised from 1- or 3-day old seedlings of Peking and Biloxi cultivars also formed galls when infected in vitro with agrobacteria carrying either the Ti or Ri plasmid. Tumor lines established from cotyledon and stem galls induced by A. tumefaciens A348 (pTiA6) exhibited the T-DNA borne traits of phytohormone-independent growth and octopine synthesis. Additionally, DNA isolated from cultured tumors hybridized with labeled T-DNA probe. PMID:16664035

Owens, L D; Cress, D E

1985-01-01

217

Investigating Agrobacterium-Mediated Transformation of Verticillium albo-atrum on Plant Surfaces  

PubMed Central

Background Agrobacterium tumefaciens has long been known to transform plant tissue in nature as part of its infection process. This natural mechanism has been utilised over the last few decades in laboratories world wide to genetically manipulate many species of plants. More recently this technology has been successfully applied to non-plant organisms in the laboratory, including fungi, where the plant wound hormone acetosyringone, an inducer of transformation, is supplied exogenously. In the natural environment it is possible that Agrobacterium and fungi may encounter each other at plant wound sites, where acetosyringone would be present, raising the possibility of natural gene transfer from bacterium to fungus. Methodology/Principal Findings We investigate this hypothesis through the development of experiments designed to replicate such a situation at a plant wound site. A. tumefaciens harbouring the plasmid pCAMDsRed was co-cultivated with the common plant pathogenic fungus Verticillium albo-atrum on a range of wounded plant tissues. Fungal transformants were obtained from co-cultivation on a range of plant tissue types, demonstrating that plant tissue provides sufficient vir gene inducers to allow A. tumefaciens to transform fungi in planta. Conclusions/Significance This work raises interesting questions about whether A. tumefaciens may be able to transform organisms other than plants in nature, or indeed should be considered during GM risk assessments, with further investigations required to determine whether this phenomenon has already occurred in nature. PMID:21060684

Knight, Claire J.; Bailey, Andy M.; Foster, Gary D.

2010-01-01

218

The promoter of T L DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector  

Microsoft Academic Search

A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also

Csaba Koncz; Jeff Schell

1986-01-01

219

Agrobacterium-mediated transformation of meadow fescue (Festuca pratensis Huds.).  

PubMed

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and beta-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. PMID:19603171

Gao, Caixia; Liu, Jinxing; Nielsen, Klaus Kristian

2009-09-01

220

Temperature effects on Agrobacterium phytochrome Agp1.  

PubMed

Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25 °C to 35 °C; at 40 °C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40 °C was nearly completely restored when the temperature returned to 25 °C. UV/visible spectroscopy indicated that the protein was not denatured up to 45 °C. At 50 °C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20-45 °C, whereas irradiated samples exhibited a clear temperature effect in the 30-40 °C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40 °C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens. PMID:22043299

Njimona, Ibrahim; Lamparter, Tilman

2011-01-01

221

Agrobacterium-Mediated Transformation of Pineapple (Ananas comosus L. Merr.) Leaf Bases with MSI99, a Magainin Analogue  

Microsoft Academic Search

Leaf bases excised from in vitro derived shoots of pineapple (Ananas comosus LMerr. cv. Queen) were transformed with Agrobacterium tumefaciens strain EHA 105 harboring the pMSI168 plasmid with MSI-99, a magainin analogue, by co-cultivating for three days in the dark on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962). The leaf bases produced callus and\\/or multiple shoots upon

Minal Mhatre; L. Nagi; T. R. Ganapathi

2009-01-01

222

Agrobacterium -mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants  

Microsoft Academic Search

Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them

Bruno Delbreil; Philippe Guerche; M. Jullien

1993-01-01

223

In planta transformation method for T-DNA transfer in orchids  

NASA Astrophysics Data System (ADS)

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro?PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1?2 pro?GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko

2014-03-01

224

Agrobacterium and Plant Biotechnology  

Microsoft Academic Search

\\u000a Agrobacterium-mediated transformation has revolutionized agriculture as well as basic research in plant molecular biology, by enabling\\u000a the genetic modification of a wide variety of plant species. Advances in binary vector design and selection strategies, coupled\\u000a with improvements in regeneration technology and gene delivery mechanisms, have dramatically extended the range of organisms,\\u000a including grains, that can be transformed. Recent innovations have

Lois M. Banta; Maywa Montenegro

225

2003-2004). Efficient genetic transformation of Lotus corniculatus L. and growth of transformed plants in the  

E-print Network

Abstract — Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capable of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1) over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42%) plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by ?-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

Radomirka Nikoli?; Nevena Miti?; Slavica Ninkovi?; Mirjana Neškovi?

226

[Subcellular localization and resistance to Gibberella fujikuroi of AtELHYPRP2 in transgenic tobacco].  

PubMed

The subcellular localization and the resistance to fungal pathogen Gibberella fujikuroi of the protein encoded by Arabidopsis AtELHYPRP2 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2, AT4G12500) were investigated using transgenic tobacco plants. The coding sequence of AtELHYPRP2 was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pCAMBIA1302 to produce a fusion expression vector, pCAMBIA1302-AtELHYPRP2-GFP. Then the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated and selected via leaf disc transformation method. RT-PCR and Western blotting analyses showed that AtELHYPRP2 expressed effectively in transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from propidium iodide staining, indicating AtELHYPRP2 is localized to cell surface. Antimicrobial experiments exhibited that the constitutive expression of AtELHYPRP2 could enhance the resistance of tobacco to fungal pathogen G. fujikuroi and the infection sites could accumulate H2O2 obviously. The basal expression levels of PR1 and the systemic expression levels of PR1 and PR5 in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2 may play a role in systemic acquired resistance. PMID:25007583

Chai, Qiuxia; Li, Benchang; Xu, Ziqin

2014-03-01

227

Fate of Agrobacterium radiobacter K84 in the environment.  

PubMed Central

Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84. To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used. Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay. K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years. K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants. Populations of K84 declined substantially in fallow soil or water over a 16-week period. K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil. In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen. These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue. PMID:8357247

Stockwell, V O; Moore, L W; Loper, J E

1993-01-01

228

pSa Causes Oncogenic Suppression of Agrobacterium by Inhibiting VirE2 Protein Export  

PubMed Central

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable ?-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010. PMID:9864329

Lee, Lan-Ying; Gelvin, Stanton B.; Kado, Clarence I.

1999-01-01

229

Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor  

Microsoft Academic Search

Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium aniso- pliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence

Yong-Sun Moon; Bruno G. G. Donzelli; Stuart B. Krasnoff; Heather McLane; Mike H. Griggs; Peter Cooke; John D. Vandenberg; Donna M. Gibson; Alice C. L. Churchill

2008-01-01

230

Agrobacterium infection and plant defense--transformation success hangs by a thread  

PubMed Central

The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to “yes” or “no.” This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner. PMID:24391655

Pitzschke, Andrea

2013-01-01

231

Agrobacterium infection and plant defense-transformation success hangs by a thread.  

PubMed

The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to "yes" or "no." This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner. PMID:24391655

Pitzschke, Andrea

2013-01-01

232

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR.  

PubMed

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples. PMID:23578959

Shams, M; Vial, L; Chapulliot, D; Nesme, X; Lavire, C

2013-07-01

233

Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)  

PubMed Central

Background Molecular farming is the production of important recombinant proteins in transgenic organisms on an agricultural scale. Interferons are proteins with antiviral and antitumor activities and can be used for viral infections and cancers treatments. Objectives This study reports the transformation of INF ?2b gene in tobacco plant for the first time in Iran. Materials and Methods Interferon ?2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINF?. pCAMINF? was transferred to E. coli strain DH5? and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon ?2b gene was confirmed by dot blotting. Conclusions Since no report of interferon alpha production in plants in Iran has been expressed yet, this research could create a field of producing this drug in tobacco, in Iran. PMID:24624166

Ahangarzadeh, Shahrzad; Daneshvar, Mohammad Hosein; Rajabi-Memari, Hamid; Galehdari, Hamid; Alamisaied, Khalil

2012-01-01

234

Transgenic pearl millet male fertility restorer line (ICMP451) and hybrid (ICMH451) expressing Brassica juncea Nonexpressor of pathogenesis related genes 1 (BjNPR1) exhibit resistance to downy mildew disease.  

PubMed

Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids. PMID:24603762

Ramineni, Ramadevi; Sadumpati, Vijayakumar; Khareedu, Venkateswara Rao; Vudem, Dashavantha Reddy

2014-01-01

235

Transgenic Pearl Millet Male Fertility Restorer Line (ICMP451) and Hybrid (ICMH451) Expressing Brassica juncea Nonexpressor of Pathogenesis Related Genes 1 (BjNPR1) Exhibit Resistance to Downy Mildew Disease  

PubMed Central

Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids. PMID:24603762

Khareedu, Venkateswara Rao; Vudem, Dashavantha Reddy

2014-01-01

236

Ecological dynamics and complex interactions of Agrobacterium megaplasmids  

PubMed Central

As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together.

Platt, Thomas G.; Morton, Elise R.; Barton, Ian S.; Bever, James D.; Fuqua, Clay

2014-01-01

237

The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].  

PubMed

An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167

Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

2003-08-01

238

Cloning, Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill.)  

PubMed Central

Background: Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used. Objectives: This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran. Materials and Methods: This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay. Results: The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay. Conclusions: This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants. PMID:24644433

Soltanmohammadi, Behnoush; Jalali-Javaran, Mokhtar; Rajabi-Memari, Hamid; Mohebodini, Mehdi

2014-01-01

239

Agrobacterium ParA\\/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions  

Microsoft Academic Search

Agrobacterium tumefaciens translocates T-DNA through a polar VirB\\/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise

Krishnamohan Atmakuri; Eric Cascales; Oliver T Burton; Lois M Banta; Peter J Christie

2007-01-01

240

Identification of Genomic Species in Agrobacterium Biovar 1 by AFLP Genomic Markers?  

PubMed Central

Biovar 1 of the genus Agrobacterium consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as Agrobacterium ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of Agrobacterium tumefaciens), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely “species-specific” markers tagging the core genomes of the species. PMID:16936063

Portier, Perrine; Fischer-Le Saux, Marion; Mougel, Christophe; Lerondelle, Catherine; Chapulliot, David; Thioulouse, Jean; Nesme, Xavier

2006-01-01

241

Horizontal gene transfer from genus agrobacterium to the plant linaria in nature.  

PubMed

Genes can be transferred horizontally between prokaryotes and eukaryotes in nature. The best-studied examples occur between Agrobacterium rhizogenes and certain Nicotiana spp. To investigate possible additional cases of horizontal gene transfer in nature between Agrobacterium and plants, a real-time polymerase chain reaction-based approach was employed to screen 127 plant species, belonging to 38 families of Dicotyledones, for the presence of oncogenes homologous to the transfer DNA fragments (T-DNA) from both A. tumefaciens and A. rhizogenes. Among all of the analyzed plant species, we found that only Linaria vulgaris contained sequences homologous to the T-DNA of A. rhizogenes. All screened L. vulgaris plants from various parts of Russia contained the same homologous sequences, including rolB, rolC, ORF13, ORF14, and mis genes. The same opine gene is found in the species of Nicotiana which contain genes of A. rhizogenes. In L. vulgaris, there are two copies of T-DNA organized as a single tandem imperfect direct repeat. The plant DNA sequence of the site of integration shows similarity to a retrotransposon. This site is most likely silent, suggesting that the T-DNA is not expressed. Attempts to demonstrate expression of the T-DNA genes were negative. Our study indicates that the frequency of gene transfer and fixation in the germline from Agrobacterium to plant hosts is rare in the natural environment. PMID:23134518

Matveeva, Tatiana V; Bogomaz, Denis I; Pavlova, Olga A; Nester, Eugene W; Lutova, Ludmila A

2012-12-01

242

from E. coli and the 3 terminator re-gion from Agrobacterium tumefaciens  

E-print Network

.M. and from the European Community (ERBIC19- CT-960089) and FAPESP (94/033581-1) to P.A. M.M., L.G.M. and N was diluted 1000-fold, we simplified our protocol: we added the bacterial colony directly to the 10 µ effect was observed at a 0.5� concentration. Finally, by decreasing the reaction volume to 10 µ

Bardwell, James

243

Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones  

PubMed Central

Temporal and spacial distribution of mannopine synthase (mas) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase luxA and luxB as reporter genes. Luciferase activity was determined by luminometry in vitro and visualized by computer-enhanced single-photon video imaging in vivo. The activity of the mas dual promoters increased basipetally in developing plants and was wound-inducible in leaf and stem tissue. Hormone bioassays with isolated plant tissues and tumors deficient in the transferred DNA (T-DNA)-encoded genes iaaM, iaaH, and ipt indicated that activity of the mas dual promoters is regulated by auxin and enhanced by cytokinin in both differentiated and tumorous plant cells. Images PMID:16594033

Langridge, W. H. R.; Fitzgerald, K. J.; Koncz, C.; Schell, J.; Szalay, A. A.

1989-01-01

244

CELL BIOLOGY & MOLECULAR GENETICS Inheritance of Agrobacterium tumefaciens-Induced Tumorigenesis of Soybean  

E-print Network

and Kozma, 1984) and pea, Pisum sativum (L.) (Robbs et al., 1991). Soybeanwasinitially considered(Maturity GroupII), Thomasfor its adaptation to the southern USA.(Maturity GroupVII), and 'Masshokutou502'(P

Parrott, Wayne

245

Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones  

Microsoft Academic Search

Temporal and spacial distribution of man- nopine synthase (mar) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase luxA and luxB as reporter genes. Luciferase activity was determined by luminometry in vifm and visualized by computer-enhanced single-photon video imaging in vivo. The activity of the mas dual promoters increased basipetally in developing plants and was

246

Effect of plant genotype on the transformation of cultivated alfalfa ( Medicago sativa ) by Agrobacterium tumefaciens  

Microsoft Academic Search

The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were

Sarah Du; Larry Erickson; Steve Bowley

1994-01-01

247

Leaf disc transformation of cultivated tomato ( L. esculentum ) using Agrobacterium tumefaciens  

Microsoft Academic Search

The leaf disc transformation\\/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon\\/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios.

Sheila McCormick; Jeanne Niedermeyer; Joyce Fry; Arlene Barnason; Robert Horsch; Robert Fraley

1986-01-01

248

Agrobacterium tumefaciens -mediated genetic transformation of the white root rot ascomycete Rosellinia necatrix  

Microsoft Academic Search

Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker

Tadanori Aimi; Hiroyuki Taguchi; Yoshikazu Tanaka; Yutaka Kitamoto; Tsutomu Morinaga

2005-01-01

249

Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea  

E-print Network

. The pathogenic nature of the organism was confirmed by a bioassay on carrot disks. The MU-hydrolyzing enzyme Rhizobium radiobacter) à l'aide de caractérisations génotypiques et phénotypiques. La nature pathogène de l) held in the MU polymer has to be hydrolyzed and converted into ammonium ions in the soil. Benefits from

Hammock, Bruce D.

250

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.  

PubMed

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

251

Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.  

PubMed

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T-DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1-10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3-3.1 ?m sec?¹ in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

2014-02-01

252

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement  

PubMed Central

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop. PMID:25309562

Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

2014-01-01

253

Early Events in Agrobacterium?mediated Genetic Transformation of Citrus Explants  

PubMed Central

• Background and Aims Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so?called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co?cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co?cultivation medium as possible enhancers of gene transfer was also studied. • Methods A highly responsive citrus genotype and well?established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co?cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. • Key Results It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium?inoculated and non?inoculated explants. Furthermore, co? cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S?phase, the stage in which cells are more prone to integrate foreign DNA. • Conclusions Use of proper co?cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants. PMID:15145796

PEÑA, LEANDRO; PÉREZ, ROSA M.; CERVERA, MAGDALENA; JUÁREZ, JOSÉ A.; NAVARRO, LUIS

2004-01-01

254

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility  

PubMed Central

Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

2014-01-01

255

Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.).  

PubMed

An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 ?M acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days). PMID:23852797

Subramanyam, Kondeti; Rajesh, Manoharan; Jaganath, Balusamy; Vasuki, Amirthalingam; Theboral, Jeevaraj; Elayaraja, Dhandapani; Karthik, Sivabalan; Manickavasagam, Markandan; Ganapathi, Andy

2013-09-01

256

Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment  

PubMed Central

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30?mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280?psi of helium pressure at 6 to 8?cm distance. PMID:25295258

Ibrahim, Evra Raunie

2014-01-01

257

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.  

PubMed

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. PMID:24612605

Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

2014-01-01

258

Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration.  

PubMed

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 microM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l(-1) was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 microM 6-benzylaminopurine, 4.5 microM thidiazuron, 50 mg l(-1) adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. PMID:19343350

Du, Ningxia; Pijut, Paula M

2009-06-01

259

Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata.  

PubMed

Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l(-1) 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l(-1) ?-naphthalene acetic acid and 6.0 mg l(-1) 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105-harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter-was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene. PMID:23873641

Ma, Kai; Hu, Chun Gen; Xu, Bing; Yao, Jia Ling

2013-09-01

260

Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-  

E-print Network

-assisted Agrobacterium- mediated transformation-treated soybean cotyledons K.R. Finer1 and J.J. Finer2 1 Department expressing green £uorescent protein (GFP). GFP provided a quick, non-destructive method to evaluate, in real time, Agrobacterium colonization of cotyledon surfaces as well as infection of internal cells. GFP

Finer, John J.

261

Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos  

PubMed Central

The tree-legume Leucaena leucocephala (leucaena) is used as a perennial fodder because of its fast-growing foliage, which is high in protein content. The use of leucaena as a fodder is however restricted due to the presence of the toxin mimosine. Improvements in the nutritional contents as well as other agronomic traits of leucaena can be accomplished through genetic transformation. The objective of this research was to develop a transformation protocol for leucaena using phosphinothricin resistance as the plant selectable marker. Explants obtained from immature zygotic embryos infected with the Agrobacterium tumefaciens strain C58C1 containing the binary plasmid pCAMBIA3201 produced four putative transformed leucaena plants. Transformation was con- firmed by PCR, RT-PCR, Southern blot, Western analyses, GUS-specific enzyme activity and herbicide leaf spraying assay. A transformation efficiency of 2% was established using this protocol. PMID:20041041

Jube, Sandro

2009-01-01

262

Agrobacterium T-DNA integration: molecules and models  

E-print Network

. Whereas extensive work has revealed the biological mechanisms governing the production, Agrobacterium-to-plant-kingdom DNA transfer [1]. Although used mainly for plant genetic engineering [2], Agrobacterium can transform the subject of numerous studies over the past several decades [8­12]. In nature, Agrobacterium causes crown-gall

Citovsky, Vitaly

263

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology  

E-print Network

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology Tzvi Tzfira1 and Vitaly Citovsky2 Agrobacterium-mediated genetic transformation is the dominant technology used.copbio.2006.01.009 Introduction Agrobacterium genetically transforms its host by transfer- ring a well

Citovsky, Vitaly

264

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays  

PubMed Central

Background Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain ‘normal’ sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or ‘stroma-filled-tubules’ emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes. Results Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation. Conclusion Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as ‘normal’ as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy. PMID:24886417

2014-01-01

265

An in vivo, luciferase-based, Agrobacterium-infiltration assay system: implications for post-transcriptional gene silencing.  

PubMed

An in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with Agrobacterium tumefaciens is described. The system makes use of A. tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 1.13.12.5) gene. Single or mixed Agrobacterium lines were infiltrated into leaf tissues (Nicotiana tabacum or Nicotiana benthamiana) through stomatal openings and leaf disks from infused areas floated on reaction buffers specific to each enzyme. Photons emitted were then measured to determine reporter gene activity. Parameters affecting assay reliability and sensitivity were tested, including: buffer composition; bacterial density; infusion location; reaction kinetics; and environmental factors (light and temperature). The resulting in vivo assay system generates results comparable to those obtained using a commercially available in vitro dual-luciferase(R) reporter gene assay, and reports relative expression levels, as well as induction characteristics, analogous to those obtained using leaf tissue from stably transformed plants harboring the same promoter::gene constructs. Light and temperature were observed to markedly impact transient reporter activities. Co-expression of viral suppressors of post-transcriptional gene silencing (PTGS), HcPro, p19 and AC2, confirms the occurrence of PTGS within infused zones, and provides a convenient mechanism for PTGS analysis. The in vivo transient assay was used to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS. PMID:16523348

Cazzonelli, Christopher Ian; Velten, Jeff

2006-08-01

266

Elevated Temperature Differentially Affects Virulence, VirB Protein Accumulation, and T-Pilus Formation in Different Agrobacterium tumefaciens and Agrobacterium vitis Strains  

Microsoft Academic Search

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly

CHRISTIAN BARON; NATALIE DOMKE; MICHAEL BEINHOFER; SIEGFRIED HAPFELMEIER

2001-01-01

267

Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.  

PubMed

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)-CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856

Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

2014-06-01

268

Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.  

PubMed

Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue. PMID:24472037

Bilichak, Andriy; Yao, Youli; Kovalchuk, Igor

2014-06-01

269

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants  

NASA Technical Reports Server (NTRS)

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

Cardoza, V.; Stewart, C. N.

2003-01-01

270

Osa Protein Constitutes a Strong Oncogenic Suppression System That Can Block vir-Dependent Transfer of IncQ Plasmids between Agrobacterium Cells and the Establishment of IncQ Plasmids in Plant Cells  

PubMed Central

The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens. osa confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems. Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms. PMID:15489437

Lee, Lan-Ying; Gelvin, Stanton B.

2004-01-01

271

Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore  

PubMed Central

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound. PMID:11157228

Penyalver, Ramon; Oger, Philippe; Lopez, Maria M.; Farrand, Stephen K.

2001-01-01

272

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.  

PubMed

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and ?-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

273

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host  

PubMed Central

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and ?-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Morera, Solange; Faure, Denis

2014-01-01

274

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.  

PubMed

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3) )]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a ?-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method. PMID:24974933

Zhang, Fuli; Chen, Can; Ge, Honglian; Liu, Jinmei; Luo, Yunling; Liu, Kun; Chen, Long; Xu, Kedong; Zhang, Yi; Tan, Guangxuan; Li, Chengwei

2014-09-01

275

High throughput Agrobacterium-mediated switchgrass transformation  

Microsoft Academic Search

Switchgrass is one of the most important biomass\\/bioenergy crops. For its improvement as a feedstock through biotechnological approach, we have developed a high throughput Agrobacterium-mediated transformation system for cv. Alamo and two new elite cultivars, Performer and Colony. Highly regenerable and transformation-competent embryogenic calli were identified and used for genetic transformation. GFP reporter gene was employed to identify transformation events

Ruyu Li; Rongda Qu

2011-01-01

276

Genetic transformation of HeLa cells by Agrobacterium  

E-print Network

Genetic transformation of HeLa cells by Agrobacterium Talya Kunik* , Tzvi Tzfira*, Yoram Kapulnik attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells it into their genome. Genetic transformation of plant cells by Agrobacterium tu- mefaciens is the only known natural

Citovsky, Vitaly

277

Marine star-shaped-aggregate-forming bacteria: Agrobacterium atlanticum sp. nov.; Agrobacterium meteori sp. nov.; Agrobacterium ferrugineum sp. nov., nom. rev.; Agrobacterium gelatinovorum sp. nov., nom. rev.; and Agrobacterium stellulatum sp. nov., nom. rev.  

PubMed

Two new species of aerobic, gram-negative, peritrichously flagellated or nonmotile marine bacteria usually forming star-shaped aggregates were isolated from northeastern Atlantic Ocean bottom sediments. These organisms resembled eight star-shaped-aggregate-forming bacterial species from the Baltic Sea originally ascribed to the genus Agrobacterium but not included on the Approved Lists of Bacterial Names because of their questionable relationships to true agrobacteria. These two sets of star-shaped-aggregate-forming bacteria were compared by means of phenotypic data, DNA base compositions, DNA-DNA relatedness, and one-dimensional electrophoretic analysis of low-molecular-weight RNAs (5S rRNA and tRNA). According to the results of genotyping, the northeastern Atlantic Ocean isolates and three of the Baltic Sea species formed a group of closely related bacteria that could not be excluded from the genus Agrobacterium with certainty. Until more genotypic data are available, these five marine species are regarded as a distinct subdivision of the genus Agrobacterium consisting of Agrobacterium atlanticum sp. nov. (type strain, 1480T = DSM 5823T), A. meteori sp. nov. (type strain, 1513T = DSM 5824T), A. ferrugineum sp. nov. nom. rev. emend. (type strain, ATCC 25652T), A. gelatinovorum sp. nov. nom. rev. emend. (type strain, ATCC 25655T), and A. stellulatum sp. nov. nom. rev. emend. (type strain, ATCC 15215T). "A. aggregatum" proved to be a later subjective synonym of A. stellulatum, which had priority. The remaining four Baltic Sea species, "A. agile," "A. kieliense," "A. luteum," and "A. sanguineum," could not be placed in the new subdivision of Agrobacterium. PMID:1371058

Rüger, H J; Höfle, M G

1992-01-01

278

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model.  

PubMed

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B; Bailey-Serres, Julia; Brady, Siobhan M

2014-10-01

279

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens  

PubMed Central

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272

Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S.; Mirza, Bushra

2014-01-01

280

Horizontal gene transfer from Agrobacterium to plants  

PubMed Central

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

Matveeva, Tatiana V.; Lutova, Ludmila A.

2014-01-01

281

Horizontal gene transfer from Agrobacterium to plants.  

PubMed

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

Matveeva, Tatiana V; Lutova, Ludmila A

2014-01-01

282

Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.  

PubMed

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 ?M of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction. PMID:23931121

Rodrigues, Maria Beatriz Calderan; Fávaro, Léia Cecília de Lima; Pallu, Ana Paula de Souza; Ferreira, Anderson; Sebastianes, Fernanda de Souza; Rodrigues, Maria Juliana Calderan; Spósito, Marcel Bellato; de Araújo, Welington Luiz; Pizzirani-Kleiner, Aline Aparecida

2013-01-01

283

High efficiency transformation of Brassica napus using Agrobacterium vectors  

Microsoft Academic Search

An efficient procedure for obtaining transgenicBrassica napus plants usingAgrobacterium binary vectors is described. The target tissue for the transformation is the cut end of cotyledonary petioles. These tissues, when cultured with their lamina intact, show a regeneration frequency of more than 80%. The cells of this cut surface, which undergo organogenesis, are very susceptible to topical infection byAgrobacterium. The cocultivation

Maurice M. Moloney; Janis M. Walker; Kiran K. Sharma

1989-01-01

284

Factors influencing successful Agrobacterium -mediated genetic transformation of wheat  

Microsoft Academic Search

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified

H. Wu; C. Sparks; B. Amoah; H. D. Jones

2003-01-01

285

[Expression of two plant agglutinin genes in transgenic tobacco plants].  

PubMed

A plant expression vector pBACG containing the DNA sequence coding for Amaranthus caudatus agglutinin (ACA) and a modified Glanthus nivalis agglutinin (GNA) gene was constructed. Leaf explants of Nicotiana tobacum cv. SRI were transformed with A. tumefaciens LBA4404 harbouring the above expression vector. Results from PCR and Southern blotting analysis showed that both the ACA and GNA gene were inserted into the genome of transformed tobacco plants. Western blottingting analysis of soluble protein isolated from transgenic plants showed that ACA and GNA were synthesized. The results from insect bioassay with peach aphids ( Myzus persicae) revealed that the transgenic plants of pBACG had acquired high resistance against peach aphids. The average aphid-inhibition rate reached up to 83.9% and 85.3% for transgenic plants (T0) and their selfed progenies (T1) respectively,indicating that the functions of these two genes were inheritable. PMID:16078746

Liu, Zhao-Hua; Zhang, Zhen-Shan; Guo, Hong-Nian; Jia, Yan-Tao; Zheng, Guang-Yu; Tian, Ying-Chuan

2005-07-01

286

The Genome of the Natural Genetic Engineer Agrobacterium  

E-print Network

agent of the plant disease crown gall (1), A. tumefaciens infects more than 90 families of dicotyledonous plants, resulting in major agronomic losses (2, 3). The gall results from the transfer,12 Maynard V. Olson,5 Eugene W. Nester1,13 § The 5.67-megabase genome of the plant pathogen

Liao, Li

287

EMBO Member's Review New insights into an old story: Agrobacterium-  

E-print Network

and integrated into the host chromosomal DNA--resulting in genetic manipulation of the host. The expression of T tumefaciens as a vector for genetic manipulation. Engineered DNA segments of interest, which are first cloned in plants by plant transformation Andrea Pitzschke1 and Heribert Hirt2,3, * 1 Department of Applied Genetics

Hirt, Heribert

288

Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09.  

PubMed

Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. ZX09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreoscilla hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide (CO) difference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physiological changes included its elevated respiration rate and cellular invertase activity. PMID:25367790

Chen, Yun-Mei; Xu, Hai-Yang; Wang, Yang; Zhang, Jian-Fa; Wang, Shi-Ming

2014-11-01

289

Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09*  

PubMed Central

Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. ZX09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreoscilla hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide (CO) difference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physiological changes included its elevated respiration rate and cellular invertase activity. PMID:25367790

Chen, Yun-mei; Xu, Hai-yang; Wang, Yang; Zhang, Jian-fa; Wang, Shi-ming

2014-01-01

290

A plant-derived edible vaccine against hepatitis B virus.  

PubMed

The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein. PMID:10506582

Kapusta, J; Modelska, A; Figlerowicz, M; Pniewski, T; Letellier, M; Lisowa, O; Yusibov, V; Koprowski, H; Plucienniczak, A; Legocki, A B

1999-10-01

291

ORIGINAL PAPER Agrobacterium-mediated genetic transformation and plant  

E-print Network

ORIGINAL PAPER Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood transcription-PCR. This transformation proto- col provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB. Keywords Fraxinus Á Genetic transformation Á Pumpkin

292

Three-dimensional Reconstruction of Agrobacterium VirE2 Protein with Single-stranded DNA*  

E-print Network

tumefaciens is the causative agent of the crown gall disease in many plant species (1, 2). To attack its host tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single proliferation and to gall formation. Within this tumor-like tissue, genes are expressed for enzymes that govern

Citovsky, Vitaly

293

Direct shoot organogenesis on hypocotyl explants with collar region from in vitro seedlings of Coffea canephora Pierre ex. Frohner cv. C × R and Agrobacterium tumefaciens -mediated transformation  

Microsoft Academic Search

Direct differentiation of shoot buds from the collar region of hypocotyl segments of Coffea canephora was obtained on Murashige and Skoog (MS) medium supplemented with 40 ?M silver nitrate (AgNO3) and growth regulators indole-3-acetic acid (IAA) and N6 benzyladenine (BA). The highest response to shoot differentiation\\u000a of 60% frequency and the maximum number of multiple shoots (2–3) per explant were obtained

V. Sridevi; P. Giridhar; P. S. Simmi; G. A. Ravishankar

2010-01-01

294

Response of chickpea genotypes to Agrobacterium-mediated delivery of Chickpea chlorotic dwarf virus (CpCDV) genome and identification of resistance source.  

PubMed

Chickpea stunt disease caused by Chickpea chlorotic dwarf virus (CpCDV) (genus Mastrevirus, family Geminiviridae) is the most important biotic stress affecting chickpea crops worldwide. A survey conducted on the incidence of stunt disease clearly revealed high incidence of the disease with severe symptom expression in both indigenous and imported genotypes. To manage the disease in a sustainable way, resistant genotypes need to be bred by adopting objective and precise assessment of the disease response of chickpea genotypes. At present, evaluation of CpCDV resistance is conducted on the basis of natural infection in the field, which is bound to be erroneous due to vagaries in vector population. To circumvent the above problems, we devised an agroinoculation technique that involves the delivery of viral genomic DNA through Agrobacterium tumefaciens. An objective scoring system assigning quantitative value to different symptoms has been evolved to assess the response of chickpea genotypes to CpCDV inoculation. Using the inoculation and scoring techniques, we screened 70 genotypes, which helped in differentiating field resistance that is more due to resistance to vector feeding than resistance to the virus. PMID:23955474

Kanakala, S; Verma, H N; Vijay, P; Saxena, D R; Malathi, V G

2013-11-01

295

Properties of the hydantoinase from agrobacterium SP. IP I-671  

Microsoft Academic Search

Summary A hydantoin-hydrolyzing enzyme has been purified from an newly isolatedAgrobacterium sp. by procedures including ammonium sulfate fractionation and ion-exchange chromatography. Kinetic studies have demonstrated that this enzyme, which is strictlyd-selective and has a broad substrate specificity exhibits remarkable stability. Microbial bioconversion at 60°C and pH 10.0, allowed complete conversion of 30 g\\/L ofd,l 5-benzylhydantoin into thedN-carbamyl derivative of phenylalanine

Serge Runser; Eric Ohleyer

1990-01-01

296

Dendrobium orchids contain an inducer of Agrobacterium virulence genes  

Microsoft Academic Search

SuccessfulAgrobacterium-mediated transformation of monocots such as orchids hinges on the induction of virulence(vir)genes in the bacterium, an event that is required for the activation of the T-DNA processing and transfer pathway. In dicots, several plant phenolic compounds, for example acetosyringone, coumaryl alcohol and sinapyl alcohol, are knownvirgene inducers, but in monocots their presence and characteristics are not well established. We

G.-L. Nan; C.-S. Tang; A. R. Kuehnle; C. I. Kado

1997-01-01

297

Agrobacterium mediated transformation of gypsophila ( Gypsophila paniculata L.)  

Microsoft Academic Search

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility\\u000a of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment\\u000a of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium

Michal Moyal Ben Zvi; Amir Zuker; Marianna Ovadis; Elena Shklarman; Hagit Ben-Meir; Shamir Zenvirt; Alexander Vainstein

2008-01-01

298

Genetic transformation of Ascochyta rabiei using Agrobacterium -mediated transformation  

Microsoft Academic Search

In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans\\u000a trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid

David White; Weidong Chen

2006-01-01

299

Transformation of Solanum nigrum L. protoplasts by Agrobacterium rhizogenes  

Microsoft Academic Search

Solanum nigrum protoplasts were co-cultivated with Agrobacterium rhizogenes harboring agropine-type Ri plasmid (pRi15834). A large number of transformed calli were obtained on Murashige and Shoog's (MS) medium lacking plant growth regulators. Frequency of transformation was about 3.5×10-3. In most of the calli, hairy roots appeared on MS medium without plant growth regulator. When the hairy roots were cut into segments

Zhi-Ming Wei; Hiroshi Kamada; Hiroshi Harada

1986-01-01

300

Abstract Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial  

E-print Network

Abstract Successful transformation of plant tissue using Agrobacterium relies on several factors Introduction Transgenic soybean [Glycine max (L.) Merrill] plants have been obtained using both Agrobacterium and McMullen 1991). DNA integration patterns in transformed plant tissue ob- tained via particle

Finer, John J.

301

Genetically engineering plants using agrobacterium, Robert HorschSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Robert Horsch DNAi Location:Manipulation>Techniques>transferring & storing>interviews Bacterial transfer Robert Horsch talks about agrobacterium as a ready-made delivery system for getting foreign DNA into plants.

2008-10-06

302

High efficiency Agrobacterium rhizogenes -mediated transformation of Saponaria vaccaria L. (Caryophyllaceae) using fluorescence selection  

Microsoft Academic Search

A highly efficient and convenient method for the Agrobacterium rhizogenes-dependent production of transformed roots of Saponaria vaccaria L. (Caryophyllaceae) is described. The parameters tested and optimized include S. vaccaria cultivar, explant type, Agrobacterium rhizogenes strain and culture conditions. For cotransformation using additional recombinant T-DNA-containing A. rhizogenes strains, use of neomycin phosphotransferase and enhanced green fluorescent protein genes as selectable markers

Janice F. Schmidt; Maria D. Moore; Lawrence E. Pelcher; Patrick S. Covello

2007-01-01

303

Agrobacterium larrymoorei sp. nov., a pathogen isolated from aerial tumours of Ficus benjamina.  

PubMed

Tumorigenic Agrobacterium strains isolated from tumours growing on pruned branches of Ficus benjamina have previously been shown to have unique opine metabolism and sufficient 16S rRNA sequence differences to suggest that they belong to a new species. DNA-DNA hybridization results confirmed that these strains represent a new species and Agrobacterium larrymoorei sp. nov. (type strain ATCC 51759T = CFBP 5473T = NCPPB 4096T) is proposed as the name for the species. PMID:11411669

Bouzar, H; Jones, J B

2001-05-01

304

Plant PhysiologyNovember 2003 Volume 133 Number 3November 2003 Volume 133 Number 3 Focus Issue: The Agrobacterium-Plant Cell InteractionFocus Issue: The Agrobacterium-Plant Cell Interaction  

E-print Network

, this bacterium causes crown gall disease in many dicotyledonous plant species by transferring a specific DNA fragPlant PhysiologyNovember 2003 · Volume 133 · Number 3November 2003 · Volume 133 · Number 3 Focus Issue: The Agrobacterium-Plant Cell InteractionFocus Issue: The Agrobacterium-Plant Cell Interaction #12

Citovsky, Vitaly

305

Abstract Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the effi-  

E-print Network

/ Revision received: 18 February 1998 / Accepted: 13 March 1998 E. R. Santarém · H. N. Trick · J. S. Essig: optimization of transient expression Communicated by J. M. Widholm E. R. Santarém1 · H. N. Trick2 · J. S. Essig · J. J. Finer Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons

Finer, John J.

306

The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection  

E-print Network

in plant development, particularly in lignin and pigment biosynthe- sis.They also provide structuralReview The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium by plants mainly for protection against stress. The functions of phenolic compounds in plant physiology

Citovsky, Vitaly

307

Agrobacterium rhizogens mediated transformation of Rauvolfia serpentina: Regeneration and alkaloid synthesis  

Microsoft Academic Search

Shoot cultures of Rauvolfia serpentina infected with Agrobacterium rhizogenes 15834 produced tumourous tissue at the site of injection, which eventually developed callus with hairy roots. Sporadic shoot formation occurred from the hairy roots. The shoots were grown to maturity in the green house. The mature transformed plants (RST) showed distinct variations in their physiological characteristics. The flowers of the transformed

B. D. Benjamin; G. Roja; M. R. Heble

1993-01-01

308

Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer  

Microsoft Academic Search

Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollina- tion from four different maize genotypes was investigated for competence for agroinfection. There was a direct corre- lation between different morphological stages of the unwounded

Michael Schlappi; Barbara Hohn

1992-01-01

309

Agrobacterium-mediated delivery of infectious maize streak virus into maize plants  

Microsoft Academic Search

Cells of certain strains of Agrobacterium colonize plants by transferring a portion of their DNA (the T-DNA) into a host plant cell, so causing it to proliferate and produce substances (opines) which the bacteria can use as food1. Most dicotyledonous plants can act as hosts, but most monocotyledonous species (including the economically important gramineae) are thought not to be susceptible2.

Nigel Grimsley; Thomas Hohn; Jeffrey W. Davies; Barbara Hohn

1987-01-01

310

An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii.  

PubMed

Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 ?M acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae. PMID:24198218

Pratheesh, P T; Vineetha, M; Kurup, G Muraleedhara

2014-06-01

311

Advances in Agrobacterium-mediated plant transformation with enphasys on soybean  

Microsoft Academic Search

Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter

Paulo Celso de Mello-Farias; Ana Lúcia Soares Chaves

2008-01-01

312

Abstract Changes in Agrobacterium colony and cell morphology were observed following co-culture of the  

E-print Network

) 20:250­255 DOI 10.1007/s002990100315 GENETIC TRANSFORMATION AND HYBRIDIZATION K.R. Finer · K only at high concentrations. Keywords Agrobacterium · Filamentous form · Swarming · Transformation that has been used extensively over the past 15 years as a vector for the transformation of plant cells

Finer, John J.

313

Author's personal copy Epigenetic control of Agrobacterium T-DNA integration  

E-print Network

Author's personal copy Review Epigenetic control of Agrobacterium T-DNA integration Shimpei Magori Control of cellular and developmental processes in plants. © 2011 Elsevier B.V. All rights reserved the ability to transform virtually anyeukaryotic species, from fungal to humancells (reviewed in [1

Citovsky, Vitaly

314

Comparative analysis of transgenic tall fescue (Festuca arundinacea Schreb.) plants obtained by Agrobacterium-mediated transformation and particle bombardment.  

PubMed

Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants. PMID:18648817

Gao, Caixia; Long, Danfeng; Lenk, Ingo; Nielsen, Klaus Kristian

2008-10-01

315

Efficient transformation of Arabidopsis thaliana : comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains  

Microsoft Academic Search

Summary  The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM:

Kazuhito Akama; Hideaki Shiraishi; Shozo Ohta; Kenzo Nakamura; Kiyotaka Okada; Yoshiro Shimura

1992-01-01

316

The use of green fluorescent protein (GFP) improves Agrobacterium -mediated transformation of ‘Spadona’ pear ( Pyrus communis L.)  

Microsoft Academic Search

An efficient and reproducible system for Agrobacterium-mediated transformation of the pear (Pyrus communis L.) cultivar Spadona was developed. Leaf explants of in vitro propagated plants were cocultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504, carrying the uidA-intron and nptII genes. Under selective conditions, 5% of the plantlets regenerated and were positively stained for GUS. However, most of

S. D. Yancheva; L. A. Shlizerman; S. Golubowicz; Z. Yabloviz; A. Perl; U. Hanania; M. A. Flaishman

2006-01-01

317

[Cloning of AHA gene from Amaranthus hypochondriacus and it's aphid inhibitory effect in transgenic tabacco plants].  

PubMed

Using total DNA isolated from Amaranthus hypochondriacus as template, Amaranthus hypochondriacus agglutinin AHA gene was amplified by PCR and cloned. Sequence analysis results showed that this gene is consisted of 2453 base pairs including one 1538 bp intron and two exons of 212 bp and 703 bp respectively. After inverse PCR amplification, coding region of AHA gene was obtained. AHA gene with it's intron (AHAg) and withou intron (AHAc) were inserted downstream of 35S promotor in the binary vector pBin438 resulting in the construction of two plant expression vectors pBAHAg and pBAHAc repectively. Leave explants of Nicotinana tabacum var. SR1 were transformed with A. tumefaciens LBA4404 harbouring the above expression vectors. Results from PCR and Southern blot analysis showed that AHA genes were inserted into the genome of transformed tobacco plants. Immunodot blot analysis indicated that AHA was expressed in transgenic plants. The results from insect bioassay with peach aphid (Mizus persicae) showed that the transgenic plants of pBAHAg and pBAHAc were aphid resistant, evidenced by a 57%-48% reduction in insect population density, some plants were more than 85%. The aphid resistance of transgenic plants transformed with AHAg gene as judged by aphid inhibition rate was higher than that of plants transormed with AHAc gene indicating that the intron in AHAg may be favorable for expression of AHA in transgenic plants. PMID:11330184

Zhou, Y G; Tian, Y C; Mang, K Q

2001-01-01

318

Salidroside production by hairy roots of Rhodiola sachalinensis obtained after transformation with Agrobacterium rhizogenes.  

PubMed

Hairy roots induced by Agrobacterium rhizogenes grow faster, and are considered as genetically stable. These hairy roots can be used as an interesting material for the production of secondary metabolites of pharmaceutical value. Salidroside has been identified as the major compounds from the roots of Rhodiola sachalinensis A. BOR. Here, we provide an update that adds new perspectives on the prospects and challenges of producing Salidroside from hairy roots induced by Agrobacterium rhizogene in Rhodiola sachalinensis A. BOR. For high salidroside production, the optimal concentration for precursor (Tyrosol, Tyrosine, and Phenylalanine) and elicitor (Aspergillus niger, Coriolus versicolor, and Ganoderma lucidum) was added in the LB liquid medium, respectively. The addition of elicitor in the liquid MS medium and the utilization of precursor from chemical feeding enhanced biomass accumulation and salidroside production. The optimal concentration for elicitor and precursor in the liquid medium was 0.05 mg/l and 1 mmol/l, respectively. PMID:17329834

Zhou, Xiaofu; Wu, Yuxia; Wang, Xingzhi; Liu, Bao; Xu, Hongwei

2007-03-01

319

Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium.  

PubMed

Presented are a set of cosmids based on the unit copy Agrobacterium plasmid, pTAR, and the high-copy-number mutant plasmid, pUCD500, of pTiC58. The addition of a par function derived from pTAR to the vectors allowed them to be stably maintained throughout the cell population in the absence of selective pressure. These vectors, designed for Agrobacterium and Rhizobium, also work in Escherichia coli. The vectors can be cotransferred to Rhizobiaceae from E. coli with the helper plasmid, pRK2013. The pTiC58 origin containing vectors, pUCD1000 and pUCD1001 were found to be incompatible with a 250-kb plasmid harbored by R. meliloti RM102Z1. RM102Z1(pUCD1000) was still capable of nodulating roots in alfalfa. PMID:3906714

Gallie, D R; Novak, S; Kado, C I

1985-09-01

320

Factors Affecting the Agrobacterium -Mediated Transient Transformation of the Wetland Monocot, Typha latifolia  

Microsoft Academic Search

An Agrobacterium-mediated transformation system, using transient transformation assays, was used to evaluate conditions influencing transformation for the wetland monocot Typha latifolia. These studies were aimed at the long-term objective of evaluating candidate genes for phytoremediation. The binary plasmid vector pCAMBIA1301\\/EHA105, containing the ß-glucuronidase coding sequence, was used in combination with factors known to affect transformation. These included callus age at

Rangaraj Nandakumar; Li Chen; Suzanne M. D. Rogers

2004-01-01

321

d- p -Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp  

Microsoft Academic Search

A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The

Serge Runser; Nicolas Chinski; Eric Ohleyer

1990-01-01

322

Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes.  

PubMed

Cultured hairy root lines resulting from infection by Agrobacterium rhizogenes are known for approximately thirty plant species. We extend this range by establishing forty original dicotyledonous hairy root lines with A. rhizogenes strain A4. Hairy roots have been cultured for at least 2-6 years on Murashige & Skoog medium. Some hairy root cultures such as Anagallis arvensis and Antirrhinum majus spontaneously regenerated whole plants. PMID:24241404

Mugnier, J

1988-01-01

323

Factors affecting Agrobacterium -mediated transformation and regeneration of sweet orange and citrange  

Microsoft Academic Search

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant

Changhe Yu; Shu Huang; Chunxian Chen; Zhanao Deng; Paul Ling; Fred G. Gmitter

2002-01-01

324

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation  

Microsoft Academic Search

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively\\u000a in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on\\u000a GFP fluorescence with a

R. Baranski; E. Klocke; G. Schumann

2006-01-01

325

Sonication-assisted Agrobacterium rhizogenes -mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation  

Microsoft Academic Search

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and

Milen I. Georgiev; Jutta Ludwig-Müller; Kalina Alipieva; Annemarie Lippert

2011-01-01

326

pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation  

Microsoft Academic Search

Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders

Roger P. Hellens; E. Anne Edwards; Nicola R. Leyland; Samantha Bean; Philip M. Mullineaux

2000-01-01

327

Root induction in pine (Pinus) and larch (Larix) spp. using Agrobacterium rhizogenes  

Microsoft Academic Search

Root induction on in vitro adventitious Pinus monticola Dougl. shoots from mature embryos was improved after 8 weeks co-cultivation with Agrobacterium rhizogenes Conn. strain A4 or the pRi transconjugant strain R1000 as compared to controls, and to naphthaleneacetic acid and indoleacetic acid treatments. An improvement in the number and quality of roots induced was observed on co-cultivated shoots as well

B. J. McAfee; E. E. White; L. E. Pelcher; M. S. Lapp

1993-01-01

328

Agrobacterium-mediated transformation of `Alpine' Fragaria vesca, and transmission of transgenes to R1 progeny  

Microsoft Academic Search

Agrobacterium-mediated transformation was used to stably introduce ?-glucuronidase (gus) and neomycin phosphotransferase (nptII) marker genes into `Alpine' Fragaria vesca FRA 197, a diploid (2n = 2x = 14) strawberry. R0 generation transformants derived from a single clump of kanamycin-resistant\\u000a callus were vegetatively propagated. The presence of the gus and nptII genes in five clonal R0 runner plants was confirmed by

K. M. Haymes; T. M. Davis

1998-01-01

329

Agrobacterium-mediated transformation of Artemisia absinthium L. (wormwood) and production of secondary metabolites  

Microsoft Academic Search

Hairy roots were obtained after infection of Artemisia absinthium shoots with Agrobacterium rhizogenes strains 1855 and LBA 9402. The susceptibility to hairy root transformation varied between plant genotypes and bacterial strains.\\u000a Hairy roots showed macroscopic differences from control root cultures. Southern blot hybridization confirmed the integration\\u000a of T-DNA from both p1855 and pBin19, while polymerase chain reaction analysis indicated the

S. Nin; A. Bennici; G. Roselli; D. Mariotti; S. Schiff; R. Magherini

1997-01-01

330

Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin-producing bacterium Agrobacterium aurantiacum  

Microsoft Academic Search

The carotenoid composition of the astaxanthin-producing bacterium Agrobacterium aurantiacum was analysed under different culture conditions. Ten kinds of carotenoids, ?-carotene, echinenone, ?-cryptoxanthin, 3-hydroxyechinenone, canthaxanthin, 3?-hydroxyechinenone, zeaxanthin, adonirubin, adonixanthin and astaxanthin, were identified by HPLC and spectroscopical techniques. A. aurantiacum synthesized astaxanthin from ?-carotene through two hydroxylation steps at C-3 and 3?, and oxidation steps at C-4 and 4?. The order

Akihiro Yokoyama; Wataru Miki

1995-01-01

331

The molecular cloning and expression of a cellobiase gene from an Agrobacterium in Escherichia coli  

Microsoft Academic Search

Summary  The ?-glucosidase from ATCC 21400, anAgrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed\\u000a phase FPLC. The partial amino acid sequence for one peptide was determined by automated Edman degradation. The sequence was\\u000a used to synthesize a mixture of oligodeoxyribonucleotides which was used as a hybridization probe to identify

Warren W. Wakarchuk; Douglas G. Kilburn; Robert C. Miller Jr; R. Antony J. Warren

1986-01-01

332

Electroporation stimulates tranformation of freshly isolated cell suspension protoplasts of Solanum dulcamara by Agrobacterium  

Microsoft Academic Search

Freshly isolated cell suspension protoplasts ofSolanum dulcamara were mixed withAgrobacterium rhizogenes, allowed to settle for 2 h, exposed to electrical pulses and further incubated for 2h. Two pulses of 600 V cm-1 for 2 msec separated by 15 sec produced transformed colonies at relative and absolute transformation frequencies which were 3–4 and 10 fold greater than those obtained by co-cultivation

P. K. Chand; E. L. Rech; T. J. Golds; J. B. Power; M. R. Davey

1989-01-01

333

Agrobacterium -mediated transformation of Nicotiana attenuata , a model ecological expression system  

Microsoft Academic Search

Summary.   Research into the genetic basis of the ecological sophistication of plants is hampered by the availability of transformable\\u000a systems with a wealth of well-described ecological interactions. We present an Agrobacterium-mediated transformation system for the model ecological expression system, Nicotiana attenuata, a native tobacco that occupies the post-fire niche in the Great Basin Desert of North America. We describe a

Tamara Krügel; Michelle Lim; Klaus Gase; Rayko Halitschke; Ian T. Baldwin

2002-01-01

334

Improved Agrobacterium -mediated transformation of sunflower ( Helianthus annuus L.): assessment of macerating enzymes and sonication  

Microsoft Academic Search

Agrobacterium -mediated transformation of shoot apices of sunflower ( Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (g-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When

S. Weber; W. Friedt; N. Landes; J. Molinier; C. Himber; P. Rousselin; G. Hahne; R. Horn

2003-01-01

335

Agrobacterium rhizogenes -mediated genetic transformation and regeneration of a conifer: Larix decidua  

Microsoft Academic Search

Summary  Gene transfer and plant regeneration systems have been developed for European larch (Larix decidua Mill.) in our laboratory. Aseptically germinated young seedlings were hypocotyl wound-inoculated withAgrobacterium rhizogenes strains 11325 containing a wild-type Ri (root-inducing) plasmid. Swollen stems appeared at infected wounds followed by either\\u000a abundant hairy roots or adventitious shoot buds that developed within 3 to 4 wk after inoculation.

Yinghua Huang; Alexander M. Diner; David F. Karnosky

1991-01-01

336

Characterization of competent cells and early events of Agrobacterium -mediated genetic transformation in Arabidopsis thaliana  

Microsoft Academic Search

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene ß-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In

Rajbir S. Sangwan; Yvan Bourgeois; Spencer Brown; Gérard Vasseur; Brigitte Sangwan-Norreel

1992-01-01

337

Agrobacterium–mediated transformation and stable expression of the green fluorescent protein in Brassica rapa  

Microsoft Academic Search

An efficient Agrobacterium–mediated method for transformation, regeneration and screening of Brassica rapa subsp. oleifera (synonym to B. campestris) was developed. For transformation of B. rapa subsp. oleifera, 5-d-old cotyledons were co-cultivated for 2 d with Agrobacteria (strain AGL1) harbouring a binary vector carrying a gene for green fluorescent protein (GFP). For regeneration, cultivation of explants in Murashige–Skoog-based media supplemented with 2 mg

Tony Wahlroos; Petri Susi; Lidia Tylkina; Svetlana Malyshenko; Svetlana Zvereva; Timo Korpela

2003-01-01

338

The effect of media composition on EDTA degradation by Agrobacterium sp  

Microsoft Academic Search

EDTA degradation by an Agrobacterium sp. has been examined by quantifying [sup 14]C-labeled CO[sub 2] produced from iron-[2-[sup 14]C] EDTA and by measured loss of nonlabeled EDTA by HPLC. Fe-EDTA degradation resulted in a rise in pH, nitrate concentration, and ammonia concentration. Addition of glycerol resulted in suppression of Fe-EDTA degradation and in a decrease in pH and NH[sub 4][sup

A. V. Palumbo; S. Y. Lee; P. Boerman

2009-01-01

339

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.  

PubMed

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

2013-01-01

340

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.  

PubMed Central

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate. PMID:8763954

Alt-Morbe, J; Stryker, J L; Fuqua, C; Li, P L; Farrand, S K; Winans, S C

1996-01-01

341

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)  

NASA Technical Reports Server (NTRS)

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

1999-01-01

342

Characterization of Plasmid-Borne and Chromosome-Encoded Traits of Agrobacterium Biovar 1, 2, and 3 Strains from France  

PubMed Central

We collected 111 Agrobacterium isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of Agrobacterium. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named Agrobacterium vitis) and of Agrobacterium grapevine strains in general. Among pathogenic strains of Agrobacterium, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine. PMID:10788345

Ride, Michel; Ride, Suzanne; Petit, Annik; Bollet, Claude; Dessaux, Yves; Gardan, Louis

2000-01-01

343

From: Methods in Molecular Biology, vol. 344: Agrobacterium Protocols, 2/e, volume 2 Edited by: Kan Wang Humana Press Inc., Totowa, NJ  

E-print Network

, and experi- mental protocols for Agrobacterium-mediated genetic transformation of yet more plant species components of the genetic transformation pathway. Key Words: Human cells; heterologous transformation system; nuclear import; plant factors. 1. Introduction Agrobacterium-mediated genetic transformation is the only

Citovsky, Vitaly

344

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2  

PubMed Central

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K2TeO3 was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K2TeO3 concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K2TeO3-amended medium. The bona fide agrobacterium colonies growing on media amended with K2TeO3 were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 103 to 104 agrobacteria · g of dry soil?1 in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains. PMID:11133429

Mougel, Christophe; Cournoyer, Benoit; Nesme, Xavier

2001-01-01

345

Production of an extracellular polysaccharide by Agrobacterium sp DS3 NRRL B14297 isolated from soil  

Microsoft Academic Search

A bacterium isolated from soil and identified asAgrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.5:2.4:1, together with 3.7% (w\\/w) pyruvic acid. Methylation analyses showed the presence of (1?3)-, (1?4)- and (1?6)-linked glucose, (1?3)- and (1?4, 1?6)-linked galactose

C T Hou; J A Ahlgren; W Brown; J J Nicholson

1996-01-01

346

The effect of media composition on EDTA degradation by Agrobacterium sp  

Microsoft Academic Search

EDTA degradation by anAgrobacterium sp. has been examined by quantifying14C-labeled CO2 produced from iron-[2-14C] EDTA and by measured loss of nonlabeled EDTA by HPLC. Fe-EDTA degradation resulted in a rise in pH, nitrate concentration,\\u000a and ammonia concentration. Addition of glycerol resulted in suppression of Fe-EDTA degradation and in a decrease in pH and\\u000a NH4\\u000a + concentration in the media. Addition

Anthony V. Palumbo; S. Y. Lee; Patrice Boerman

1994-01-01

347

pBECKS. A flexible series of binary vectors for Agrobacterium-mediated plant transformation.  

PubMed

A series of binary T-DNA vectors (pBECKS) has been created for use in the Agrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19 vector; the deleterious mutation within the coding sequence of nptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combinations of the marker genes gusA, C1/Lc, nptII, hph, and bar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors has been designed to facilitate the insertion and transfer of novel gene sequences by providing a nptII-linked plant expression cassette or lacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors in order to facilitate their selection across the range of Agrobacterium virulence strains. PMID:9438254

McCormac, A C; Elliott, M C; Chen, D F

1997-12-01

348

Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection  

PubMed Central

An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and ?-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15?mgL?1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600?nm) of 0.3 for 72?h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth. PMID:24376383

Alvarez, Jose M.; Ordas, Ricardo J.

2013-01-01

349

Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche  

PubMed Central

Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L) 6-benzylaminopurine and (0.08 mg/L) naphthaleneacetic acid, we have achieved the highest frequency (90%) for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0) and an infection time (20–30 min). According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP) marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30%) than older leaves (10%). PMID:23354481

Han, Xue; Ma, Shurong; Kong, Xianghui; Takano, Tetsuo; Liu, Shenkui

2013-01-01

350

Identification of Strains of Alcaligenes and Agrobacterium by a Polyphasic Approach  

PubMed Central

The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing. PMID:11526136

Clermont, Dominique; Harmant, Christine; Bizet, Chantal

2001-01-01

351

Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum  

Microsoft Academic Search

Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial

Robert Konieczny; Bohuš Obert; Juraj Bleho; Ond?ej Novák; Claudia Heym; Monika Tuleja; Jens Müller; Miroslav Strnad; Diedrik Menzel; Jozef Šamaj

2011-01-01

352

Triphysaria root transformation with Agrobacterium rhizogenes The root transformation procedure is described in Bandaranayake et al. (2010) Plant Cell 22,  

E-print Network

Yoder lab 2010 Triphysaria root transformation with Agrobacterium rhizogenes The root transformation procedure is described in Bandaranayake et al. (2010) Plant Cell 22, 1404. The technique was originally described for transforming Medicago truncatula roots for rhizobium assays (Boisson-Dernieret al

Yoder, John I.

353

Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L. and the regeneration of transformed plants.  

PubMed

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 microM 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 microM BA. Following elongation on MS medium supplemented with 1 microM BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four. PMID:14745503

Zdravkovi?-Kora?, S; Muhovski, Y; Druart, P; Cali?, D; Radojevi?, L

2004-04-01

354

Transport of gamma-butyrobetaine in an Agrobacterium species isolated from soil.  

PubMed Central

An Agrobacterium sp. isolated from soil by selective growth on gamma-butyrobetaine (gamma-trimethylaminobutyrate) as the sole source of both carbon and nitrogen has been shown to possess an inducible transport system for this growth substrate. This transport system has a Kt of 0.5 microM and a maximal velocity of 3.8 nmol/min per mg (dry weight). The influx of gamma-butyrobetaine is optimal at pH 8.5 and operates against a concentration gradient. The transport system shows a high specificity for trimethylamine carboxylic acid molecules of defined chain length. gamma-Butyrobetaine uptake was significantly reduced in osmotically shocked cells and a gamma-butyrobetaine binding activity was detected in the crude shock fluid. This suggests a transport mechanism involving a periplasmic gamma-butyrobetaine binding protein. PMID:3782024

Nobile, S; Deshusses, J

1986-01-01

355

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers.  

PubMed

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige-Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T(0) generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T(1) seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T(1) generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use. PMID:21400224

Nanasato, Yoshihiko; Konagaya, Ken-ichi; Okuzaki, Ayako; Tsuda, Mai; Tabei, Yutaka

2011-08-01

356

Structural elucidation of a novel core oligosaccharide backbone of the lipopolysaccharide from the new bacterial species Agrobacterium larrymoorei.  

PubMed

Agrobacterium larrymoorei is a Gram-negative phytopathogenic bacterium, which produces tumours on Ficus benjamina plants and differs from other Agrobacteria both genetically and biochemically. The lipopolysaccharide (LPS) plays an important role in the pathogenesis of Agrobacteria. The present paper is the first report on the molecular primary structure of the core region of an Agrobacterium LPS. The following structure of the core and lipid A carbohydrate backbone of an R-form LPS of A. larrymoorei was determined by chemical degradations and 1D and 2D NMR spectroscopy: [carbohydrate structure: see text] All sugars are alpha-D-pyranoses if not stated otherwise, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Qui3NAcyl is 3,6-dideoxy-3-(3-hydroxy-2,3-dimethyl-5-oxoprolylamino)glucose, GlcAN and GalAN are amides of GlcA and GalA. PMID:14670730

Molinaro, Antonio; De Castro, Cristina; Lanzetta, Rosa; Parrilli, Michelangelo; Raio, Aida; Zoina, Astolfo

2003-11-14

357

Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes : iridoid and phenylethanoid glycoside accumulation in hairy root cultures  

Microsoft Academic Search

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types\\u000a of explants and acetosyringone (AS) (200 ?M). The highest frequency of transformation (over 50% of explants forming roots\\u000a at the infected sites after 6 weeks of culture on Lloyd and McCown

Renata Gr?bkowska; Aleksandra Królicka; Wojciech Mielicki; Marzena Wielanek; Halina Wysoki?ska

2010-01-01

358

Agrobacterium -mediated transformed transgenic triploid bermudagrass ( Cynodon dactylon × C. transvaalensis ) plants are highly resistant to the glufosinate herbicide Liberty  

Microsoft Academic Search

Glufosinate resistance gene isolated from Streptomyces hygromicinroscopicus (bar) that confers the resistance of herbicide Liberty, a broad-spectrum grass and broadleaf contact herbicide widely used for weed control, was introduced into triploid bermudagrass by Agrobacterium-mediated transformation. Embryogenic calluses derived from stolonous nodal segment were co-cultured with the disarmed strain EHA105 harboring the binary vector pBG1300H containing the bar gene under the

Fanrong Hu; Lei Zhang; Xueyan Wang; Jie Ding; Dianxing Wu

2005-01-01

359

agronomie: plant genetics and breeding tude srologique des souches marocaines  

E-print Network

agronomie: plant genetics and breeding �tude s�rologique des souches marocaines d'Agrobacterium tumefaciens, agent de la galle du collet des rosac�es fruiti�res A Benjama N Alami EM Saadaoui 1 Laboratoire of Moroccan strains of Agrobacterium tumefaciens, the causal agent of crown- gall in stone-fruit trees

Paris-Sud XI, Université de

360

Transformation of Catalpa ovata by Agrobacterium rhizogenes and phenylethanoid glycosides production in transformed root cultures.  

PubMed

Transformed root cultures of Catalpa ovata were established following shoots infection with four agropine strains of Agrobacterium rhizogenes. Frequency of root formation was dependent on the bacterial strain and the presence of acetosyringone in the incubation medium. It is the first report concerning the possibility of transforming Catalpa ovata by A. rhizogenes. Both transformed and untransformed root cultures of C. ovata were studied for their growth and phenylethanoid glycoside production. As with the roots of intact plants, cis- and trans-verbascoside as well as martynoside were produced in transformed and untransformed root cultures of C. ovata. In hairy roots, total (cis + trans) verbascoside production could be stimulated up to three-fold of that of roots of 6-month-old plants grown in a greenhouse, by using an appropriate root line cultured in liquid 1/2 B5 Gamborg medium containing indole-3-butyric acid (0.1 mg/l) in the dark but not light conditions. Transformed and untransformed root cultures of C. ovata were also found to have 10 times higher martynoside production than roots of intact plants. PMID:11421453

Wysoki?ska, H; Lisowska, K; Floryanowicz-Czekalska, K

2001-01-01

361

Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation.  

PubMed

Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products. PMID:17899095

Lu, Chaofu; Kang, Jinling

2008-02-01

362

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.  

PubMed

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including ?-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis. PMID:25320471

Sharafi, Ali; Sohi, Haleh Hashemi; Mirzaee, Hooman; Azadi, Pejman

2014-10-01

363

An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).  

PubMed

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-?M 6-benzyladenine and 2.0-?M ?-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-?M acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient ?-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed ?-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and ?-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin. PMID:23813408

Pandey, Sonika; Mishra, Avinash; Patel, Manish Kumar; Jha, Bhavanath

2013-09-01

364

Explant and cultivar effects on genetic transformation of petunia  

E-print Network

. The most useful method foz plant transformation uses Agrobacterium tumefaciens. This is a soil borne bacterium that infects many dicotyledonous plant species through a wound. The infection forms a tumozous growth commonly called a crown-gall through...). Agrobacterium tumefaciens is a soil bacterium that infects many plant species, causing a tumozous growth commonly known as crown gall. The genus belongs to the family Rhizobiaceae, being closely related to the genus Rhizobium. The different Agrobacterium...

Ulian, Eugenio Cesar

2012-06-07

365

Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting  

PubMed Central

Background Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. Results An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. Conclusions This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications. PMID:21595986

2011-01-01

366

Agrobacterium-mediated infection of whole plants by yellow dwarf viruses.  

PubMed

Barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPV (CYDV-RPV) are only transmitted between host plants by aphid vectors and not by mechanical transmission. This presents a severe limitation for the use of a reverse genetics approach to analyze the effects of mutations in these viruses on plant infection and aphid transmission. Here we describe the use of agroinfection to infect plants with BYDV-PAV and CYDV-RPV. The cDNAs corresponding to the complete RNA genomes of BYDV-PAV and CYDV-RPV were cloned into a binary vector under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal. The self-cleaving ribozyme from hepatitis virus D was included to produce a transcript in planta with a 3' terminus identical to the natural viral RNA. ELISA and RT-PCR analysis showed that the replicons of BYDV-PAV and CYDV-RPV introduced by Agrobacterium into Nicotiana benthamiana and N. clevelandii gave rise to a local infection in the infiltrated mesophyll cells. After several weeks systemic infection of phloem tissue was detected, although no systemic symptoms were observed. Three heterologous virus silencing suppressors increased the efficiency of agroinfection and accumulation of BYDV-PAV and CYDV-RPV in the two Nicotiana species. The progeny viruses purified from infiltrated tissues were successfully transmitted to oat plants by aphids, and typical yellow dwarf symptoms were observed. This study reports the first agroinfection of eudicot plants using BYDV-PAV and CYDV-RPV. PMID:21763366

Yoon, Ju-Yeon; Choi, Seung-Kook; Palukaitis, Peter; Gray, Stewart M

2011-09-01

367

[Hairy root induction and plant regeneration of crownvetch (Coronilla varia L.) transformed by Agrobacterium rhizogenes].  

PubMed

An efficient system of genetic transformation and plant regeneration via somatic embryogenesis was established in crownvetch (Coronilla varia L.) by infecting the segments of cotyledons and hypocotyls of 15d-old seedlings with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants or via calluses on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or liquid MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency (87.4%) was achieved by preculturing cotyledons for 2d and pre-treating the A. rhizogenes with suitable concentration of acetosyringone at logarithmic phase (OD600 = 0.8). The embryogenic calluses with 100% induction frequency were induced from hairy roots on MS medium containing 0.2mg/L 2,4-D, 0.5mg/L NAA and 0.5mg/L KT. Globular-, heart-, torpedo-, and cotyledon shaped somatic embryos were produced orderly and developed into plantlets when transferred the embryogenic calluses on MS medium supplemented with 0.5mg/L KT, 0.2mg/L IBA and 300mg/L proline. The transformed plants did not show differences in morphology except abundant lateral root branches compared to the non-transformed plants. However, the contents of 3-nitropropanic acid in hairy roots and leaves of one of 5 transformed clones were 57.68% and 58.17% in roots and leaves of untransformed plants, respectively. Opine paper electrophoresis revealed the integration and expression of TR-DNA. PCR analysis confirmed that the TL-DNA including 654 bp rol B sequence was inserted into the genome of transformed hairy roots and their regenerated plants. PMID:16572849

Han, Xiao-Ling; Bu, Huai-Yu; Hao, Jian-Guo; Zhao, Yu-Wei; Jia, Jing-Fen

2006-01-01

368

Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.  

PubMed

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses. PMID:23386302

Gao, Xiquan; Shan, Libo

2013-01-01

369

Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway  

PubMed Central

Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

Guleria, Praveen; Yadav, Sudesh Kumar

2013-01-01

370

A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis  

PubMed Central

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

371

Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.  

PubMed Central

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8637884

Zupan, J R; Citovsky, V; Zambryski, P

1996-01-01

372

Transport of DNA into the nuclei of xenopus oocytes by a modified VirE2 protein of Agrobacterium.  

PubMed Central

We used Agrobacterium T-DNA nuclear transport to examine the specificity of nuclear targeting between plants and animals and the nuclear import of DNA by a specialized transport protein. Two karyophilic Agrobacterium virulence (Vir) proteins, VirD2 and VirE2, which presumably associate with the transported T-DNA and function in many plant species, were microinjected into Drosophila embryos and Xenopus oocytes. In both animal systems, VirD2 localized to the cell nuclei and VirE2 remained exclusively cytoplasmic, suggesting that VirE2 nuclear localization signals may be plant specific. Repositioning one amino acid residue within VirE2 nuclear localization signals enabled them to function in animal cells. The modified VirE2 protein bound DNA and actively transported it into the nuclei of Xenopus oocytes. These observations suggest a functional difference in nuclear import between animals and plants and show that DNA can be transported into the cell nucleus via a protein-specific pathway. PMID:8721747

Guralnick, B; Thomsen, G; Citovsky, V

1996-01-01

373

Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995  

SciTech Connect

Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

Marton, L.

1996-02-01

374

Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting  

Microsoft Academic Search

To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple cultivars (‘Delicious’, ‘Golden Delicious’, ‘Royal Gala’ and ‘Greensleeves’) were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection

Siela N. Maximova; Abhaya M. Dandekar; Mark J. Guiltinan

1998-01-01

375

Conversion of a putative Agrobacterium sugar-binding protein into a FRET sensor with high selectivity for sucrose.  

PubMed

Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes. Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose nanosensors. Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 mum affinity. This FLIPsuc-4mu protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose. Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4mu for sucrose, maltose, and glucose. Mutant nanosensors showed up to 50- and 11-fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells. PMID:16912038

Lager, Ida; Looger, Loren L; Hilpert, Melanie; Lalonde, Sylvie; Frommer, Wolf B

2006-10-13

376

In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro.  

PubMed

Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans' AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22-25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes. PMID:23572898

Sankara Rao, K; Sreevathsa, Rohini; Sharma, Pinakee D; Keshamma, E; Udaya Kumar, M

2008-10-01

377

Agrobacterium -Mediated Transformation of cry 1Ac Gene into Shoot-Tip Meristem of Diploid Cotton Gossypium arboreum cv. RG8 and Regeneration of Transgenic Plants  

Microsoft Academic Search

An Agrobacterium-mediated gene transfer protocol was developed for the diploid cotton Gossypium arboreum using meristematic cells of shoot tips, followed by direct shoot organogenesis or multiple shoot induction of putative transformants.\\u000a Seven-day- old shoot tips of in vitro-germinated seedlings of G. arboreum cv. RG8 were excised by removing cotyledonary leaves and providing “V”-shaped oblique cuts on either side of explants.

S. B. Nandeshwar; Sandhya Moghe; P. K. Chakrabarty; M. K. Deshattiwar; Keshav Kranthi; P. Anandkumar; C. D. Mayee; B. M. Khadi

2009-01-01

378

Analysis of TR-DNA\\/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4  

Microsoft Academic Search

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with

Lise Jouanin; David Bouchez; Roger F. Drong; David Tepfer; Jerry L. Slightom

1989-01-01

379

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of Agrobacterium sp. ATCC 31749  

Microsoft Academic Search

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and\\u000a 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and\\u000a kinetic characterization studies were conducted in a scale-down system. The

Hong-Tao Zhang; Xiao-Bei Zhan; Zhi-Yong Zheng; Jian-Rong Wu; Nike English; Xiao-Bin Yu; Chi-Chung Lin

380

Mat ( M ulti- A uto- T ransformation) vector system. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants  

Microsoft Academic Search

Summary  We have developed a new transformation method called MATVS (Multi-Auto-Transformation Vector System). The oncogenes (ipt or rol genes) of Agrobacterium are used as selectable markers to regenerate transgenic cells and to select marker-free transgenic plants in the MATVS. The\\u000a chimeric ipt gene or the rol genes are combined withthe site-specific recombination R\\/RS system to remove the oncogenes from the transgenic

H. Ebinuma; A. Komamine

2001-01-01

381

Deletion Analysis of the 5?-Upstream Region of the Agrobacterium rhizogenes Ri Plasmid rolC Gene Required for Tissue-Specific Expression 1  

PubMed Central

The cis-acting elements located in the ?848 and +23 region of the 5?-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid were investigated. The cis-acting DNA region required for phloem-specific expression was found within the ?153 region, whereas a minimum region needed for the expression in the seed embryo was located at position ?120. ImagesFigure 2 PMID:16668908

Sugaya, Sumiko; Uchimiya, Hirofumi

1992-01-01

382

Occurrence of tetraploidy in Nicotiana attenuata plants after Agrobacterium -mediated transformation is genotype specific but independent of polysomaty of explant tissue  

Microsoft Academic Search

Genotypes of Nicotiana attenuata collected from Utah and Arizona were transformed with 17 different vectors (14 unpublished vectors based on 3 new backbone\\u000a vectors) using an Agrobacterium-mediated procedure to functionally analyze genes important for plant–insect interactions. None of the 51 T1–T3 transgenic\\u000a Utah lines analyzed by the flow cytometry were tetraploid, as opposed to 18 of 33 transgenic Arizona lines

Ben Bubner; Klaus Gase; Beatrice Berger; Dirk Link; Ian T. Baldwin

2006-01-01

383

Genetic transformation of oilseed rape ( Brassica napus ) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype  

Microsoft Academic Search

Genetically transformed repeseed (Brassica napus) roots were obtained by in vitro inoculation of excised stem segments with Agrobacterium rhizogenes. Axenic root organ clones were established and they exhibited a phenotype characteristic of transformed roots: rapid growth, reduced apical dominance and root plagiotropism. Stem regeneration was induced by exposing root fragments to 2,4-dichloroacetic acid (2,4-D) in liquid medium, followed by transfer

P. Guerche; L. Jouanin; D. Tepfer; G. Pelletier

1987-01-01

384

Role of the host cell cycle in the Agrobacterium -mediated genetic transformation of Petunia : Evidence of an S-phase control mechanism for T-DNA transfer  

Microsoft Academic Search

Chimeric ß-glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and\\/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone

Estelle Villemont; Frédéric Dubois; Rajbir S. Sangwan; Gérard Vasseur; Yvan Bourgeois; Brigitte S. Sangwan-Norreel

1997-01-01

385

Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid  

Microsoft Academic Search

A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two

Eva Zyprian; Clarence I. Kado

1990-01-01

386

The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA-binding site.  

PubMed

Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants. PMID:25212215

Maes, Michal; Amit, Einav; Danieli, Tsafi; Lebendiker, Mario; Loyter, Abraham; Friedler, Assaf

2014-11-01

387

Robert Horsch, still imageSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Image of Dr. Robert Horsch As a plant biologist for Monsanto, Robert Horsch and two other colleagues published a paper about a soil bacterium, Agrobacterium tumefaciens, that detailed its natural genetic engineering abilities.

2008-10-06

388

Robert Horsch, still imageSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Image of Robert Horsch. As a plant biologist for Monsanto, Robert Horsch and two other colleagues published a paper about a soil bacterium, Agrobacterium tumefaciens, that detailed its natural genetic engineering abilities.

2008-10-06

389

Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation.  

PubMed

Purple-colored transgenic creeping bentgrass (Agrostis stolonifera L.) plants were developed for ornamental purpose by means of Agrobacterium-mediated transformation. Embryogenic creeping bentgrass calli were transformed with the pCAMBIA 3301 vector harboring maize (Zea mays) flavonoid/anthocyanin biosynthetic pathway transcription factor genes, Lc (Leaf color) and Pl (Purple leaf), individually and in combination, and three types of putative transgenic plants (Lc, Pl, and Lc + Pl) were generated. Genomic integration and expression of the transgenes were confirmed by Southern and northern blot analyses, respectively. The transgenic creeping bentgrass plants expressing both Lc and Pl genes were entirely purple, whereas those expressing Pl alone had purple stems and those expressing Lc alone lacked purple pigmentation in adult plants. The anthocyanin content was estimated in all the three types of transgenic plant and correlated well with the degree of purple coloration observed. These results suggest that both Lc and Pl genes are necessary and sufficient to confer purple coloration to creeping bentgrass. PMID:19050897

Han, Yun-Jeong; Kim, Yong-Min; Lee, Jee-Yeon; Kim, Soo Jung; Cho, Kyu-Chang; Chandrasekhar, Thummala; Song, Pill-Soon; Woo, Young-Min; Kim, Jeong-Il

2009-03-01

390

Expression and inheritance of inserted markers in binary vector carrying Agrobacterium rhizogenes-transformed potato (Solanum tuberosum L.).  

PubMed

Transgenic shoots were regenerated from eight diploid potato hairy root clones obtained by transformation with Agrobacterium rhizogenes harboring next to its wild-type Ri-plasmid a binary vector containing the neomycin phosphotransferase and the ?-glucuronidase genes. The plants exhibited the typical hairy root phenotype. Of the plants isolated, 58% were tetraploid and 38% were diploid. Flowering and tuberization was much better in the diploid than in the tetraploid plants. Transgenic plants formed a significantly larger root system when grown on kanamycin-containing medium as compared to growth on kanamycin-free medium. Direct evidence for genetic transformation was obtained by opine, neomycin phosphotransferase and ?-glucuronidase assays, and by molecular hybridization. Fourteen flowering diploid plants were reciprocally crossed with untransformed S. tuberosum plants, but only six were successful. Seedlings obtained from four crosses showed that all traits were transmitted to the offspring. Molecular analysis confirmed the presence of multiple integrations (copies) of both vector T-DNA and Ri-T-DNA. The genetic data, furthermore, suggest that the traits derived from Ri-T-DNA and binary vector T-DNA are linked, as no recombination between the different traits was observed. PMID:24225832

Visser, R G; Hesseling-Meinders, A; Jacobsen, E; Nijdam, H; Witholt, B; Feenstra, W J

1989-11-01

391

Production of triterpenoid anti-cancer compound taraxerol in Agrobacterium-transformed root cultures of butterfly pea (Clitoria ternatea L.).  

PubMed

Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T(L)-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T(R)-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, (1)H-NMR, and (13)C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species. PMID:22843061

Swain, Swasti S; Rout, Kedar K; Chand, Pradeep K

2012-10-01

392

Cloning and nucleotide sequence of the Bartonella bacilliformis gene: alaS and leuS, which encode aminoacyl tRNA synthetases; pyrF, which encodes orotidine 5' monophosphate decarboxylase; and txpA, an ABC transporter-like protein similar to the Agrobacterium tumefaciens chvA gene  

E-print Network

, and flagellar filament. Synthesis of functional flagella requires many genes for the ordered assembly of these domains, many of which occur in clusters (26). Flagellin is the structural filament protein coded by the late genes of flagellar biosynthesis (26...). Many flagellated bacteria contain two or more similar, but not identical, flagellin proteins, coded by separate Jkr genes (16, 26). The nucleotide sequence of flaA &om B. bacilli fonni s has been determined This thesis follows the style of Journal...

Upeslacis, Erik

2012-06-07

393

Transformation of sunflower ( Helianthus annuus L.) following wounding with glass beads  

Microsoft Academic Search

A procedure was developed for transformation of Helianthus annuus (sunflower) using Agrobacterium tumefaciens. Cotyledons were removed from young seedlings, and the remaining tissue was uniformly wounded by shaking with glass beads. The wounded tissue was then co-cultivated with a hypervirulent strain of Agrobacterium tumefaciens harboring the binary plasmid pCNL56. Minimal use of defined medium was required, and no callus was

W. Scott Grayburn; Brady A. Vick

1995-01-01

394

Design, construction and cloning of pCAMBIA-MiAMP1 vector for enhancing disease resistance in plants using Agrobacterium mediated transformation  

Microsoft Academic Search

Objective: To design, construct and clone a pCAMBIA-MiAMP1 vector that can be used to enhance disease resistance in plants through Agrobacterium-mediated transformation. Methodology and results: The cDNA sequence encoding MiAMP1 antimicrobial peptide as a defense resistance gene, which had been cloned into the cloning site of pGEM-T T-vector, was obtained from Australia. The pGEM-T-MiAMP1 was transformed into E.coli and the

Ghasemi Bezdi Kamal; Sheveloukha Victor Stepanovich; Karlov Gennady Illich

395

Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.  

PubMed

Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum. PMID:24023833

Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

2013-01-01

396

Biodegradation of Atrazine by Agrobacterium radiobacter J14a and Use of This Strain in Bioremediation of Contaminated Soil  

PubMed Central

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 ?g of [14C-U-ring]atrazine ml?1 in 72 h with a concurrent increase in the population size from 7.9 × 105 to 5.0 × 107 cells ml?1. Under these conditions cells mineralized the [ethyl-14C]atrazine and incorporated approximately 30% of the 14C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 105 J14a cells g?1 into soil with a low indigenous population of atrazine degraders treated with 50 and 200 ?g of atrazine g?1 soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (105 cells g?1) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization. PMID:9726884

Struthers, J. K.; Jayachandran, K.; Moorman, T. B.

1998-01-01

397

agronomie: plant genetics and breeding Behaviour of Prunus cultivars and hybrids towards  

E-print Network

agronomie: plant genetics and breeding Behaviour of Prunus cultivars and hybrids towards to the telluric bacteria Agrobacterium tumefaciens that induces crown gall. The results show that: i) for Prunus tumefaciens / crown gall / Prunus / rootstock / susceptibility R�sum� — Comportement d'hybrides et de

Paris-Sud XI, Université de

398

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).  

PubMed

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764

Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

2013-08-01

399

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots  

PubMed Central

Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene ?-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens. PMID:22559055

2012-01-01

400

TrendsGenet.January2006Vol.22No.1,pp.xxxxxxISSN0168-9525 On a chromosome far, far away  

E-print Network

's long been known that Agrobacterium tumefaciens can transfer its tumor-inducing (Ti) plasmid into plant genomes to cause crown gall disease. However, it's recently been shown that Agrobacterium can transfer its of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality

Citovsky, Vitaly

401

Genetic transformation in higher plants  

Microsoft Academic Search

Successful transformation of plant cells has been obtained utilizing vectors and DNA delivery methods derived from the plant pathogen, Agrobacterium tumefaciens. This soil bacterium is capable of transferring a DNA segment (T?DNA), located between specific nucleotide border sequences, from its large tumor inducing (Ti) plasmid into the nuclear DNA of infected plant cells. The exploitation of the Agrobacterium\\/Ti plasmid system

Robert T. Fraley; Stephen G. Rogers; Robert B. Horsch; Stanton B. Gelvin

1986-01-01

402

Agrobacterium rhizogenes-transformed Roots of Coffee (Coffea arabica): Conditions for Long-term Proliferation, and Morphological and Molecular Characterization  

PubMed Central

Background and Aims The aims of this study were to set up proliferation conditions for hairy roots of Coffea arabica regenerated after transformation by Agrobacterium rhizogenes strain A4-RS, and to carry out the morphological and molecular characterization of hairy root clones maintained over the long term. Methods Auxin supply, light conditions and sucrose concentration were modified with the aim of establishing efficient root proliferation conditions. The morphological variability among 62 established hairy root clones was phenotyped by scanning the roots and analysing the images using ‘whinRHIZO’ software procedures. PCR analysis of integration in transformed root cells of rol and aux oncogenes from the T-DNA of the Ri plasmid was used to study the molecular variability among clones. Key Results Auxin supply was necessary to obtain and stimulate growth and branching, and IBA applied at 0·5 µm was the most efficient auxin. Significant differences were shown among the 62 clones for total root length and for the percentage of fine roots. These variables were stable across subcultures and could hence be used for efficient characterization of hairy root clones. The majority of hairy root clones (86 %) exhibited non-significant phenotype differences with non-transformed roots. Eight clones were significantly different from the non-transformed controls in that they possessed a low proportion of fine roots. Two other hairy root clones grew significantly faster than the other clones. The PCR analysis revealed a low variability in the integration of rol and aux oncogenes in transformed root cells. The TR-DNA was never integrated as aux1 and aux2 genes were not found, although rolB and rolC genes from the TL-DNA were always present. Conclusions The discovery of low morphological variability among coffee hairy roots together with the identification of morphological variables allowing easy identification of phenotypically altered clones represent two important results. They make hairy roots a possible, and efficient, tool for functional-genomic studies of coffee root genes. PMID:18316320

Alpizar, E.; Dechamp, E.; Lapeyre-Montes, F.; Guilhaumon, C.; Bertrand, B.; Jourdan, C.; Lashermes, P.; Etienne, H.

2008-01-01

403

Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum.  

PubMed Central

DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases. PMID:8979363

Herlache, T C; Hotchkiss, A T; Burr, T J; Collmer, A

1997-01-01

404

Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy  

Microsoft Academic Search

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA ß-glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by

Ove Nilsson; C. H. Anthony Little; Göran Sandberg; Olof Olsson

1996-01-01

405

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation 1  

PubMed Central

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of ?-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis. PMID:16666706

Campell, Bruce R.; Su, Ling-Yuan; Pengelly, William L.

1989-01-01

406

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH).  

PubMed

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH. PMID:25104333

Pedroso, Marcelo M; Ely, Fernanda; Miti?, Nataša; Carpenter, Margaret C; Gahan, Lawrence R; Wilcox, Dean E; Larrabee, James L; Ollis, David L; Schenk, Gerhard

2014-12-01

407

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of Agrobacterium sp. ATCC 31749.  

PubMed

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45-60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD(+), NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20-50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process. PMID:21739265

Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; English, Nike; Yu, Xiao-Bin; Lin, Chi-Chung

2012-01-01

408

Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.  

PubMed

A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited. PMID:2103448

Zyprian, E; Kado, C I

1990-08-01

409

One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi.  

PubMed

Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Previously we reported a method called DelsGate for rapid preparation of deletion constructs for protoplast-mediated fungal transformation systems, which is based on Gateway® technology. However, over the past several years Agrobacteriumtumefaciens-mediated transformation (ATMT) has become the preferred genetic transformation method for an increasing number of fungi. Therefore, we developed a method for One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), to rapidly create deletion constructs for ATMT systems. The OSCAR methodology involves PCR amplification of the upstream and downstream flanks of the gene of interest, using gene specific primers each with a 5' extension containing one of four different attB recombination sites, modified from the Invitrogen MultiSite Gateway® system. Amplified gene flanks are then mixed with specifically designed marker and binary vectors and treated with BP clonase, generating the deletion construct in a single cloning step. The entire process of deletion construct preparation can be accomplished in just 2days. Using OSCAR we generated eight targeted deletion constructs and used two of them to generate deletion mutants in Verticillium dahliae by ATMT. In summary, OSCAR methodology combines PCR and Gateway® technology to rapidly and robustly generate precise deletion constructs for fungal ATMT and homologous gene replacement. PMID:21362493

Paz, Zahi; García-Pedrajas, María D; Andrews, David L; Klosterman, Steven J; Baeza-Montañez, Lourdes; Gold, Scott E

2011-07-01

410

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining theSolanum tuberosum and an evolvedAgrobacterium radiobacter AD1 epoxide hydrolases  

Microsoft Academic Search

Solubleepoxidehydrolase(EH)fromthepotato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were pur- ified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2- ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselec- tivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethane- diol (2.0 ? 0.2

Li Cao; Jintae Lee; Wilfred Chen; Thomas K. Wood

2006-01-01

411

Potential of a 16S rRNA-Based Taxonomic Microarray for Analyzing the Rhizosphere Effects of Maize on Agrobacterium spp. and Bacterial Communities†  

PubMed Central

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems. PMID:16751545

Sanguin, Herve; Remenant, Benoit; Dechesne, Arnaud; Thioulouse, Jean; Vogel, Timothy M.; Nesme, Xavier; Moenne-Loccoz, Yvan; Grundmann, Genevieve L.

2006-01-01

412

Regeneration of tobacco plants from crown gall tumors  

Microsoft Academic Search

Summary  Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall\\u000a strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from\\u000a these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the

John W. Einset; Anne Cheng

1979-01-01

413

Cloning and Characterization of the Phosphatidylserine Synthase Gene of Agrobacterium sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1->3)-?-d-Glucan (Curdlan)  

PubMed Central

Genes involved in the production of the extracellular (1?3)-?-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pssAG, encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the PssAG protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pssAG::TnphoA mutation was complemented by the intact pssAG gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport. PMID:12107128

Karnezis, Tara; Fisher, Helen C.; Neumann, Gregory M.; Stone, Bruce A.; Stanisich, Vilma A.

2002-01-01

414

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N -methyltransferase is methyl jasmonate-dependent  

Microsoft Academic Search

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced\\u000a into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root

Lei Zhang; Bin Yang; Beibei Lu; Guoyin Kai; Zinan Wang; Yang Xia; Ruxian Ding; Hanming Zhang; Xiaofen Sun; Wansheng Chen; Kexuan Tang

2007-01-01

415

Genetic transformation of grapevine cells  

Microsoft Academic Search

Biovar 1 strains ofAgrobacterium tumefaciens have been used to transform a cell suspension culture ofVitis vinifera cv. Cabernet Sauvignon. Cocultivation of cultures withAgrobacterium strains bearing either the cointegrate pGV3850::1103neo, or the binary vector pGA474-68, each gave rise to kanamycin resistant tissue. The stable integration and expression of the neomycin phosphotransferase gene was confirmed by Southern blotting and enzymic assay, respectively.

T. J. Baribault; K. G. M. Skene; N. Steele Scott

1989-01-01

416

Plant Transformation by Lux+ Agrobacterium  

NSDL National Science Digital Library

This resource provides protocols for conducting a labortory exercise in Recombinant DNA technology. Students learn how to introduce foreign genes into a plant host. This exercise can also be used to generate genetically modified plant strains for use in other laboratory experiments (e.g. herbicide or herbivore resistant strains for controlled ecological studies).

William J. Page (University of Alberta;); Anna Szenthe (University of Alberta;)

1998-01-01

417

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants  

Microsoft Academic Search

An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying

J. Puonti-Kaerlas; T. Eriksson; P. Engström

1992-01-01

418

Transformation of sunflower ( Helianthus annuus L.): a reliable protocol  

Microsoft Academic Search

A reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A

Nathalie Knittel; Véronique Gruber; Günther Hahne; Philippe Lénée

1994-01-01

419

Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe  

Microsoft Academic Search

The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO). The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean. The endogenous, soybean-specific, lectin gene was also detected for confirmation of

Kyriaki Glynou; Penelope C. Ioannou; Theodore K. Christopoulos

2004-01-01

420

ReseaRch PaPeR www.landesbioscience.com RNa Biology 1  

E-print Network

hundreds of sRNAs in the plant pathogen Agrobacterium tumefaciens.36,37 This bacterium is able to induce tumors (crown galls) upon transfer of a DNA fragment (T-DNA) from its tumor-inducing (Ti) plasmid to the nuclear genome of the host plant.38,39 In the transformed plant cells, expression of T-DNA encoded growth

Will, Sebastian

421

Extraction of lipids from fermentation biomass using near-critical dimethylether  

Microsoft Academic Search

The extraction of lipids from both wet and dry biomass produced by fermentation has been carried out using near-critical dimethylether (DME) as the extraction solvent. Fermentations were carried out from a shake flask up to a 300L scale using the microorganism Mortierella alpina, and up to a 20L scale for Phaffia rhodozyma and Agrobacterium tumefaciens. The lipids extracted at a

Owen Catchpole; Jason Ryan; Yin Zhu; Kristina Fenton; John Grey; Mikhail Vyssotski; Andrew MacKenzie; Eduard Nekrasov; Kevin Mitchell

2010-01-01

422

Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana  

Microsoft Academic Search

Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore,

Amanda M Davis; Anthony Hall; Andrew J Millar; Chiarina Darrah; Seth J Davis

2009-01-01

423

Stephan Sudowe and Angelika B. Reske-Kunz (eds.), Biolistic DNA Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 940, DOI 10.1007/978-1-62703-110-3_2, Springer Science+Business Media, LLC 2013  

E-print Network

, Methods in Molecular Biology, vol. 940, DOI 10.1007/978-1-62703-110-3_2, © Springer Science+Business Media-mediated transformation of proto- plasts, infiltration of Agrobacterium tumefaciens (agroinfiltration), and biolistic bombardment. PEG-mediated and electroporation- mediated transformation of protoplasts work efficiently in some

Citovsky, Vitaly

424

Comparative anatomy of gall development on Gypsophila paniculata induced by bacteria with different mechanisms of pathogenicity  

Microsoft Academic Search

Galls induced on Gypsophila paniculata by Pantoea agglomerans pv. gypsophilae (Pag) and Agrobacterium tumefaciens (At), bacteria with different mechanisms of pathogenicity, were compared morphologically and anatomically. The pathogenicity of Pag is dependent on the presence of an indigenous plasmid that harbors hrp gene cluster, genes encoding Hop virulence proteins and biosynthetic genes for auxin (IAA) and cytokinins (CKs), whereas that

L. Chalupowicz; I. Barash; M. Schwartz; R. Aloni; S. Manulis

2006-01-01

425

GhSEM-1 Marker Potentially Associated with Regeneration Ability in Cotton  

Microsoft Academic Search

A marker protein for embryogénie potential could be useful in determining if target tissue for Agrobacterium tumefaciens or microprojectile bombardment has the ability to regenerate plants. Certain varieties of cotton, especially Coker 312, are known to form somatic embryos readily, while others are more recalcitrant. Callus induced from Coker 312–17wa s found to be more embryogénie than that from TM-1.

M. Altaf-Khan; H. J. Kim; G O. Myers; B. A. Triple

2006-01-01

426

The Plant Cell, Vol. 3, 573-582, June 1991 O 1991American Society of Plant Physiologists Phytochrome Control of the tms2 Gene in Transgenic  

E-print Network

Phytochrome Control of the tms2 Gene in Transgenic Arabidopsis: A Strategy for Selecting Mutants in the-1606 lntroduction of the tms2 gene from Agrobacterium tumefaciens into Arabidopsis thaliana yields transgenic concentrations of auxin amide substrates that do not significantly affect wild-type seedlings. The tms2 gene

Tobin, Elaine

427

Regeneration of transgenic shape Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis  

Microsoft Academic Search

Different approaches to producing transgenic grapevines based on regeneration via embryogenesis were investigated. Embryogenic callus was initiated from anther tissue of Vitis vinifera cv. Sultana and three embryogenic culture types (embryogenic callus, tissue type I; proliferating embryos, tissue type II; and a suspension) were established. The three culture types were incolucaled with Agrobacterium tumefaciens harbouring a binary vector which contained

Tricia Franks; Ding Gang He; Mark Thomas

1998-01-01

428

Comparison of different transformation methods for Aspergillus giganteus  

Microsoft Academic Search

Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus, the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens-mediated transformation. Electroporation and biolistic transformation were found to be inappropriate

Vera Meyer; Dirk Mueller; Till Strowig; Ulf Stahl

2003-01-01

429

Water content, temperature and biocide effects on the growth kinetics of bacteria isolated from JP-8 aviation fuel storage tanks  

Microsoft Academic Search

Three bacterial strains isolated from JP-8 aviation fuel storage tanks were used to examine their ability to utilize the fuel as their sole source of carbon and energy. The isolates were Staphylococcus epidermidis, Agrobacterium tumefaciens and Ralstonia picketii. The purpose of this study was to determine the effect of temp