Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

Bioinformatics Analysis of the Complete Genome Sequence of the Mango Tree Pathogen Pseudomonas syringae pv. syringae UMAF0158 Reveals Traits Relevant to Virulence and Epiphytic Lifestyle

PubMed Central

Arrebola, Eva; Carrión, Víctor J.; Gutiérrez-Barranquero, José Antonio; Pérez-García, Alejandro; Ramos, Cayo; Cazorla, Francisco M.; de Vicente, Antonio

2015-01-01

The genome sequence of more than 100 Pseudomonas syringae strains has been sequenced to date; however only few of them have been fully assembled, including P. syringae pv. syringae B728a. Different strains of pv. syringae cause different diseases and have different host specificities; so, UMAF0158 is a P. syringae pv. syringae strain related to B728a but instead of being a bean pathogen it causes apical necrosis of mango trees, and the two strains belong to different phylotypes of pv.syringae and clades of P. syringae. In this study we report the complete sequence and annotation of P. syringae pv. syringae UMAF0158 chromosome and plasmid pPSS158. A comparative analysis with the available sequenced genomes of other 25 P. syringae strains, both closed (the reference genomes DC3000, 1448A and B728a) and draft genomes was performed. The 5.8 Mb UMAF0158 chromosome has 59.3% GC content and comprises 5017 predicted protein-coding genes. Bioinformatics analysis revealed the presence of genes potentially implicated in the virulence and epiphytic fitness of this strain. We identified several genetic features, which are absent in B728a, that may explain the ability of UMAF0158 to colonize and infect mango trees: the mangotoxin biosynthetic operon mbo, a gene cluster for cellulose production, two different type III and two type VI secretion systems, and a particular T3SS effector repertoire. A mutant strain defective in the rhizobial-like T3SS Rhc showed no differences compared to wild-type during its interaction with host and non-host plants and worms. Here we report the first complete sequence of the chromosome of a pv. syringae strain pathogenic to a woody plant host. Our data also shed light on the genetic factors that possibly determine the pathogenic and epiphytic lifestyle of UMAF0158. This work provides the basis for further analysis on specific mechanisms that enable this strain to infect woody plants and for the functional analysis of host specificity in the P. syringae complex. PMID:26313942

Distinct regions of the Pseudomonas syringae coiled-coil effector AvrRps4 are required for activation of immunity

PubMed Central

Sohn, Kee Hoon; Hughes, Richard K.; Piquerez, Sophie J.; Jones, Jonathan D. G.; Banfield, Mark J.

2012-01-01

Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding–leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4-dependent immunity in resistant accessions of Arabidopsis. To better understand the molecular basis of AvrRps4-triggered immunity, we determined the crystal structure of processed AvrRps4 (AvrRps4C, residues 134–221), revealing that it forms an antiparallel α-helical coiled coil. Structure-informed mutagenesis reveals an electronegative surface patch in AvrRps4C required for recognition by RPS4; mutations in this region can also uncouple triggering of the hypersensitive response from disease resistance. This uncoupling may result from a lower level of defense activation, sufficient for avirulence but not for triggering a hypersensitive response. Natural variation in AvrRps4 reveals distinct recognition specificities that involve a surface-exposed residue. Recently, a direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 has been implicated in activation of immunity. However, we were unable to detect direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 after coexpression in Nicotiana benthamiana or in yeast cells. How intracellular plant immune receptors activate defense upon effector perception remains an unsolved problem. The structure of AvrRps4C, and identification of functionally important residues for its activation of plant immunity, advances our understanding of these processes in a well-defined model pathosystem. PMID:22988101

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not.

PubMed

Wang, Li; Eggenberger, Alan; Hill, John; Bogdanove, Adam J

2006-03-01

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments[OPEN

PubMed Central

2017-01-01

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. PMID:28619883

Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

PubMed

Ham, Jong Hyun; Majerczak, Doris R; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L

2009-06-01

The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well.

A receptor-like cytoplasmic kinase phosphorylates the host target RIN4, leading to the activation of a plant innate immune receptor.

PubMed

Liu, Jun; Elmore, James Mitch; Lin, Zuh-Jyh Daniel; Coaker, Gitta

2011-02-17

Plants have evolved sophisticated surveillance systems to recognize pathogen effectors delivered into host cells. RPM1 is an NB-LRR immune receptor that recognizes the Pseudomonas syringae effectors AvrB and AvrRpm1. Both effectors associate with and affect the phosphorylation of RIN4, an immune regulator. Although the kinase and the specific mechanisms involved are unclear, it has been hypothesized that RPM1 recognizes phosphorylated RIN4. Here, we identify RIPK as a RIN4-interacting receptor-like protein kinase that phosphorylates RIN4. In response to bacterial effectors, RIPK phosphorylates RIN4 at amino acid residues T21, S160, and T166. RIN4 phosphomimetic mutants display constitutive activation of RPM1-mediated defense responses and RIN4 phosphorylation is induced by AvrB and AvrRpm1 during P. syringae infection. RIPK knockout lines exhibit reduced RIN4 phosphorylation and blunted RPM1-mediated defense responses. Taken together, our results demonstrate that the RIPK kinase associates with and modifies an effector-targeted protein complex to initiate host immunity. Copyright © 2011 Elsevier Inc. All rights reserved.

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 family member, is needed to produce L-allo-isoleucine, a precursor for the phytotoxin coronatine

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae pathovar tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors, however it promotes chlorosis in the model plant...

Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

PubMed Central

Romanchuk, Artur; Chang, Jeff H.; Mukhtar, M. Shahid; Cherkis, Karen; Roach, Jeff; Grant, Sarah R.; Jones, Corbin D.; Dangl, Jeffery L.

2011-01-01

Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species. PMID:21799664

Pseudomonas syringae Type III Effector HopBB1 Promotes Host Transcriptional Repressor Degradation to Regulate Phytohormone Responses and Virulence.

PubMed

Yang, Li; Teixeira, Paulo José Pereira Lima; Biswas, Surojit; Finkel, Omri M; He, Yijian; Salas-Gonzalez, Isai; English, Marie E; Epple, Petra; Mieczkowski, Piotr; Dangl, Jeffery L

2017-02-08

Independently evolved pathogen effectors from three branches of life (ascomycete, eubacteria, and oomycete) converge onto the Arabidopsis TCP14 transcription factor to manipulate host defense. However, the mechanistic basis for defense control via TCP14 regulation is unknown. We demonstrate that TCP14 regulates the plant immune system by transcriptionally repressing a subset of the jasmonic acid (JA) hormone signaling outputs. A previously unstudied Pseudomonas syringae (Psy) type III effector, HopBB1, interacts with TCP14 and targets it to the SCF COI1 degradation complex by connecting it to the JA signaling repressor JAZ3. Consequently, HopBB1 de-represses the TCP14-regulated subset of JA response genes and promotes pathogen virulence. Thus, HopBB1 fine-tunes host phytohormone crosstalk by precisely manipulating part of the JA regulon to avoid pleiotropic host responses while promoting pathogen proliferation. Copyright © 2017 Elsevier Inc. All rights reserved.

The bacterial type III-secreted protein AvrRps4 is a bipartite effector

PubMed Central

Spears, Benjamin J.; Garner, Christopher M.; Rogan, Conner J.; Su, Jianbin; Bhattacharjee, Saikat

2018-01-01

Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the Pseudomonas syringae pv. pisi effector AvrRps4. AvrRps4 is processed in planta into AvrRps4N (133 amino acids), homologous to the N-termini of other effectors including the native P. syringae pv. tomato strain DC3000 effector HopK1, and AvrRps4C (88 amino acids). Previous data suggested that AvrRps4C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4C from DC3000, but not from a DC3000 hopK1- strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4C in tandem with AvrRps4N, or as a chimera with HopK1N, fully complements AvrRps4-triggered immunity. AvrRps4N in the absence of AvrRps4C enhances virulence in Col-0. In addition, AvrRps4N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4C, further supporting the role of AvrRps4N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4C to AvrRps4N may have counteracted recognition of AvrRps4N, and that the plant RPS4/RRS1 resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties. PMID:29601603

The bacterial type III-secreted protein AvrRps4 is a bipartite effector.

PubMed

Halane, Morgan K; Kim, Sang Hee; Spears, Benjamin J; Garner, Christopher M; Rogan, Conner J; Okafor, Elizabeth C; Su, Jianbin; Bhattacharjee, Saikat; Gassmann, Walter

2018-03-01

Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the Pseudomonas syringae pv. pisi effector AvrRps4. AvrRps4 is processed in planta into AvrRps4N (133 amino acids), homologous to the N-termini of other effectors including the native P. syringae pv. tomato strain DC3000 effector HopK1, and AvrRps4C (88 amino acids). Previous data suggested that AvrRps4C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4C from DC3000, but not from a DC3000 hopK1- strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4C in tandem with AvrRps4N, or as a chimera with HopK1N, fully complements AvrRps4-triggered immunity. AvrRps4N in the absence of AvrRps4C enhances virulence in Col-0. In addition, AvrRps4N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4C, further supporting the role of AvrRps4N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4C to AvrRps4N may have counteracted recognition of AvrRps4N, and that the plant RPS4/RRS1 resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties.

CML8, an Arabidopsis Calmodulin-Like Protein, Plays a Role in Pseudomonas syringae Plant Immunity.

PubMed

Zhu, Xiaoyang; Robe, Eugénie; Jomat, Lucile; Aldon, Didier; Mazars, Christian; Galaud, Jean-Philippe

2017-02-01

Calcium is a universal second messenger involved in various cellular processes including plant development and stress responses. Its conversion into biological responses requires the presence of calcium sensor relays such as calmodulin (CaM) and calmodulin-like (CML) proteins. While the role of CaM is well described, the functions CML proteins remain largely uncharacterized. Here, we show that Arabidopsis CML8 expression is strongly and transiently induced by Pseudomonas syringae, and reverse genetic approaches indicated that the overexpression of CML8 confers on plants a better resistance to pathogenic bacteria compared with wild-type, knock-down and knock-out lines, indicating that CML8 participates as a positive regulator in plant immunity. However, this difference disappeared when inoculations were performed using bacteria unable to inject effectors into a plant host cell or deficient for some effectors known to target the salicylic acid (SA) signaling pathway. SA content and PR1 protein accumulation were altered in CML8 transgenic lines, supporting a role for CML8 in SA-dependent processes. Pathogen-associated molecular pattern (PAMP) treatments with flagellin and elf18 peptides have no effects on CML8 gene expression and do not modify root growth of CML8 knock-down and overexpressing lines compared with wild-type plants. Collectively, our results support a role for CML8 in plant immunity against P. syringae. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

PubMed

Brown, I R; Mansfield, J W; Taira, S; Roine, E; Romantschuk, M

2001-03-01

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean1[C][W][OPEN

PubMed Central

Wang, Jialin; Shine, M.B.; Gao, Qing-Ming; Navarre, Duroy; Jiang, Wei; Liu, Chunyan; Chen, Qingshan; Hu, Guohua; Kachroo, Aardra

2014-01-01

Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector. PMID:24872380

Specific resistances against Pseudomonas syringae effectors AvrB and AvrRpm1 have evolved differently in common bean, soybean, and Arabidopsis

PubMed Central

Chen, Nicolas W. G.; Sévignac, Mireille; Thareau, Vincent; Magdelenat, Ghislaine; David, Perrine; Ashfield, Tom; Innes, Roger W.; Geffroy, Valérie

2010-01-01

Summary In plants, the evolution of specific resistance is poorly understood. Pseudomonas syringae effectors AvrB and AvrRpm1 are recognized by phylogenetically distinct resistance (R) proteins in Arabidopsis (Brassicaceae) and soybean (Glycine max, Fabaceae). In soybean, these resistances are encoded by two tightly linked R genes Rpg1-b and Rpg1-r. To study the evolution of these specific resistances, we investigated AvrB- and AvrRpm1-induced responses in common bean (Phaseolus vulgaris, Fabaceae).Common bean genotypes of various geographical origins were inoculated with P. syringae strains expressing AvrB or AvrRpm1. A common bean recombinant-inbred-line (RIL) population was used to map R genes to AvrRpm1.No common bean genotypes recognized AvrB. By contrast, multiple genotypes responded to AvrRpm1, and two independent R genes conferring AvrRpm1-specific resistance were mapped to the ends of linkage group B11 (Rpsar-1) and B8 (Rpsar-2). Rpsar-1 is located in a region syntenic with the soybean Rpg1 cluster. However, mapping of specific Rpg1 homologous genes suggests that AvrRpm1 recognition evolved independently in common bean and soybean.The conservation of genomic position of AvrRpm1-specific genes between soybean and common bean suggests a model whereby specific clusters of R genes are predisposed to evolve recognition of the same effector molecules. PMID:20561214

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

PubMed

Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

2017-01-01

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

PubMed

Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

2012-05-01

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression. © 2012 Blackwell Publishing Ltd.

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

PubMed

Sharma, Shailendra; Sharma, Shiveta; Hirabuchi, Akiko; Yoshida, Kentaro; Fujisaki, Koki; Ito, Akiko; Uemura, Aiko; Terauchi, Ryohei; Kamoun, Sophien; Sohn, Kee Hoon; Jones, Jonathan D G; Saitoh, Hiromasa

2013-05-01

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

The tomato calcium sensor Cbl10 and its interacting protein kinase Cipk6 define a signaling pathway in plant immunity.

PubMed

de la Torre, Fernando; Gutiérrez-Beltrán, Emilio; Pareja-Jaime, Yolanda; Chakravarthy, Suma; Martin, Gregory B; del Pozo, Olga

2013-07-01

Ca(2+) signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virus-induced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca(2+)-signaling system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens. Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca(2+), suggesting that tomato Cbl10 and Cipk6 constitute a Ca(2+)-regulated signaling module. Overexpression of tomato Cipk6 in N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca(2+) and ROS signaling in plant immunity.

Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants.

PubMed

Anderson, Ryan G; Casady, Megan S; Fee, Rachel A; Vaughan, Martha M; Deb, Devdutta; Fedkenheuer, Kevin; Huffaker, Alisa; Schmelz, Eric A; Tyler, Brett M; McDowell, John M

2012-12-01

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR-like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non-pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's host. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Evolutionary Relationship of Disease Resistance Genes in Soybean and Arabidopsis Specific for the Pseudomonas syringae Effectors AvrB and AvrRpm11[W][OPEN

PubMed Central

Ashfield, Tom; Redditt, Thomas; Russell, Andrew; Kessens, Ryan; Rodibaugh, Natalie; Galloway, Lauren; Kang, Qing; Podicheti, Ram; Innes, Roger W.

2014-01-01

In Arabidopsis (Arabidopsis thaliana), the Pseudomonas syringae effector proteins AvrB and AvrRpm1 are both detected by the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) disease resistance (R) protein. By contrast, soybean (Glycine max) can distinguish between these effectors, with AvrB and AvrRpm1 being detected by the Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r R proteins, respectively. We have been using these genes to investigate the evolution of R gene specificity and have previously identified RPM1 and Rpg1b. Here, we report the cloning of Rpg1r, which, like RPM1 and Rpg1b, encodes a coiled-coil (CC)-nucleotide-binding (NB)-leucine-rich repeat (LRR) protein. As previously found for Rpg1b, we determined that Rpg1r is not orthologous with RPM1, indicating that the ability to detect both AvrB and AvrRpm1 evolved independently in soybean and Arabidopsis. The tightly linked soybean Rpg1b and Rpg1r genes share a close evolutionary relationship, with Rpg1b containing a recombination event that combined a NB domain closely related to Rpg1r with CC and LRR domains from a more distantly related CC-NB-LRR gene. Using structural modeling, we mapped polymorphisms between Rpg1b and Rpg1r onto the predicted tertiary structure of Rpg1b, which revealed highly polymorphic surfaces within both the CC and LRR domains. Assessment of chimeras between Rpg1b and Rpg1r using a transient expression system revealed that AvrB versus AvrRpm1 specificity is determined by the C-terminal portion of the LRR domain. The P. syringae effector AvrRpt2, which targets RPM1 INTERACTOR4 (RIN4) proteins in both Arabidopsis and soybean, partially blocked recognition of both AvrB and AvrRpm1 in soybean, suggesting that both Rpg1b and Rpg1r may detect these effectors via modification of a RIN4 homolog. PMID:25034017

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion

USDA-ARS?s Scientific Manuscript database

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae...

Virulence of Pseudomonas syringae pv. tomato DC3000 Is Influenced by the Catabolite Repression Control Protein Crc.

PubMed

Chakravarthy, Suma; Butcher, Bronwyn G; Liu, Yingyu; D'Amico, Katherine; Coster, Matthew; Filiatrault, Melanie J

2017-04-01

Pseudomonas syringae infects diverse plant species and is widely used as a model system in the study of effector function and the molecular basis of plant diseases. Although the relationship between bacterial metabolism, nutrient acquisition, and virulence has attracted increasing attention in bacterial pathology, it is largely unexplored in P. syringae. The Crc (catabolite repression control) protein is a putative RNA-binding protein that regulates carbon metabolism as well as a number of other factors in the pseudomonads. Here, we show that deletion of crc increased bacterial swarming motility and biofilm formation. The crc mutant showed reduced growth and symptoms in Arabidopsis and tomato when compared with the wild-type strain. We have evidence that the crc mutant shows delayed hypersensitive response (HR) when infiltrated into Nicotiana benthamiana and tobacco. Interestingly, the crc mutant was more susceptible to hydrogen peroxide, suggesting that, in planta, the mutant may be sensitive to reactive oxygen species generated during pathogen-associated molecular pattern-triggered immunity (PTI). Indeed, HR was further delayed when PTI-induced tissues were challenged with the crc mutant. The crc mutant did not elicit an altered PTI response in plants compared with the wild-type strain. We conclude that Crc plays an important role in growth and survival during infection.

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

PubMed

Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

2014-01-01

Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv. tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis.

PubMed

Wei, Hai-Lei; Zhang, Wei; Collmer, Alan

2018-05-08

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a complex repertoire of type III secretion effector (T3E) proteins. Effectorless polymutant DC3000D36E was used with a modularized system for native delivery of the 29 DC3000 T3Es singly and in pairs. Assays of the performance of this T3E library in N. benthamiana leaves revealed a matrix of T3E interplay, with six T3Es eliciting death and eight others variously suppressing the death activity of the six. The T3E library was also interrogated for effects on DC3000D36E elicitation of a reactive oxygen species burst, for growth in planta, and for T3Es that reversed these effects. Pseudomonas fluorescens and Agrobacterium tumefaciens heterologous delivery systems yielded notably different sets of death-T3Es. The DC3000D36E T3E library system highlights the importance of 13 T3Es and their interplay in interactions with N. benthamiana. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life

PubMed Central

Weßling, Ralf; Epple, Petra; Altmann, Stefan; He, Yijian; Yang, Li; Henz, Stefan R.; McDonald, Nathan; Wiley, Kristin; Bader, Kai Christian; Gläßer, Christine; Mukhtar, M. Shahid; Haigis, Sabine; Ghamsari, Lila; Stephens, Amber E.; Ecker, Joseph R.; Vidal, Marc; Jones, Jonathan D. G.; Mayer, Klaus F. X.; van Themaat, Emiel Ver Loren; Weigel, Detlef; Schulze-Lefert, Paul; Dangl, Jeffery L.; Panstruga, Ralph; Braun, Pascal

2014-01-01

SUMMARY While conceptual principles governing plant immunity are becoming clear, its systems-level organization and the evolutionary dynamic of the host-pathogen interface are still obscure. We generated a systematic protein-protein interaction network of virulence effectors from the ascomycete pathogen Golovinomyces orontii and Arabidopsis thaliana host proteins. We combined this dataset with corresponding data for the eubacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. The resulting network identifies host proteins onto which intraspecies and interspecies pathogen effectors converge. Phenotyping of 124 Arabidopsis effector-interactor mutants revealed a correlation between intra- and interspecies convergence and several altered immune response phenotypes. The effectors and most heavily targeted host protein co-localized in sub-nuclear foci. Products of adaptively selected Arabidopsis genes are enriched for interactions with effector targets. Our data suggest the existence of a molecular host-pathogen interface that is conserved across Arabidopsis accessions, while evolutionary adaptation occurs in the immediate network neighborhood of effector targets. PMID:25211078

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae1[OPEN

PubMed Central

Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-01-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. PMID:26265775

Redox proteomics of tomato in response to Pseudomonas syringae infection

PubMed Central

Balmant, Kelly Mayrink; Parker, Jennifer; Yoo, Mi-Jeong; Zhu, Ning; Dufresne, Craig; Chen, Sixue

2015-01-01

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation–reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses. PMID:26504582

A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner.

PubMed

Li, Qi; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Chen, Yanyu; Zhou, Jian-Min; Dou, Daolong

2016-05-31

Oomycete pathogens produce a large number of effectors to promote infection. Their mode of action are largely unknown. Here we show that a Phytophthora sojae effector, PsCRN63, suppresses flg22-induced expression of FRK1 gene, a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation, indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arabidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici. The callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants. Genes involved in PTI were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms an dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed, the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63.

Network Analysis Reveals a Common Host-Pathogen Interaction Pattern in Arabidopsis Immune Responses.

PubMed

Li, Hong; Zhou, Yuan; Zhang, Ziding

2017-01-01

Many plant pathogens secrete virulence effectors into host cells to target important proteins in host cellular network. However, the dynamic interactions between effectors and host cellular network have not been fully understood. Here, an integrative network analysis was conducted by combining Arabidopsis thaliana protein-protein interaction network, known targets of Pseudomonas syringae and Hyaloperonospora arabidopsidis effectors, and gene expression profiles in the immune response. In particular, we focused on the characteristic network topology of the effector targets and differentially expressed genes (DEGs). We found that effectors tended to manipulate key network positions with higher betweenness centrality. The effector targets, especially those that are common targets of an individual effector, tended to be clustered together in the network. Moreover, the distances between the effector targets and DEGs increased over time during infection. In line with this observation, pathogen-susceptible mutants tended to have more DEGs surrounding the effector targets compared with resistant mutants. Our results suggest a common plant-pathogen interaction pattern at the cellular network level, where pathogens employ potent local impact mode to interfere with key positions in the host network, and plant organizes an in-depth defense by sequentially activating genes distal to the effector targets.

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae.

PubMed

Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-09-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. © 2015 American Society of Plant Biologists. All Rights Reserved.

Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha

USDA-ARS?s Scientific Manuscript database

Programmed cell death (PCD) is triggered when Pto, a serine-threonine protein kinase recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv. tomato. This PCD requires MAPKKKalpha as a positive regulator in tomato and Nicotiana benthamiana. To examine how PCD-eliciting activi...

Recombineering Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants.

PubMed

DebRoy, Sruti; Thilmony, Roger; Kwack, Yong-Bum; Nomura, Kinya; He, Sheng Yang

2004-06-29

Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants. Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen. It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the Delta CEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp. stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms. We found that the loss of virulence in Delta CEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants. The Delta CEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis. This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants. The hopPtoM and avrE genes in the CEL of P. syringae were found to encode suppressors of this SA-dependent basal defense. The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants.

Recombineering using RecET from Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

miRNA863-3p sequentially targets negative regulators—atypical receptor-like Pseudokinases—and serrate, a positive regulator of plant immunity upon infection

USDA-ARS?s Scientific Manuscript database

Plant small RNAs (sRNAs) play important roles in regulating gene expression during pathogen infection. We identified miR863-3p, which is specifically induced by the avirulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 carrying the effector avrRpt2. During early infection stages, miR86...

Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity.

PubMed

Shan, Libo; He, Ping; Li, Jianming; Heese, Antje; Peck, Scott C; Nürnberger, Thorsten; Martin, Gregory B; Sheen, Jen

2008-07-17

Successful pathogens have evolved strategies to interfere with host immune systems. For example, the ubiquitous plant pathogen Pseudomonas syringae injects two sequence-distinct effectors, AvrPto and AvrPtoB, to intercept convergent innate immune responses stimulated by multiple microbe-associated molecular patterns (MAMPs). However, the direct host targets and precise molecular mechanisms of bacterial effectors remain largely obscure. We show that AvrPto and AvrPtoB bind the Arabidopsis receptor-like kinase BAK1, a shared signaling partner of both the flagellin receptor FLS2 and the brassinosteroid receptor BRI1. This targeting interferes with ligand-dependent association of FLS2 with BAK1 during infection. It also impedes BAK1-dependent host immune responses to diverse other MAMPs and brassinosteroid signaling. Significantly, the structural basis of AvrPto-BAK1 interaction appears to be distinct from AvrPto-Pto association required for effector-triggered immunity. These findings uncover a unique strategy of bacterial pathogenesis where virulence effectors block signal transmission through a key common component of multiple MAMP-receptor complexes.

The Fusarium oxysporum effector Six6 contributes to virulence and suppresses I-2-mediated cell death.

PubMed

Gawehns, F; Houterman, P M; Ichou, F Ait; Michielse, C B; Hijdra, M; Cornelissen, B J C; Rep, M; Takken, F L W

2014-04-01

Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death.

Pseudomonas caspiana sp. nov., a citrus pathogen in the Pseudomonas syringae phylogenetic group.

PubMed

Busquets, Antonio; Gomila, Margarita; Beiki, Farid; Mulet, Magdalena; Rahimian, Heshmat; García-Valdés, Elena; Lalucat, Jorge

2017-07-01

In a screening by multilocus sequence analysis of Pseudomonas strains isolated from diverse origins, 4 phylogenetically closely related strains (FBF58, FBF102 T , FBF103, and FBF122) formed a well-defined cluster in the Pseudomonas syringae phylogenetic group. The strains were isolated from citrus orchards in northern Iran with disease symptoms in the leaves and stems and its pathogenicity against citrus plants was demonstrated. The whole genome of the type strain of the proposed new species (FBF102 T =CECT 9164 T =CCUG 69273 T ) was sequenced and characterized. Comparative genomics with the 14 known Pseudomonas species type strains of the P. syringae phylogenetic group demonstrated that this strain belonged to a new genomic species, different from the species described thus far. Genome analysis detected genes predicted to be involved in pathogenesis, such as an atypical type 3 secretion system and two type 6 secretion systems, together with effectors and virulence factors. A polyphasic taxonomic characterization demonstrated that the 4 plant pathogenic strains represented a new species, for which the name Pseudomonas caspiana sp. nov. is proposed. Copyright © 2017 Elsevier GmbH. All rights reserved.

Population-genomic insights into emergence, crop adaptation and dissemination of Pseudomonas syringae pathogens

PubMed Central

Monteil, Caroline L.; Yahara, Koji; Studholme, David J.; Mageiros, Leonardos; Méric, Guillaume; Swingle, Bryan; Morris, Cindy E.

2016-01-01

Many bacterial pathogens are well characterized but, in some cases, little is known about the populations from which they emerged. This limits understanding of the molecular mechanisms underlying disease. The crop pathogen Pseudomonas syringae sensu lato has been widely isolated from the environment, including wild plants and components of the water cycle, and causes disease in several economically important crops. Here, we compared genome sequences of 45 P. syringae crop pathogen outbreak strains with 69 closely related environmental isolates. Phylogenetic reconstruction revealed that crop pathogens emerged many times independently from environmental populations. Unexpectedly, differences in gene content between environmental populations and outbreak strains were minimal with most virulence genes present in both. However, a genome-wide association study identified a small number of genes, including the type III effector genes hopQ1 and hopD1, to be associated with crop pathogens, but not with environmental populations, suggesting that this small group of genes may play an important role in crop disease emergence. Intriguingly, genome-wide analysis of homologous recombination revealed that the locus Psyr 0346, predicted to encode a protein that confers antibiotic resistance, has been frequently exchanged among lineages and thus may contribute to pathogen fitness. Finally, we found that isolates from diseased crops and from components of the water cycle, collected during the same crop disease epidemic, form a single population. This provides the strongest evidence yet that precipitation and irrigation water are an overlooked inoculum source for disease epidemics caused by P. syringae. PMID:28348830

Population-genomic insights into emergence, crop adaptation and dissemination of Pseudomonas syringae pathogens.

PubMed

Monteil, Caroline L; Yahara, Koji; Studholme, David J; Mageiros, Leonardos; Méric, Guillaume; Swingle, Bryan; Morris, Cindy E; Vinatzer, Boris A; Sheppard, Samuel K

2016-10-01

Many bacterial pathogens are well characterized but, in some cases, little is known about the populations from which they emerged. This limits understanding of the molecular mechanisms underlying disease. The crop pathogen Pseudomonas syringae sensu lato has been widely isolated from the environment, including wild plants and components of the water cycle, and causes disease in several economically important crops. Here, we compared genome sequences of 45 P. syringae crop pathogen outbreak strains with 69 closely related environmental isolates. Phylogenetic reconstruction revealed that crop pathogens emerged many times independently from environmental populations. Unexpectedly, differences in gene content between environmental populations and outbreak strains were minimal with most virulence genes present in both. However, a genome-wide association study identified a small number of genes, including the type III effector genes hopQ1 and hopD1 , to be associated with crop pathogens, but not with environmental populations, suggesting that this small group of genes may play an important role in crop disease emergence. Intriguingly, genome-wide analysis of homologous recombination revealed that the locus Psyr 0346, predicted to encode a protein that confers antibiotic resistance, has been frequently exchanged among lineages and thus may contribute to pathogen fitness. Finally, we found that isolates from diseased crops and from components of the water cycle, collected during the same crop disease epidemic, form a single population. This provides the strongest evidence yet that precipitation and irrigation water are an overlooked inoculum source for disease epidemics caused by P. syringae .

Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity.

PubMed

Popov, Georgy; Fraiture, Malou; Brunner, Frederic; Sessa, Guido

2016-08-01

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast-based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid-inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

Differential Regulation of Two-Tiered Plant Immunity and Sexual Reproduction by ANXUR Receptor-Like Kinases.

PubMed

Mang, Hyunggon; Feng, Baomin; Hu, Zhangjian; Boisson-Dernier, Aurélien; Franck, Christina M; Meng, Xiangzong; Huang, Yanyan; Zhou, Jinggeng; Xu, Guangyuan; Wang, Taotao; Shan, Libo; He, Ping

2017-12-01

Plants have evolved two tiers of immune receptors to detect infections: cell surface-resident pattern recognition receptors (PRRs) that sense microbial signatures and intracellular nucleotide binding domain leucine-rich repeat (NLR) proteins that recognize pathogen effectors. How PRRs and NLRs interconnect and activate the specific and overlapping plant immune responses remains elusive. A genetic screen for components controlling plant immunity identified ANXUR1 (ANX1), a malectin-like domain-containing receptor-like kinase, together with its homolog ANX2, as important negative regulators of both PRR- and NLR-mediated immunity in Arabidopsis thaliana ANX1 constitutively associates with the bacterial flagellin receptor FLAGELLIN-SENSING2 (FLS2) and its coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). Perception of flagellin by FLS2 promotes ANX1 association with BAK1, thereby interfering with FLS2-BAK1 complex formation to attenuate PRR signaling. In addition, ANX1 complexes with the NLR proteins RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2) and RESISTANCE TO P. SYRINGAE PV MACULICOLA1. ANX1 promotes RPS2 degradation and attenuates RPS2-mediated cell death. Surprisingly, a mutation that affects ANX1 function in plant immunity does not disrupt its function in controlling pollen tube growth during fertilization. Our study thus reveals a molecular link between PRR and NLR protein complexes that both associate with cell surface-resident ANX1 and uncovers uncoupled functions of ANX1 and ANX2 during plant immunity and sexual reproduction. © 2017 American Society of Plant Biologists. All rights reserved.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

PubMed

Hatsugai, Noriyuki; Igarashi, Daisuke; Mase, Keisuke; Lu, You; Tsuda, Yayoi; Chakravarthy, Suma; Wei, Hai-Lei; Foley, Joseph W; Collmer, Alan; Glazebrook, Jane; Katagiri, Fumiaki

2017-09-15

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses. © 2017 The Authors.

Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development

PubMed Central

Xiao, Fangming; Mark Goodwin, S; Xiao, Yanmei; Sun, Zhaoyu; Baker, Douglas; Tang, Xiaoyan; Jenks, Matthew A; Zhou, Jian-Min

2004-01-01

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces. PMID:15241470

Hexanoic acid is a resistance inducer that protects tomato plants against Pseudomonas syringae by priming the jasmonic acid and salicylic acid pathways.

PubMed

Scalschi, Loredana; Vicedo, Begonya; Camañes, Gemma; Fernandez-Crespo, Emma; Lapeña, Leonor; González-Bosch, Carmen; García-Agustín, Pilar

2013-05-01

Hexanoic acid-induced resistance (Hx-IR) is effective against several pathogens in tomato plants. Our study of the mechanisms implicated in Hx-IR against Pseudomonas syringae pv. tomato DC3000 suggests that hexanoic acid (Hx) treatment counteracts the negative effect of coronatine (COR) and jasmonyl-isoleucine (JA-Ile) on the salicylic acid (SA) pathway. In Hx-treated plants, an increase in the expression of jasmonic acid carboxyl methyltransferase (JMT) and the SA marker genes PR1 and PR5 indicates a boost in this signalling pathway at the expense of a decrease in JA-Ile. Moreover, Hx treatment potentiates 12-oxo-phytodienoic acid accumulation, which suggests that this molecule might play a role per se in Hx-IR. These results support a positive relationship between the SA and JA pathways in Hx-primed plants. Furthermore, one of the mechanisms of virulence mediated by COR is stomatal re-opening on infection with P. syringae. In this work, we observed that Hx seems to inhibit stomatal opening in planta in the presence of COR, which suggests that, on infection in tomato, this treatment suppresses effector action to prevent bacterial entry into the mesophyll. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Overlapping Yet Response-Specific Transcriptome Alterations Characterize the Nature of Tobacco-Pseudomonas syringae Interactions.

PubMed

Bozsó, Zoltán; Ott, Péter G; Kámán-Tóth, Evelin; Bognár, Gábor F; Pogány, Miklós; Szatmári, Ágnes

2016-01-01

In this study transcriptomic alterations of bacterially induced pattern triggered immunity (PTI) were compared with other types of tobacco-Pseudomonas interactions. In addition, using pharmacological agents we blocked some signal transduction pathways (Ca(2+) influx, kinases, phospholipases, proteasomic protein degradation) to find out how they contribute to gene expression during PTI. PTI is the first defense response of plant cells to microbes, elicited by their widely conserved molecular patterns. Tobacco is an important model of Solanaceae to study resistance responses, including defense mechanisms against bacteria. In spite of these facts the transcription regulation of tobacco genes during different types of plant bacterial interactions is not well-described. In this paper we compared the tobacco transcriptomic alterations in microarray experiments induced by (i) PTI inducer Pseudomonas syringae pv. syringae type III secretion mutant (hrcC) at earlier (6 h post inoculation) and later (48 hpi) stages of defense, (ii) wild type P. syringae (6 hpi) that causes effector triggered immunity (ETI) and cell death (HR), and (iii) disease-causing P. syringae pv. tabaci (6 hpi). Among the different treatments the highest overlap was between the PTI and ETI at 6 hpi, however, there were groups of genes with specifically altered activity for either type of defenses. Instead of quantitative effects of the virulent P. tabaci on PTI-related genes it influenced transcription qualitatively and blocked the expression changes of a special set of genes including ones involved in signal transduction and transcription regulation. P. tabaci specifically activated or repressed other groups of genes seemingly not related to either PTI or ETI. Kinase and phospholipase A inhibitors had highest impacts on the PTI response and effects of these signal inhibitors on transcription greatly overlapped. Remarkable interactions of phospholipase C-related pathways with the proteasomal system were also observable. Genes specifically affected by virulent P. tabaci belonged to various previously identified signaling routes, suggesting that compatible pathogens may modulate diverse signaling pathways of PTI to overcome plant defense.

Cellulose production in Pseudomonas syringae pv. syringae: a compromise between epiphytic and pathogenic lifestyles.

PubMed

Arrebola, Eva; Carrión, Víctor J; Gutiérrez-Barranquero, José Antonio; Pérez-García, Alejandro; Rodríguez-Palenzuela, Pablo; Cazorla, Francisco M; de Vicente, Antonio

2015-07-01

Genome sequencing and annotation have revealed a putative cellulose biosynthetic operon in the strain Pseudomonas syringae pv. syringae UMAF0158, the causal agent of bacterial apical necrosis. Bioinformatics analyses and experimental methods were used to confirm the functionality of the cellulose biosynthetic operon. In addition, the results showed the contribution of the cellulose operon to important aspects of P. syringae pv. syringae biology, such as the formation of biofilms and adhesion to the leaf surface of mango, suggesting that this operon increases epiphytic fitness. However, based on the incidence and severity of the symptoms observed in tomato leaflets, cellulose expression reduces virulence, as cellulose-deficient mutants increased the area of necrosis, whereas the cellulose-overproducing strain decreased the area of necrosis compared with the wild type. In conclusion, the results of this study show that the epiphytic and pathogenic stages of the P. syringae pv. syringae UMAF0158 lifestyle are intimately affected by cellulose production. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antagonism between salicylic and abscisic acid reflects early host-pathogen conflict and moulds plant defence responses.

PubMed

de Torres Zabala, Marta; Bennett, Mark H; Truman, William H; Grant, Murray R

2009-08-01

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant-pathogen interactions. Recently it has been demonstrated that abscisic acid signalling pathways are utilized by the bacterial phytopathogen Pseudomonas syringae to promote pathogenesis. In this study, we examined the dynamics, inter-relationship and impact of three key acidic phytohormones, salicylic acid, abscisic acid and jasmonic acid, and the bacterial virulence factor, coronatine, during progression of P. syringae infection of Arabidopsis thaliana. We show that levels of SA and ABA, but not JA, appear to play important early roles in determining the outcome of the infection process. SA is required in order to mount a full innate immune responses, while bacterial effectors act rapidly to activate ABA biosynthesis. ABA suppresses inducible innate immune responses by down-regulating SA biosynthesis and SA-mediated defences. Mutant analyses indicated that endogenous ABA levels represent an important reservoir that is necessary for effector suppression of plant-inducible innate defence responses and SA synthesis prior to subsequent pathogen-induced increases in ABA. Enhanced susceptibility due to loss of SA-mediated basal resistance is epistatically dominant over acquired resistance due to ABA deficiency, although ABA also contributes to symptom development. We conclude that pathogen-modulated ABA signalling rapidly antagonizes SA-mediated defences. We predict that hormonal perturbations, either induced or as a result of environmental stress, have a marked impact on pathological outcomes, and we provide a mechanistic basis for understanding priming events in plant defence.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae.

PubMed

Nemchinov, Lev G; Shao, Jonathan; Lee, Maya N; Postnikova, Olga A; Samac, Deborah A

2017-01-01

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae

PubMed Central

Shao, Jonathan; Lee, Maya N.; Postnikova, Olga A.; Samac, Deborah A.

2017-01-01

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties. PMID:29244864

Host target modification as a strategy to counter pathogen hijacking of the jasmonate hormone receptor

DOE PAGES

Zhang, Li; Yao, Jian; Withers, John; ...

2015-11-02

In the past decade, characterization of the host targets of pathogen virulence factors took a center stage in the study of pathogenesis and disease susceptibility in plants and humans. However, the impressive knowledge of host targets has not been broadly exploited to inhibit pathogen infection. In this paper, we show that host target modification could be a promising new approach to “protect” the disease-vulnerable components of plants. In particular, recent studies have identified the plant hormone jasmonate (JA) receptor as one of the common targets of virulence factors from highly evolved biotrophic/hemibiotrophic pathogens. Strains of the bacterial pathogen Pseudomonas syringae,more » for example, produce proteinaceous effectors, as well as a JA-mimicking toxin, coronatine (COR), to activate JA signaling as a mechanism to promote disease susceptibility. Guided by the crystal structure of the JA receptor and evolutionary clues, we succeeded in modifying the JA receptor to allow for sufficient endogenous JA signaling but greatly reduced sensitivity to COR. Transgenic Arabidopsis expressing this modified receptor not only are fertile and maintain a high level of insect defense, but also gain the ability to resist COR-producing pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. Finally, our results provide a proof-of-concept demonstration that host target modification can be a promising new approach to prevent the virulence action of highly evolved pathogens.« less

AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000

USDA-ARS?s Scientific Manuscript database

Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and is active while the bacteria are associa...

Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7.

PubMed

Nicaise, Valerie; Joe, Anna; Jeong, Byeong-ryool; Korneli, Christin; Boutrot, Freddy; Westedt, Isa; Staiger, Dorothee; Alfano, James R; Zipfel, Cyril

2013-03-06

Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III-secreted effector HopU1 is a mono-ADP-ribosyltransferase required for full virulence on Arabidopsis thaliana. HopU1 targets several RNA-binding proteins including GRP7, whose role in immunity is still unclear. Here, we show that GRP7 associates with translational components, as well as with the pattern recognition receptors FLS2 and EFR. Moreover, GRP7 binds specifically FLS2 and EFR transcripts in vivo through its RNA recognition motif. HopU1 does not affect the protein-protein associations between GRP7, FLS2 and translational components. Instead, HopU1 blocks the interaction between GRP7 and FLS2 and EFR transcripts in vivo. This inhibition correlates with reduced FLS2 protein levels upon Pseudomonas infection in a HopU1-dependent manner. Our results reveal a novel virulence strategy used by a microbial effector to interfere with host immunity.

Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses

PubMed Central

Dudnik, Alexey; Dudler, Robert

2014-01-01

The Pseudomonas syringae species complex has recently been named the number one plant pathogen, due to its economic and environmental impacts, as well as for its role in scientific research. The bacterium has been repeatedly reported to cause outbreaks on bean, cucumber, stone fruit, kiwi and olive tree, as well as on other crop and non-crop plants. It also serves as a model organism for research on the Type III secretion system (T3SS) and plant-pathogen interactions. While most of the current work on this pathogen is either carried out on one of three model strains found on dicot plants with completely sequenced genomes or on isolates obtained from recent outbreaks, not much is known about strains isolated from grasses (Poaceae). Here, we use comparative genomics in order to identify putative virulence-associated genes and other Poaceae-specific adaptations in several newly available genome sequences of strains isolated from grass species. All strains possess only a small number of known Type III effectors, therefore pointing to the importance of non-Type III secreted virulence factors. The implications of this finding are discussed. PMID:25437611

Identification of the CvsSR regulon in Pseudomonas syringae reveals overlap with the Type-III secretion and AlgU regulons

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae pv. tomato DC3000 (Pto) lives epiphytically and endophytically during its infection cycle. Two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors are used by Pto to sense environmental changes within the leaf apoplast during pathogenesis. The TCS, CvsSR i...

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 Family Member, Is Needed To Produce l-allo-Isoleucine, a Precursor for the Phytotoxin Coronatine

PubMed Central

Worley, Jay N.; Russell, Alistair B.; Wexler, Aaron G.; Bronstein, Philip A.; Kvitko, Brian H.; Krasnoff, Stuart B.; Munkvold, Kathy R.; Swingle, Bryan

2013-01-01

Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 μg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family. PMID:23144243

Leaf shedding as an anti-bacterial defense in Arabidopsis cauline leaves

PubMed Central

2017-01-01

Plants utilize an innate immune system to protect themselves from disease. While many molecular components of plant innate immunity resemble the innate immunity of animals, plants also have evolved a number of truly unique defense mechanisms, particularly at the physiological level. Plant’s flexible developmental program allows them the unique ability to simply produce new organs as needed, affording them the ability to replace damaged organs. Here we develop a system to study pathogen-triggered leaf abscission in Arabidopsis. Cauline leaves infected with the bacterial pathogen Pseudomonas syringae abscise as part of the defense mechanism. Pseudomonas syringae lacking a functional type III secretion system fail to elicit an abscission response, suggesting that the abscission response is a novel form of immunity triggered by effectors. HAESA/HAESA-like 2, INFLORESCENCE DEFICIENT IN ABSCISSION, and NEVERSHED are all required for pathogen-triggered abscission to occur. Additionally phytoalexin deficient 4, enhanced disease susceptibility 1, salicylic acid induction-deficient 2, and senescence-associated gene 101 plants with mutations in genes necessary for bacterial defense and salicylic acid signaling, and NahG transgenic plants with low levels of salicylic acid fail to abscise cauline leaves normally. Bacteria that physically contact abscission zones trigger a strong abscission response; however, long-distance signals are also sent from distal infected tissue to the abscission zone, alerting the abscission zone of looming danger. We propose a threshold model regulating cauline leaf defense where minor infections are handled by limiting bacterial growth, but when an infection is deemed out of control, cauline leaves are shed. Together with previous results, our findings suggest that salicylic acid may regulate both pathogen- and drought-triggered leaf abscission. PMID:29253890

The ECF sigma factor, PSPTO_1043, in Pseudomonas syringae pv. tomato DC3000 is induced by oxidative stress and regulates genes involved in oxidative stress response

PubMed Central

Butcher, Bronwyn G.; Bao, Zhongmeng; Wilson, Janet; Swingle, Bryan; Filiatrault, Melanie; Schneider, David; Cartinhour, Samuel

2017-01-01

The bacterial plant pathogen Pseudomonas syringae adapts to changes in the environment by modifying its gene expression profile. In many cases, the response is mediated by the activation of extracytoplasmic function (ECF) sigma factors that direct RNA polymerase to transcribe specific sets of genes. In this study we focus on PSPTO_1043, one of ten ECF sigma factors in P. syringae pv. tomato DC3000 (DC3000). PSPTO_1043, together with PSPTO_1042, encode an RpoERsp/ChrR-like sigma/anti-sigma factor pair. Although this gene pair is unique to the P. syringae group among the pseudomonads, homologous genes can be found in photosynthetic genera such as Rhodospirillum, Thalassospira, Phaeospirillum and Parvibaculum. Using ChIP-Seq, we detected 137 putative PSPTO_1043 binding sites and identified a likely promoter motif. We characterized 13 promoter candidates, six of which regulate genes that appear to be found only in P. syringae. PSPTO_1043 responds to the presence of singlet oxygen (1O2) and tert-butyl hydroperoxide (tBOOH) and several of the genes regulated by PSPTO_1043 appear to be involved in response to oxidative stress. PMID:28700608

The ECF sigma factor, PSPTO_1043, in Pseudomonas syringae pv. tomato DC3000 is induced by oxidative stress and regulates genes involved in oxidative stress response.

PubMed

Butcher, Bronwyn G; Bao, Zhongmeng; Wilson, Janet; Stodghill, Paul; Swingle, Bryan; Filiatrault, Melanie; Schneider, David; Cartinhour, Samuel

2017-01-01

The bacterial plant pathogen Pseudomonas syringae adapts to changes in the environment by modifying its gene expression profile. In many cases, the response is mediated by the activation of extracytoplasmic function (ECF) sigma factors that direct RNA polymerase to transcribe specific sets of genes. In this study we focus on PSPTO_1043, one of ten ECF sigma factors in P. syringae pv. tomato DC3000 (DC3000). PSPTO_1043, together with PSPTO_1042, encode an RpoERsp/ChrR-like sigma/anti-sigma factor pair. Although this gene pair is unique to the P. syringae group among the pseudomonads, homologous genes can be found in photosynthetic genera such as Rhodospirillum, Thalassospira, Phaeospirillum and Parvibaculum. Using ChIP-Seq, we detected 137 putative PSPTO_1043 binding sites and identified a likely promoter motif. We characterized 13 promoter candidates, six of which regulate genes that appear to be found only in P. syringae. PSPTO_1043 responds to the presence of singlet oxygen (1O2) and tert-butyl hydroperoxide (tBOOH) and several of the genes regulated by PSPTO_1043 appear to be involved in response to oxidative stress.

Investigation of Intercellular Salicylic Acid Accumulation during Compatible and Incompatible Arabidopsis-Pseudomonas syringae Interactions Using a Fast Neutron-Generated Mutant Allele of EDS5 Identified by Genetic Mapping and Whole-Genome Sequencing

PubMed Central

Catana, Vasile; Golding, Brian; Weretilnyk, Elizabeth A.; Cameron, Robin K.

2014-01-01

A whole-genome sequencing technique developed to identify fast neutron-induced deletion mutations revealed that iap1-1 is a new allele of EDS5 (eds5-5). RPS2-AvrRpt2-initiated effector-triggered immunity (ETI) was compromised in iap1-1/eds5-5 with respect to in planta bacterial levels and the hypersensitive response, while intra- and intercellular free salicylic acid (SA) accumulation was greatly reduced, suggesting that SA contributes as both an intracellular signaling molecule and an antimicrobial agent in the intercellular space during ETI. During the compatible interaction between wild-type Col-0 and virulent Pseudomonas syringae pv. tomato (Pst), little intercellular free SA accumulated, which led to the hypothesis that Pst suppresses intercellular SA accumulation. When Col-0 was inoculated with a coronatine-deficient strain of Pst, high levels of intercellular SA accumulation were observed, suggesting that Pst suppresses intercellular SA accumulation using its phytotoxin coronatine. This work suggests that accumulation of SA in the intercellular space is an important component of basal/PAMP-triggered immunity as well as ETI to pathogens that colonize the intercellular space. PMID:24594657

Powdery mildews on lilac in western North America include Phyllactinia syringae, sp. nov.

PubMed

Larsen, Harold J; Braun, Uwe; Blomquist, Cheryl; Woods, Patrick; Mohan, S Krishna

2017-01-01

Two powdery mildews, Erysiphe syringae and the previously undescribed Phyllactinia syringae, sp. nov., occur on lilac in western North America. Phyllactinia syringae is found on common lilac, whereas E. syringae is found on Chinese lilac and, occasionally, common lilac. Infection by P. syringae is extremely unobtrusive until formation of a hypophyllous mycelial mat with chasmothecia in late fall. Infection by E. syringae in late summer is conspicuous, with its thick, superficial mycelial mat on the leaf upper surface detracting from the aesthetic appearance of the bush.

Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis

PubMed Central

Cunty, A.; Cesbron, S.; Poliakoff, F.; Jacques, M.-A.

2015-01-01

The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level. PMID:26209667

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

PubMed

Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

2011-07-01

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

An Extracytoplasmic Function Sigma Factor-Mediated Cell Surface Signaling System in Pseudomonas syringae pv. tomato DC3000 Regulates Gene Expression in Response to Heterologous Siderophores ▿ †

PubMed Central

Markel, Eric; Maciak, Charlene; Butcher, Bronwyn G.; Myers, Christopher R.; Stodghill, Paul; Bao, Zhongmeng; Cartinhour, Sam; Swingle, Bryan

2011-01-01

The diversity of regulatory systems encoded by bacteria provides an indication of the variety of stresses and interactions that these organisms encounter in nature. We have been investigating how the plant pathogen Pseudomonas syringae pv. tomato DC3000 responds to iron limitation and have focused on the iron starvation (IS) sigma factors to identify regulon members and to explore the mechanistic details of genetic control for this class of regulators. In the study described in this report, we used chromatin immunoprecipitation paired with high-throughput sequencing (ChIP-Seq) to screen the genome for locations associated with binding of the P. syringae IS sigma factor PSPTO_1203. We used multiple methods to demonstrate differential regulation of two genes identified in the ChIP-Seq screen and characterize the promoter elements that facilitate PSPTO_1203-dependent regulation. The genes regulated by PSPTO_1203 encode a TonB-dependent transducer (PSPTO_1206) and a cytoplasmic membrane protein (PSPTO_2145), which is located in the P. syringae pyoverdine cluster. Additionally, we identified siderophores that induce the activity of PSPTO_1203 and used this information to investigate the functional components of the signal transduction cascade. PMID:21840980

Novel Positive Regulatory Role for the SPL6 Transcription Factor in the N TIR-NB-LRR Receptor-Mediated Plant Innate Immunity

PubMed Central

Padmanabhan, Meenu S.; Ma, Shisong; Burch-Smith, Tessa M.; Czymmek, Kirk; Huijser, Peter; Dinesh-Kumar, Savithramma P.

2013-01-01

Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the Nicotiana TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to Tobacco mosaic virus. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against Pseudomonas syringae carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5. PMID:23516366

Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

PubMed Central

Mesarich, Carl H.; Rees-George, Jonathan; Gardner, Paul P.; Ghomi, Fatemeh Ashari; Gerth, Monica L.; Andersen, Mark T.; Rikkerink, Erik H. A.; Fineran, Peter C.

2017-01-01

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a ‘phenotype of interest’ (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with ‘Fuzzy-Spreader’-like morphologies were also identified through a visual screen. The second, a ‘mutant of interest’ (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid. PMID:28249011

Genomic Analysis of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Provides Insight into the Origins of an Emergent Plant Disease

PubMed Central

McCann, Honour C.; Rikkerink, Erik H. A.; Bertels, Frederic; Fiers, Mark; Lu, Ashley; Rees-George, Jonathan; Andersen, Mark T.; Gleave, Andrew P.; Haubold, Bernhard; Wohlers, Mark W.; Guttman, David S.; Wang, Pauline W.; Straub, Christina; Vanneste, Joel; Rainey, Paul B.; Templeton, Matthew D.

2013-01-01

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries – even millennia – ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease. PMID:23935484

The release of alginate lyase from growing Pseudomonas syringae pathovar phaseolicola

NASA Technical Reports Server (NTRS)

Ott, C. M.; Day, D. F.; Koenig, D. W.; Pierson, D. L.

2001-01-01

Pseudomonas syringae pathovar phaseolicola, which produces alginate during stationary growth phase, displayed elevated extracellular alginate lyase activity during both mid-exponential and late-stationary growth phases of batch growth. Intracellular activity remained below 22% of the total activity during exponential growth, suggesting that alginate lyase has an extracellular function for this organism. Extracellular enzyme activity in continuous cultures, grown in either nutrient broth or glucose-simple salts medium, peaked at 60% of the washout rate, although nutrient broth-grown cultures displayed more than twice the activity per gram of cell mass. These results imply that growth rate, nutritional composition, or both initiate a release of alginate lyase from viable P. syringae pv. phaseolicola, which could modify its entrapping biofilm.

Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato.

PubMed

Richter, Carolin; Dirks, Mareike E; Gronover, Christian Schulze; Prüfer, Dirk; Moerschbacher, Bruno M

2012-02-01

Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and β-glucuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P. syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis.

PubMed

Cunty, A; Cesbron, S; Poliakoff, F; Jacques, M-A; Manceau, C

2015-10-01

The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse.

PubMed

Martín-Sanz, Alberto; de la Vega, Marcelino Pérez; Murillo, Jesús; Caminero, Constantino

2013-07-01

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

Comparative genomics reveals genes significantly associated with woody hosts in the plant pathogen Pseudomonas syringae

PubMed Central

Laue, Bridget E.; Sharp, Paul M.; Green, Sarah

2016-01-01

Summary The diversification of lineages within Pseudomonas syringae has involved a number of adaptive shifts from herbaceous hosts onto various species of tree, resulting in the emergence of highly destructive diseases such as bacterial canker of kiwi and bleeding canker of horse chestnut. This diversification has involved a high level of gene gain and loss, and these processes are likely to play major roles in the adaptation of individual lineages onto their host plants. In order to better understand the evolution of P. syringae onto woody plants, we have generated de novo genome sequences for 26 strains from the P. syringae species complex that are pathogenic on a range of woody species, and have looked for statistically significant associations between gene presence and host type (i.e. woody or herbaceous) across a phylogeny of 64 strains. We have found evidence for a common set of genes associated with strains that are able to colonize woody plants, suggesting that divergent lineages have acquired similarities in genome composition that may form the genetic basis of their adaptation to woody hosts. We also describe in detail the gain, loss and rearrangement of specific loci that may be functionally important in facilitating this adaptive shift. Overall, our analyses allow for a greater understanding of how gene gain and loss may contribute to adaptation in P. syringae. PMID:27145446

Characterization of pyoverdine and achromobactin in Pseudomonas syringae pv. phaseolicola 1448a

PubMed Central

2011-01-01

Background Pseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation P. syringae 1448a secretes siderophores for active uptake of iron. The primary siderophore of P. syringae 1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of Pseudomonas often exhibit structural variations in the pyoverdine produced by different strains, all P. syringae pathovars previously tested have been found to make an identical pyoverdine molecule. P. syringae 1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different P. syringae pathovars. Results Five putative pyoverdine NRPS genes in P. syringae 1448a were characterized in-silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from P. syringae 1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other P. syringae pathovars, but surprisingly P. syringae 1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of P. syringae 1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions. Conclusions This study provides the first evidence of a P. syringae pathovar producing a side chain variant form of pyoverdine. We also describe novel IC50 and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for P. syringae 1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies. PMID:21967163

Diversity and Abundance of Ice Nucleating Strains of Pseudomonas syringae in a Freshwater Lake in Virginia, USA.

PubMed

Pietsch, Renée B; Vinatzer, Boris A; Schmale, David G

2017-01-01

The bacterium Pseudomonas syringae is found in a variety of terrestrial and aquatic environments. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as Ice+) allowing them to catalyze the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in France, little is known about the genetic diversity of P. syringae in natural aquatic habitats in North America. We collected samples of freshwater from three different depths in Claytor Lake, Virginia, USA between November 2015 and June 2016. Samples were plated on non-selective medium (TSA) and on medium selective for Pseudomonas (KBC) and closely related species to estimate the total number of culturable bacteria and of Pseudomonas , respectively. A droplet freezing assay was used to screen colonies for the Ice+ phenotype. Ice+ colonies were then molecularly identified based on the cts (citrate synthase) gene and the 16S rDNA gene. Phylogenetic analysis of cts sequences showed a surprising diversity of phylogenetic subgroups of P. syringae . Frequencies of Ice+ isolates on P. syringae selective medium ranged from 0 to 15% per sample with the highest frequency being found in spring. Our work shows that freshwater lakes can be a significant reservoir of Ice+ P. syringae . Future work is needed to determine the contribution of P. syringae from freshwater lakes to the P. syringae populations present in the atmosphere and on plants and, in particular, if freshwater lakes could be an inoculum source of P. syringae -caused plant disease outbreaks.

Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae

PubMed Central

Deng, Xin; Liang, Haihua; Chen, Kai; He, Chuan; Lan, Lefu; Tang, Xiaoyan

2014-01-01

Pseudomonas syringae uses the two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanisms and the regulons of RhpRS have yet to be fully elucidated. Here, we show that RhpS functions as a kinase and a phosphatase on RhpR and as an autokinase upon itself. RhpR is phosphorylated by the small phosphodonor acetyl phosphate. A specific RhpR-binding site containing the inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by a DNase I footprint analysis. Electrophoretic mobility shift assay indicated that P-RhpR has a higher binding affinity to the IR motif than RhpR. To identify additional RhpR targets in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) and detected 167 enriched loci including the hrpR promoter, suggesting the direct regulation of T3SS cascade genes by RhpR. A genome-wide microarray analysis showed that, in addition to the T3SS cascade genes, RhpR differentially regulates a large set of genes with various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes by coordinating T3SS expression and many other biological processes. PMID:25249629

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2014 CFR

2014-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2012 CFR

2012-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2013 CFR

2013-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2010 CFR

2010-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

Characterization of Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast of Mandarin in Montenegro.

PubMed

Ivanović, Žarko; Perović, Tatjana; Popović, Tatjana; Blagojević, Jovana; Trkulja, Nenad; Hrnčić, Snježana

2017-02-01

Citrus blast caused by bacterium Pseudomonas syringae is a very important disease of citrus occuring in many areas of the world, but with few data about genetic structure of the pathogen involved. Considering the above fact, this study reports genetic characterization of 43 P. syringae isolates obtained from plant tissue displaying citrus blast symptoms on mandarin ( Citrus reticulata ) in Montenegro, using multilocus sequence analysis of gyrB , rpoD , and gap1 gene sequences. Gene sequences from a collection of 54 reference pathotype strains of P. syringae from the Plant Associated and Environmental Microbes Database (PAMDB) was used to establish a genetic relationship with our isolates obtained from mandarin. Phylogenetic analyses of gyrB , rpoD , and gap1 gene sequences showed that P. syringae pv. syringae causes citrus blast in mandarin in Montenegro, and belongs to genomospecies 1. Genetic homogeneity of isolates suggested that the Montenegrian population might be clonal which indicates a possible common source of infection. These findings may assist in further epidemiological studies of this pathogen and for determining mandarin breeding strategies for P. syringae control.

Characterization of Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast of Mandarin in Montenegro

PubMed Central

Ivanović, Žarko; Perović, Tatjana; Popović, Tatjana; Blagojević, Jovana; Trkulja, Nenad; Hrnčić, Snježana

2017-01-01

Citrus blast caused by bacterium Pseudomonas syringae is a very important disease of citrus occuring in many areas of the world, but with few data about genetic structure of the pathogen involved. Considering the above fact, this study reports genetic characterization of 43 P. syringae isolates obtained from plant tissue displaying citrus blast symptoms on mandarin (Citrus reticulata) in Montenegro, using multilocus sequence analysis of gyrB, rpoD, and gap1 gene sequences. Gene sequences from a collection of 54 reference pathotype strains of P. syringae from the Plant Associated and Environmental Microbes Database (PAMDB) was used to establish a genetic relationship with our isolates obtained from mandarin. Phylogenetic analyses of gyrB, rpoD, and gap1 gene sequences showed that P. syringae pv. syringae causes citrus blast in mandarin in Montenegro, and belongs to genomospecies 1. Genetic homogeneity of isolates suggested that the Montenegrian population might be clonal which indicates a possible common source of infection. These findings may assist in further epidemiological studies of this pathogen and for determining mandarin breeding strategies for P. syringae control. PMID:28167885

Cloning of genes required for hypersensitivity and pathogenicity in Pseudomonas syringae pv. aptata.

PubMed

Minardi, P

1995-01-01

A genomic library of Pseudomonas syringae pv. aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv. syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P. syringae pv. aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis of EcoRI-digested genomic DNA of P. syringae pv. aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P. syringae pv. aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11. These results indicate that P. syringae pv. aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P. syringae pv. syringae.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae

USDA-ARS?s Scientific Manuscript database

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L.) in the central and western U.S. and has been reported in Australia and Europe. The disease is not always recognized because symptoms are often associated with frost damage. Two culti...

Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity

PubMed Central

Fabro, Georgina; Steinbrenner, Jens; Coates, Mary; Ishaque, Naveed; Baxter, Laura; Studholme, David J.; Körner, Evelyn; Allen, Rebecca L.; Piquerez, Sophie J. M.; Rougon-Cardoso, Alejandra; Greenshields, David; Lei, Rita; Badel, Jorge L.; Caillaud, Marie-Cecile; Sohn, Kee-Hoon; Van den Ackerveken, Guido; Parker, Jane E.; Beynon, Jim; Jones, Jonathan D. G.

2011-01-01

Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (∼70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation. PMID:22072967

[Erwinia amylovora--the fire blight pathogen of trees in Ukraine].

PubMed

Iakovleva, L M; Moroz, S N; Shcherbina, T N; Ogorodnik, L E; Gvozdiak, R I; Patyka, V F

2014-01-01

Niduses of fire blight of fruit and ornamental trees have been found in the Kyiv and Vinnitsa regions of Ukraine. Pathogen Erwinia amylovora was isolated between April and October. The pathogen was often accompanied by bacteria Pseudomonas syringae pv. syringae. Artificial infection with a mixture of bacteria E. amylovora and P. syringae pv. syringae accelerates and enhances the disease process in the laboratory.

Molecular signatures in Arabidopsis thaliana in response to insect attack and bacterial infection.

PubMed

Barah, Pankaj; Winge, Per; Kusnierczyk, Anna; Tran, Diem Hong; Bones, Atle M

2013-01-01

Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species Arabidopsis thaliana (A. thaliana henceforth) during insect Brevicoryne brassicae (B. brassicae henceforth) and bacterial Pseudomonas syringae pv. tomato strain DC3000 (P. syringae henceforth) attacks. The main aim of this study was to identify the attacker-specific and general defence response signatures in A. thaliana when attacked by phloem-feeding aphids or pathogenic bacteria. The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as WRKY, MYB, ERF, BHLH and bZIP, could be crucial for stress-specific defence regulation in Arabidopsis during aphid and P. syringae attack. The defence response pathways, signalling pathways and metabolic processes associated with aphid attack and P. syringae infection partially overlapped. Components of several important biosynthesis and signalling pathways, such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and glucosinolates, were differentially affected during the two the treatments. Several stress-regulated transcription factors were known to be associated with stress-inducible microRNAs. The differentially regulated gene sets included many signature transcription factors, and our co-expression analysis showed that they were also strongly co-expressed during 69 other biotic stress experiments. Defence responses and functional networks that were unique and specific to aphid or P. syringae stresses were identified. Furthermore, our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and, thus gives indicates a new direction for conducting large-scale targeted experiments to explore the detailed regulatory links between them. The presented results provide a comparative understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at the transcriptomic level.

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity1[OPEN

PubMed Central

Connor, Richard A.

2017-01-01

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. PMID:27909045

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity.

PubMed

Zhou, Bangjun; Mural, Ravi V; Chen, Xuanyang; Oates, Matt E; Connor, Richard A; Martin, Gregory B; Gough, Julian; Zeng, Lirong

2017-02-01

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. © 2017 American Society of Plant Biologists. All Rights Reserved.

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against Pseudomonas syringae Pathogens

PubMed Central

Lee, Seonghee; Rojas, Clemencia M.; Ishiga, Yasuhiro; Pandey, Sona; Mysore, Kirankumar S.

2013-01-01

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae. PMID:24349286

Gene-for-gene relationship in the host-pathogen system Malus × robusta 5-Erwinia amylovora.

PubMed

Vogt, Isabelle; Wöhner, Thomas; Richter, Klaus; Flachowsky, Henryk; Sundin, George W; Wensing, Annette; Savory, Elizabeth A; Geider, Klaus; Day, Brad; Hanke, Magda-Viola; Peil, Andreas

2013-03-01

Fire blight is a destructive bacterial disease caused by Erwinia amylovora affecting plants in the family Rosaceae, including apple. Host resistance to fire blight is present mainly in accessions of Malus spp. and is thought to be quantitative in this pathosystem. In this study we analyzed the importance of the E. amylovora effector avrRpt2(EA) , a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2(EA) was able to overcome the fire blight resistance of Mr5. One single nucleotide polymorphism (SNP), resulting in an exchange of cysteine to serine in the encoded protein, was detected in avrRpt2(EA) of several Erwinia strains differing in virulence to Mr5. E. amylovora strains encoding serine (S-allele) were able to overcome resistance of Mr5, whereas strains encoding cysteine (C-allele) were not. Allele specificity was also observed in a coexpression assay with Arabidopsis thaliana RIN4 in Nicotiana benthamiana. A homolog of RIN4 has been detected and isolated in Mr5. These results suggest a system similar to the interaction of RPS2 from A. thaliana and AvrRpt2 from P. syringae with RIN4 as guard. Our data are suggestive of a gene-for-gene relationship for the host-pathogen system Mr5 and E. amylovora. No claim to original US government works. New Phytologist © 2013 New Phytologist Trust.

40 CFR 180.1145 - Pseudomonas syringae; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... FOOD Exemptions From Tolerances § 180.1145 Pseudomonas syringae; exemption from the requirement of a tolerance. Pseudomonas syringae is exempted from the requirement of a tolerance on all raw agricultural...

40 CFR 180.1145 - Pseudomonas syringae; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... FOOD Exemptions From Tolerances § 180.1145 Pseudomonas syringae; exemption from the requirement of a tolerance. Pseudomonas syringae is exempted from the requirement of a tolerance on all raw agricultural...

Analyses of natural variation indicates that the absence of RPS4/RRS1 and amino acid change in RPS4 cause loss of their functions and resistance to pathogens.

PubMed

Narusaka, Mari; Iuchi, Satoshi; Narusaka, Yoshihiro

2017-03-04

A pair of Arabidopsis thaliana resistance proteins, RPS4 and RRS1, recognizes the cognate Avr effector from the bacterial pathogens Pseudomonas syringae pv. tomato expressing avrRps4 (Pst-avrRps4), Ralstonia solanacearum, and the fungal pathogen Colletotrichum higginsianum and leads to defense signaling activation against the pathogens. In the present study, we analyzed 14 A. thaliana accessions for natural variation in Pst-avrRps4 and C. higginsianum susceptibility, and found new compatible and incompatible Arabidopsis-pathogen interactions. We first found that A. thaliana accession Cvi-0 is susceptible to Pst-avrRps4. Interestingly, the genome sequence assembly indicated that Cvi-0 lost both RPS4 and RRS1, but not RPS4B and RRS1B, compared to the reference genome sequence from A. thaliana accession Col-0. On the other hand, the natural variation analysis of RPS4 alleles from various Arabidopsis accessions revealed that one amino-acid change, Y950H, is responsible for the loss of resistance to Pst-avrRps4 and C. higginsianum in RLD-0. Our data indicate that the amino acid change, Y950H, in RPS4 resulted in the loss of both RPS4 and RRS1 functions and resistance to pathogens.

The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.

PubMed

Morris, Cindy E; Sands, David C; Vinatzer, Boris A; Glaux, Catherine; Guilbaud, Caroline; Buffière, Alain; Yan, Shuangchun; Dominguez, Hélène; Thompson, Brian M

2008-03-01

Pseudomonas syringae is a plant pathogen well known for its capacity to grow epiphytically on diverse plants and for its ice-nucleation activity. The ensemble of its known biology and ecology led us to postulate that this bacterium is also present in non-agricultural habitats, particularly those associated with water. Here, we report the abundance of P. syringae in rain, snow, alpine streams and lakes and in wild plants, in addition to the previously reported abundance in epilithic biofilms. Each of these substrates harbored strains that corresponded to P. syringae in terms of biochemical traits, pathogenicity and pathogenicity-related factors and that were ice-nucleation active. Phylogenetic comparisons of sequences of four housekeeping genes of the non-agricultural strains with strains of P. syringae from disease epidemics confirmed their identity as P. syringae. Moreover, strains belonging to the same clonal lineage were isolated from snow, irrigation water and a diseased crop plant. Our data suggest that the different substrates harboring P. syringae modify the structure of the associated populations. Here, we propose a comprehensive life cycle for P. syringae--in agricultural and non-agricultural habitats--driven by the environmental cycle of water. This cycle opens the opportunity to evaluate the importance of non-agricultural habitats in the evolution of a plant pathogen and the emergence of virulence. The ice-nucleation activity of all strains from snow, unlike from other substrates, strongly suggests that P. syringae plays an active role in the water cycle as an ice nucleus in clouds.

Biophysical characterization of soluble Pseudomonas syringae ice nucleation protein InaZ fragments.

PubMed

Han, Yu Jin; Song, HyoJin; Lee, Chang Woo; Ly, Nguyễn Hoàng; Joo, Sang-Woo; Lee, Jun Hyuck; Kim, Soon-Jong; Park, SangYoun

2017-01-01

Ice nucleation protein (INP) with its functional domain consisting of multiple 48-residue repeat units effectively induces super-cooled water into ice. Circular dichroism and infrared deconvolution analyses on a soluble 240-residue fragment of Pseudomonas syringae InaZ (InaZ240) containing five 48-residue repeat units indicated that it is mostly composed of β-sheet and random coil. Analytical ultracentrifugation suggested that InaZ240 behaves as a monomer of an elongated ellipsoid. However, InaZ240 showed only minimum ice binding compared to anti-freeze proteins. Other P. syringae InaZ proteins with more 48-residue repeat units were made, in which the largest soluble fragment obtainable was an InaZ with twelve 48-residue repeat units. Size-exclusion chromatography analyses further suggested that the overall shape of the expressed InaZ fragments is pH-dependent, which becomes compact as the numbers of 48-residue repeat unit increase. Copyright © 2016 Elsevier B.V. All rights reserved.

Bactericidal Compounds Controlling Growth of the Plant Pathogen Pseudomonas syringae pv. actinidiae, Which Forms Biofilms Composed of a Novel Exopolysaccharide

PubMed Central

Ghods, Shirin; Sims, Ian M.; Moradali, M. Fata

2015-01-01

Pseudomonas syringae pv. actinidiae is the major cause of bacterial canker and is a severe threat to kiwifruit production worldwide. Many aspects of the disease caused by P. syringae pv. actinidiae, such as the pathogenicity-relevant formation of a biofilm composed of extracellular polymeric substances (EPSs), are still unknown. Here, a highly virulent strain of P. syringae pv. actinidiae, NZ V-13, was studied with respect to biofilm formation and architecture using a flow cell system combined with confocal laser scanning microscopy. The biofilm formed by P. syringae pv. actinidiae NZ V-13 was heterogeneous, consisting of a thin cellular base layer 5 μm thick and microcolonies with irregular structures. The major component of the EPSs produced by P. syringae pv. actinidiae NZ V-13 bacteria was isolated and identified to be an exopolysaccharide. Extensive compositional and structural analysis showed that rhamnose, fucose, and glucose were the major constituents, present at a ratio of 5:1.5:2. Experimental evidence that P. syringae pv. actinidiae NZ V-13 produces two polysaccharides, a branched α-d-rhamnan with side chains of terminal α-d-Fucf and an α-d-1,4-linked glucan, was obtained. The susceptibility of the cells in biofilms to kasugamycin and chlorine dioxide was assessed. About 64 and 73% of P. syringae pv. actinidiae NZ V-13 cells in biofilms were killed when kasugamycin and chlorine dioxide were used at 5 and 10 ppm, respectively. Kasugamycin inhibited the attachment of P. syringae pv. actinidiae NZ V-13 to solid surfaces at concentrations of 80 and 100 ppm. Kasugamycin was bacteriostatic against P. syringae pv. actinidiae NZ V-13 growth in the planktonic mode, with the MIC being 40 to 60 ppm and a bactericidal effect being found at 100 ppm. Here we studied the formation, architecture, and composition of P. syringae pv. actinidiae biofilms as well as used the biofilm as a model to assess the efficacies of bactericidal compounds. PMID:25841017

Image-Based Quantification of Plant Immunity and Disease.

PubMed

Laflamme, Bradley; Middleton, Maggie; Lo, Timothy; Desveaux, Darrell; Guttman, David S

2016-12-01

Measuring the extent and severity of disease is a critical component of plant pathology research and crop breeding. Unfortunately, existing visual scoring systems are qualitative, subjective, and the results are difficult to transfer between research groups, while existing quantitative methods can be quite laborious. Here, we present plant immunity and disease image-based quantification (PIDIQ), a quantitative, semi-automated system to rapidly and objectively measure disease symptoms in a biologically relevant context. PIDIQ applies an ImageJ-based macro to plant photos in order to distinguish healthy tissue from tissue that has yellowed due to disease. It can process a directory of images in an automated manner and report the relative ratios of healthy to diseased leaf area, thereby providing a quantitative measure of plant health that can be statistically compared with appropriate controls. We used the Arabidopsis thaliana-Pseudomonas syringae model system to show that PIDIQ is able to identify both enhanced plant health associated with effector-triggered immunity as well as elevated disease symptoms associated with effector-triggered susceptibility. Finally, we show that the quantitative results provided by PIDIQ correspond to those obtained via traditional in planta pathogen growth assays. PIDIQ provides a simple and effective means to nondestructively quantify disease from whole plants and we believe it will be equally effective for monitoring disease on excised leaves and stems.

LSU network hubs integrate abiotic and biotic stress responses via interaction with the superoxide dismutase FSD2

PubMed Central

Garcia-Molina, Antoni; Altmann, Melina; Alkofer, Angela; Epple, Petra M.; Dangl, Jeffery L.

2017-01-01

Abstract In natural environments, plants often experience different stresses simultaneously, and adverse abiotic conditions can weaken the plant immune system. Interactome mapping revealed that the LOW SULPHUR UPREGULATED (LSU) proteins are hubs in an Arabidopsis protein interaction network that are targeted by virulence effectors from evolutionarily diverse pathogens. Here we show that LSU proteins are up-regulated in several abiotic and biotic stress conditions, such as nutrient depletion or salt stress, by both transcriptional and post-translational mechanisms. Interference with LSU expression prevents chloroplastic reactive oxygen species (ROS) production and proper stomatal closure during sulphur stress. We demonstrate that LSU1 interacts with the chloroplastic superoxide dismutase FSD2 and stimulates its enzymatic activity in vivo and in vitro. Pseudomonas syringae virulence effectors interfere with this interaction and preclude re-localization of LSU1 to chloroplasts. We demonstrate that reduced LSU levels cause a moderately enhanced disease susceptibility in plants exposed to abiotic stresses such as nutrient deficiency, high salinity, or heavy metal toxicity, whereas LSU1 overexpression confers significant disease resistance in several of these conditions. Our data suggest that the network hub LSU1 plays an important role in co-ordinating plant immune responses across a spectrum of abiotic stress conditions. PMID:28207043

Soil mixture composition alters Arabidopsis susceptibility to Pseudomonas syringae infection

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae is a Gram-negative bacterial pathogen that causes disease on more than 100 different plant species, including the model plant Arabidopsis thaliana. Dissection of the Arabidopsis thaliana-Pseudomonas syringae pathosystem has identified many factors that contribute to successful ...

Tomato–Pseudomonas syringae interactions under elevated CO2 concentration: the role of stomata

PubMed Central

Li, Xin; Sun, Zenghui; Shao, Shujun; Zhang, Shuai; Ahammed, Golam Jalal; Zhang, Guanqun; Jiang, Yuping; Zhou, Jie; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Shi, Kai

2015-01-01

Increasing atmospheric CO2 concentrations ([CO2]) in agricultural and natural ecosystems is known to reduce plant stomatal opening, but it is unclear whether these CO2-induced stomatal alterations are associated with foliar pathogen infections. In this study, tomato plants were grown under ambient and elevated [CO2] and inoculated with Pseudomonas syringae pv. tomato strain DC3000, a strain that is virulent on tomato plants. We found that elevated [CO2] enhanced tomato defence against P. syringae. Scanning electron microscopy analysis revealed that stomatal aperture of elevated [CO2] plants was considerably smaller than their ambient counterparts, which affected the behaviour of P. syringae bacteria on the upper surface of epidermal peels. Pharmacological experiments revealed that nitric oxide (NO) played a role in elevated [CO2]-induced stomatal closure. Silencing key genes involved in NO generation and stomatal closing, nitrate reductase (NR) and guard cell slow-type anion channel 1 (SLAC1), blocked elevated [CO2]-induced stomatal closure and resulted in significant increases in P. syringae infection. However, the SLAC1-silenced plants, but not the NR-silenced plants, still had significantly higher defence under elevated [CO2] compared with plants treated with ambient [CO2]. Similar results were obtained when the stomata-limiting factor for P. syringae entry was excluded by syringe infiltration inoculation. These results indicate that elevated [CO2] induces defence against P. syringae in tomato plants, not only by reducing the stomata-mediated entry of P. syringae but also by invoking a stomata-independent pathway to counteract P. syringae. This information is valuable for designing proper strategies against bacterial pathogens under changing agricultural and natural ecosystems. PMID:25336683

Ornithine Transcarbamylase ArgK Plays a Dual role for the Self-defense of Phaseolotoxin Producing Pseudomonas syringae pv. phaseolicola.

PubMed

Chen, Li; Li, Pin; Deng, Zixin; Zhao, Changming

2015-08-10

Pseudomonas syringae is a phytopathogenic bacterium widely spread on terrestrial plants. Sulfodiaminophosphinyl tripeptide Phaseolotoxins (PHTs), produced by P. syringae pv. phaseolicola and P. syringae pv. actinidiae, represent a kind of antimetabolic phytotoxins. PHTs inhibit host cell Ornithine transcarbamylase (OTCase) activity and induce Arginine auxotrophic phenotype. The biosynthesis of PHT is temperature dependent, being optically produced at around 18 °C, while blocked above 28 °C. PHT resistant OTCase ArgK acts as a functional replacement of housekeeping OTCase ArgF, which is the acting target of PHT, to confer PHT producers with self-resistance. It was postulated that argK might be regulated directly by a PHT biosynthetic precursor and indirectly by temperature with an unknown manner. Neither transcriptional regulator nor thermal regulation related protein encoding gene was detected from PHT biosynthetic gene cluster. The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report. Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates. It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers' self-defense.

Ornithine Transcarbamylase ArgK Plays a Dual role for the Self-defense of Phaseolotoxin Producing Pseudomonas syringae pv. phaseolicola

PubMed Central

chen, Li; li, Pin; deng, Zixin; zhao, Changming

2015-01-01

Pseudomonas syringae is a phytopathogenic bacterium widely spread on terrestrial plants. Sulfodiaminophosphinyl tripeptide Phaseolotoxins (PHTs), produced by P. syringae pv. phaseolicola and P. syringae pv. actinidiae, represent a kind of antimetabolic phytotoxins. PHTs inhibit host cell Ornithine transcarbamylase (OTCase) activity and induce Arginine auxotrophic phenotype. The biosynthesis of PHT is temperature dependent, being optically produced at around 18 °C, while blocked above 28 °C. PHT resistant OTCase ArgK acts as a functional replacement of housekeeping OTCase ArgF, which is the acting target of PHT, to confer PHT producers with self-resistance. It was postulated that argK might be regulated directly by a PHT biosynthetic precursor and indirectly by temperature with an unknown manner. Neither transcriptional regulator nor thermal regulation related protein encoding gene was detected from PHT biosynthetic gene cluster. The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report. Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates. It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers’ self-defense. PMID:26256666

Characterization of Pseudomonas syringae from blueberry fields in Oregon and Washington

USDA-ARS?s Scientific Manuscript database

Bacterial canker, caused by Pseudomonas syringae, is a common disease that kills buds and stems in blueberry fields in Oregon and western Washington. Management is primarily through application of copper; antibiotics are not registered for blueberry. Little is known about the diversity of P. syringa...

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

PubMed

Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

2015-01-01

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery. Copyright © 2015 Elsevier GmbH. All rights reserved.

Management of Bacterial Blight of Lilac Caused by Pseudomonas syringae by Growing Plants under Plastic Shelters

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae pv. syringae causes some of the most economically-important bacterial diseases affecting woody perennials grown by the nursery industry in the Pacific Northwest of the United States. In this study, we evaluated a cultural control practice, placement of plants in plastic shelter...

Diel Variation in Population Size and Ice Nucleation Activity of Pseudomonas syringae on Snap Bean Leaflets.

PubMed

Hirano, S S; Upper, C D

1989-03-01

The extent to which diel changes in the physical environment affect changes in population size and ice nucleation activity of Pseudomonas syringae on snap bean leaflets was determined under field conditions. To estimate bacterial population size and ice nucleation activity, bean leaflets were harvested at 2-h intervals during each of three 26-h periods. A tube nucleation test was used to assay individual leaflets for ice nuclei. Population sizes of P. syringae were determined by dilution plating of leaflet homogenates. The overall diel changes in P. syringae population sizes differed during each of the 26-h periods. In one 26-h period, there was a continuous increase in the logarithm of P. syringae population size despite intense solar radiation, absence of free moisture on leaf surfaces, and low relative humidity during the day. A mean doubling time of approximately 4.9 h was estimated for the 28-fold increase in P. syringae population size that occurred from 0900 to 0900 h during the 26-h period. However, doubling times of 3.3 and 1.9 h occurred briefly during this period from 1700 to 2300 h and from 0100 to 0700 h, respectively. Thus, growth rates of P. syringae in association with leaves in the field were of the same order of magnitude as optimal rates measured in the laboratory. The frequency with which leaflets bore ice nuclei active at -2.0, -2.2, and -2.5 degrees C varied greatly within each 26-h period. These large diel changes were inversely correlated primarily with the diel changes in air temperature and reflected changes in nucleation frequency rather than changes in population size of P. syringae. Thus, the response of bacterial ice nucleation activity to the physical environment was distinct from the changes in population size of ice nucleation-active P. syringae.

The Facultative Symbiont Rickettsia Protects an Invasive Whitefly against Entomopathogenic Pseudomonas syringae Strains

PubMed Central

Hunter, Martha S.; Baltrus, David A.

2014-01-01

Facultative endosymbionts can benefit insect hosts in a variety of ways, including context-dependent roles, such as providing defense against pathogens. The role of some symbionts in defense may be overlooked, however, when pathogen infection is transient, sporadic, or asymptomatic. The facultative endosymbiont Rickettsia increases the fitness of the sweet potato whitefly (Bemisia tabaci) in some populations through mechanisms that are not yet understood. In this study, we investigated the role of Rickettsia in mediating the interaction between the sweet potato whitefly and Pseudomonas syringae, a common environmental bacterium, some strains of which are pathogenic to aphids. Our results show that P. syringae multiplies within whiteflies, leading to host death, and that whiteflies infected with Rickettsia show a decreased rate of death due to P. syringae. Experiments using plants coated with P. syringae confirmed that whiteflies can acquire the bacteria at a low rate while feeding, leading to increased mortality, particularly when the whiteflies are not infected with Rickettsia. These results suggest that P. syringae may affect whitefly populations in nature and that Rickettsia can ameliorate this effect. This study highlights the possible importance of interactions among opportunistic environmental pathogens and endosymbionts of insects. PMID:25217020

LSU network hubs integrate abiotic and biotic stress responses via interaction with the superoxide dismutase FSD2.

PubMed

Garcia-Molina, Antoni; Altmann, Melina; Alkofer, Angela; Epple, Petra M; Dangl, Jeffery L; Falter-Braun, Pascal

2017-02-01

In natural environments, plants often experience different stresses simultaneously, and adverse abiotic conditions can weaken the plant immune system. Interactome mapping revealed that the LOW SULPHUR UPREGULATED (LSU) proteins are hubs in an Arabidopsis protein interaction network that are targeted by virulence effectors from evolutionarily diverse pathogens. Here we show that LSU proteins are up-regulated in several abiotic and biotic stress conditions, such as nutrient depletion or salt stress, by both transcriptional and post-translational mechanisms. Interference with LSU expression prevents chloroplastic reactive oxygen species (ROS) production and proper stomatal closure during sulphur stress. We demonstrate that LSU1 interacts with the chloroplastic superoxide dismutase FSD2 and stimulates its enzymatic activity in vivo and in vitro. Pseudomonas syringae virulence effectors interfere with this interaction and preclude re-localization of LSU1 to chloroplasts. We demonstrate that reduced LSU levels cause a moderately enhanced disease susceptibility in plants exposed to abiotic stresses such as nutrient deficiency, high salinity, or heavy metal toxicity, whereas LSU1 overexpression confers significant disease resistance in several of these conditions. Our data suggest that the network hub LSU1 plays an important role in co-ordinating plant immune responses across a spectrum of abiotic stress conditions. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The lilac cultivar Syringa ‘Charisma’ is a new host for ‘Candidatus Phytoplasma pruni’, the group 16SrIII, subgroup A, phytoplasma

USDA-ARS?s Scientific Manuscript database

The lilac cultivar ‘Charisma’ (Syringa x prestoniae ‘Charisma’) was derived by the propagation of a witches’ broom cutting taken from the Preston Lilac ‘Royalty’ (Syringa x prestoniae ‘Royalty). Because lilac witches’ broom disease has previously been shown to be associated with infection by ‘Candi...

Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

PubMed Central

Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

2016-01-01

Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

Pseudomonas syringae pv. actinidiae: a re-emerging, multi-faceted, pandemic pathogen.

PubMed

Scortichini, Marco; Marcelletti, Simone; Ferrante, Patrizia; Petriccione, Milena; Firrao, Giuseppe

2012-09-01

Pseudomonas syringae pv. actinidiae is the causal agent of bacterial canker of green-fleshed kiwifruit (Actinidia deliciosa) and yellow-fleshed kiwifruit (A. chinensis). A recent, sudden, re-emerging wave of this disease has occurred, almost contemporaneously, in all of the main areas of kiwifruit production in the world, suggesting that it can be considered as a pandemic disease. Recent in-depth genetic studies performed on P. syringae pv. actinidiae strains have revealed that this pathovar is composed of four genetically different populations which, to different extents, can infect crops of the genus Actinidia worldwide. Genome comparisons of these strains have revealed that this pathovar can gain and lose the phaseolotoxin gene cluster, as well as mobile genetic elements, such as plasmids and putative prophages, and that it can modify the repertoire of the effector gene arrays. In addition, the strains currently causing worldwide severe economic losses display an extensive set of genes related to the ecological fitness of the bacterium in planta, such as copper and antibiotic resistance genes, multiple siderophore genes and genes involved in the degradation of lignin derivatives and other phenolics. This pathogen can therefore easily colonize hosts throughout the year. Bacteria; Proteobacteria, gamma subdivision; Order Pseudomonadales; Family Pseudomonadaceae; Genus Pseudomonas; Pseudomonas syringae species complex, genomospecies 8; Pathovar actinidiae. Gram-negative, aerobic, motile, rod-shaped, polar flagella, oxidase-negative, arginine dihydrolase-negative, DNA 58.5-58.8 mol.% GC, elicits the hypersensitive response on tobacco leaves. Primarily studied as the causal agent of bacterial canker of green-fleshed kiwifruit (Actinidia deliciosa), it has also been isolated from yellow-fleshed kiwifruit (A. chinensis). In both species, it causes severe economic losses worldwide. It has also been isolated from wild A. arguta and A. kolomikta. In green-fleshed and yellow-fleshed kiwifruits, the symptoms include brown-black leaf spots often surrounded by a chlorotic margin, blossom necrosis, extensive twig die-back, reddening of the lenticels, extensive cankers along the main trunk and leader, and bleeding cankers on the trunk and the leader with a whitish to orange ooze. Pseudomonas syringae pv. actinidiae can effectively colonize its host plants throughout the year. Bacterial exudates can disperse a large amount of inoculum within and between orchards. In the spring, temperatures ranging from 12 to 18 °C, together with humid conditions, can greatly favour the multiplication of the bacterium, allowing it to systemically move from the leaf to the young shoots. During the summer, very high temperatures can reduce the multiplication and dispersal of the bacterium. Some agronomical techniques, as well as frost, wind, rain and hail storms, can contribute to further spreading. An integrated approach that takes into consideration precise scheduled spray treatments with effective and environmentally friendly bactericides and equilibrated plant nutrition, coupled with preventive measures aimed at drastically reducing the bacterial inoculum, currently seems to be the possible best solution for coexistence with the disease. The development of resistant cultivars and pollinators, effective biocontrol agents, including bacteriophages, and compounds that induce the systemic activation of plant defence mechanisms is in progress. Up-to-date information on bacterial canker research progress and on the spread of the disease in New Zealand can be found at: http://www.kvh.org.nz. Daily information on the spread of the disease and on the research being performed worldwide can be found at: http://www.freshplaza.it. © 2012 The Authors. Molecular Plant Pathology © 2012 BSPP and Blackwell Publishing Ltd.

The Facultative Symbiont Rickettsia Protects an Invasive Whitefly against Entomopathogenic Pseudomonas syringae Strains.

PubMed

Hendry, Tory A; Hunter, Martha S; Baltrus, David A

2014-12-01

Facultative endosymbionts can benefit insect hosts in a variety of ways, including context-dependent roles, such as providing defense against pathogens. The role of some symbionts in defense may be overlooked, however, when pathogen infection is transient, sporadic, or asymptomatic. The facultative endosymbiont Rickettsia increases the fitness of the sweet potato whitefly (Bemisia tabaci) in some populations through mechanisms that are not yet understood. In this study, we investigated the role of Rickettsia in mediating the interaction between the sweet potato whitefly and Pseudomonas syringae, a common environmental bacterium, some strains of which are pathogenic to aphids. Our results show that P. syringae multiplies within whiteflies, leading to host death, and that whiteflies infected with Rickettsia show a decreased rate of death due to P. syringae. Experiments using plants coated with P. syringae confirmed that whiteflies can acquire the bacteria at a low rate while feeding, leading to increased mortality, particularly when the whiteflies are not infected with Rickettsia. These results suggest that P. syringae may affect whitefly populations in nature and that Rickettsia can ameliorate this effect. This study highlights the possible importance of interactions among opportunistic environmental pathogens and endosymbionts of insects. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Effector CD4 cell tolerization is mediated through functional inactivation and involves preferential impairment of TNF-α and IFN-γ expression potentials

PubMed Central

Long, Meixiao; Higgins, Amy D.; Mihalyo, Marianne A.; Adler, Adam J.

2010-01-01

It has recently been shown that effector/memory T cells can undergo peripheral tolerization in response to self-antigen. In the present study, we found that within 24 h self-antigen profoundly impairs the ability of CD4 effectors to express TNF-α (and to a lesser extent IFN-γ); however, several days of self-antigen exposure is required to impair non-effector functions such as IL-2 expression and proliferation. Since only half of the initial effector CD4 cell population expresses effector cytokines following brief antigenic stimulation, tolerization might have been mediated either through functional inactivation of effector-competent cells, or alternatively by the selective deletion of competent and expansion of non-competent cells. When briefly stimulated effectors were fractionated based on their expression of IFN-γ, the IFN-γ− sub-population was able to express IFN-γ following secondary stimulation, indicating that all effector CD4 cells are functionally competent. Furthermore, both IFN-γ+ and IFN-γ− sub-populations underwent tolerization in response to self-HA (although the former was slightly more prone to deletion at later time points). Thus, effector CD4 cell tolerization is mediated primarily through the functional inactivation of effector-competent cells. PMID:14609577

Pathovars of Pseudomonas syringae Causing Bacterial Brown Spot and Halo Blight in Phaseolus vulgaris L. Are Distinguishable by Ribotyping

PubMed Central

González, Ana J.; Landeras, Elena; Mendoza, M. Carmen

2000-01-01

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported. PMID:10653764

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana.

PubMed

Sohn, Kee Hoon; Segonzac, Cécile; Rallapalli, Ghanasyam; Sarris, Panagiotis F; Woo, Joo Yong; Williams, Simon J; Newman, Toby E; Paek, Kyung Hee; Kobe, Bostjan; Jones, Jonathan D G

2014-10-01

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific "avirulent" pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.

The Nuclear Immune Receptor RPS4 Is Required for RRS1SLH1-Dependent Constitutive Defense Activation in Arabidopsis thaliana

PubMed Central

Sarris, Panagiotis F.; Woo, Joo Yong; Williams, Simon J.; Newman, Toby E.; Paek, Kyung Hee; Kobe, Bostjan; Jones, Jonathan D. G.

2014-01-01

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector. PMID:25340333

Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity

PubMed Central

Heidrich, Katharina; Tsuda, Kenichi; Blanvillain-Baufumé, Servane; Wirthmueller, Lennart; Bautor, Jaqueline; Parker, Jane E.

2013-01-01

In plant effector-triggered immunity (ETI), intracellular nucleotide binding-leucine rich repeat (NLR) receptors are activated by specific pathogen effectors. The Arabidopsis TIR (Toll-Interleukin-1 receptor domain)-NLR (denoted TNL) gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst) strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4, and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal “WRKY” transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4Ws/RRS1Ws allelic pair governs resistance to Pst/AvrRps4 accompanied by host programed cell death (pcd). In accession Col-0, RPS4Col/RRS1Col effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4Col (in a 35S:RPS4-HS line) confers temperature-conditioned EDS1-dependent auto-immunity. Here we show that a high (28°C, non-permissive) to moderate (19°C, permissive) temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 mutants, we establish that RPS4Col auto-immunity depends entirely on EDS1 and partially on RRS1Col. Examination of gene expression microarray data over 24 h after temperature shift reveals a mainly quantitative RRS1Col contribution to up- or down-regulation of a small subset of RPS4Col-reprogramed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1Col contributes to temperature-conditioned RPS4Col auto-immunity and are consistent with activated RPS4Col engaging RRS1Col for resistance signaling. PMID:24146667

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

PubMed

Bender, C L; Palmer, D A; Peñaloza-Vázquez, A; Rangaswamy, V; Ullrich, M

1998-01-01

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

Repeat-containing protein effectors of plant-associated organisms

PubMed Central

Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

Repeat-containing protein effectors of plant-associated organisms.

PubMed

Mesarich, Carl H; Bowen, Joanna K; Hamiaux, Cyril; Templeton, Matthew D

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

PubMed Central

Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

Inhibitory effect of Thymus vulgaris and Origanum vulgare essential oils on virulence factors of phytopathogenic Pseudomonas syringae strains.

PubMed

Carezzano, M E; Sotelo, J P; Primo, E; Reinoso, E B; Paletti Rovey, M F; Demo, M S; Giordano, W F; Oliva, M de Las M

2017-07-01

Pseudomonas syringae is a phytopathogenic bacterium that causes lesions in leaves during the colonisation process. The damage is associated with production of many virulence factors, such as biofilm and phytotoxins. The essential oils of Thymus vulgaris (thyme) and Origanum vulgare (oregano) have been demonstrated to inhibit P. syringae. The aim of this study was to investigate the effects of T. vulgaris and O. vulgare essential oils on production of virulence factors of phytopathogenic P. syringae strains, including anti-biofilm and anti-toxins activities. The broth microdilution method was used for determination of MIC and biofilm inhibition assays. Coronatine, syringomycin and tabtoxin were pheno- and genotypically evaluated. Both oils showed good inhibitory activity against P. syringae, with MIC values from 1.43 to 11.5 mg·ml -1 for thyme and 5.8 to 11.6 mg·ml -1 for oregano. Biofilm formation, production of coronatine, syringomycin and tabtoxin were inhibited by thyme and oregano essential oil in most strains. The results presented here are promising, demonstrating the bactericidal activity and reduction of virulence factor production after treatment with thyme and oregano oil, providing insight into how they exert their antibacterial activity. These natural products could be considered in the future for the control of diseases caused by P. syringae. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Bacterial canker on kiwifruit in Italy: anatomical changes in the wood and in the primary infection sites.

PubMed

Renzi, Marsilio; Copini, Paul; Taddei, Anna R; Rossetti, Antonio; Gallipoli, Lorenzo; Mazzaglia, Angelo; Balestra, Giorgio M

2012-09-01

The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae is a severe threat to kiwifruit production worldwide. Many aspects of P. syringae pv. actinidiae biology and epidemiology still require in-depth investigation. The infection by and spread of P. syringae pv. actinidiae in xylem and phloem was investigated by carrying out artificial inoculation experiments with histological and dendrochronological analyses of naturally diseased plants in Italy. We found that the bacterium can infect host plants by entering natural openings and lesions. In naturally infected kiwifruit plants, P. syringae pv. actinidiae is present in the lenticels as well as in the dead phloem tissue beneath the lenticels, surrounded by a lesion in the periderm which appears to indicate the importance of lenticels to kiwifruit infection. Biofilm formation was observed outside and inside plants. In cases of advanced stages of P. syringae pv. actinidiae infection, neuroses of the phloem occur, which are followed by cambial dieback and most likely by infection of the xylem. Anatomical changes in wood such as reduced ring width, a drastic reduction in vessel size, and the presence of tyloses were observed within several infected sites. In the field, these changes occur only a year after the first leaf symptoms are observed suggesting a significant time lapse between primary and secondary symptoms. It was possible to study the temporal development of P. syringae pv. actinidiae-induced cambial dieback by applying dendrochronology methods which revealed that cambial dieback occurs only during the growing season.

Draft Whole Genome Sequence Analyses on Pseudomonas syringae pv. actinidiae Hypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples.

PubMed

Biondi, Enrico; Zamorano, Alan; Vega, Ernesto; Ardizzi, Stefano; Sitta, Davide; De Salvador, Flavio Roberto; Campos-Vargas, Reinaldo; Meneses, Claudio; Perez, Set; Bertaccini, Assunta; Fiore, Nicola

2018-05-01

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

Bacteria in the Leaf Ecosystem with Emphasis on Pseudomonas syringae—a Pathogen, Ice Nucleus, and Epiphyte

PubMed Central

Hirano, Susan S.; Upper, Christen D.

2000-01-01

The extremely large number of leaves produced by terrestrial and aquatic plants provide habitats for colonization by a diversity of microorganisms. This review focuses on the bacterial component of leaf microbial communities, with emphasis on Pseudomonas syringae—a species that participates in leaf ecosystems as a pathogen, ice nucleus, and epiphyte. Among the diversity of bacteria that colonize leaves, none has received wider attention than P. syringae, as it gained notoriety for being the first recombinant organism (Ice− P. syringae) to be deliberately introduced into the environment. We focus on P. syringae to illustrate the attractiveness and somewhat unique opportunities provided by leaf ecosystems for addressing fundamental questions of microbial population dynamics and mechanisms of plant-bacterium interactions. Leaf ecosystems are dynamic and ephemeral. The physical environment surrounding phyllosphere microbes changes continuously with daily cycles in temperature, radiation, relative humidity, wind velocity, and leaf wetness. Slightly longer-term changes occur as weather systems pass. Seasonal climatic changes impose still a longer cycle. The physical and physiological characteristics of leaves change as they expand, mature, and senesce and as host phenology changes. Many of these factors influence the development of populations of P. syringae upon populations of leaves. P. syringae was first studied for its ability to cause disease on plants. However, disease causation is but one aspect of its life strategy. The bacterium can be found in association with healthy leaves, growing and surviving for many generations on the surfaces of leaves as an epiphyte. A number of genes and traits have been identified that contribute to the fitness of P. syringae in the phyllosphere. While still in their infancy, such research efforts demonstrate that the P. syringae-leaf ecosystem is a particularly attractive system with which to bridge the gap between what is known about the molecular biology of genes linked to pathogenicity and the ecology and epidemiology of associated diseases as they occur in natural settings, the field. PMID:10974129

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

PubMed

Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

2016-04-01

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au. © 2015 CSIRO New Phytologist © 2015 New Phytologist Trust.

Pathogen Infection and MORC Proteins Affect Chromatin Accessibility of Transposable Elements and Expression of Their Proximal Genes in Arabidopsis.

PubMed

Bordiya, Yogendra; Zheng, Yi; Nam, Ji-Chul; Bonnard, April C; Choi, Hyong Woo; Lee, Bum-Kyu; Kim, Jonghwan; Klessig, Daniel F; Fei, Zhangjun; Kang, Hong-Gu

2016-09-01

To assess the role of MORC1 in epigenetics in relation to plant immunity, genome-wide chromatin accessibility was compared between mock- or Pseudomonas syringae pv. tomato-inoculated wild type (WT) Arabidopsis, the morc1/2 double mutant, or both. Most changes in chromatin accessibility, scored by DNase I hypersensitive sites (DHSs), were located in the promoters of genes and transposable elements (TEs). Comparisons between morc1/2 and WT receiving the same treatment revealed differential DHSs (dDHSs) predominantly associated with heterochromatic TEs. By contrast, comparisons between mock- and P. syringae pv. tomato-inoculated plants from the same genotype showed dDHSs associated with biotic and abiotic stress-related genes; a smaller but significant population was in TEs. Moreover, many defense genes, including PR-1, PR-2, and PR-5, were proximal to P. syringae pv. tomato-induced, TE-associated dDHSs. A random subset of these defense genes showed moderately delayed or reduced expression or both in P. syringae pv. tomato-infected morc1/2 as compared with WT. MORC1 was physically bound to chromatin in a P. syringae pv. tomato infection-responsive manner at sites dispersed throughout the genome. Notably, silencing of TE-associated dDHSs proximal to these infection-induced, MORC1-interacting sites led to significant suppression of P. syringae pv. tomato-induced transcription of adjacent defense genes, including PR-1. These results provide evidence that MORC1 is associated with TEs and suggest that a subset of these TEs may help regulate their proximal defense genes.

The widespread plant-colonizing bacterial species Pseudomonas syringae detects and exploits an extracellular pool of choline in hosts.

PubMed

Chen, Chiliang; Li, Shanshan; McKeever, Dana R; Beattie, Gwyn A

2013-09-01

The quaternary ammonium compound (QAC) choline is a major component of membrane lipids in eukaryotes and, if available to microbial colonists of plants, could provide benefits for growth and protection from stress. Free choline is found in homogenized plant tissues, but its subcellular location and availability to plant microbes are not known. Whole-cell bacterial bioreporters of the phytopathogen Pseudomonas syringae were constructed that couple a QAC-responsive transcriptional fusion with well-characterized bacterial QAC transporters. These bioreporters demonstrated the presence of abundant free choline compounds released from germinating seeds and seedlings of the bean Phaseolus vulgaris, and a smaller but consistently detectable amount of QACs, probably choline, from leaves. The localization of P. syringae bioreporter cells to the surface and intercellular sites of plant tissues demonstrated the extracellular location of these QAC pools. Moreover, P. syringae mutants that were deficient in the uptake of choline compounds exhibited reduced fitness on leaves, highlighting the importance of extracellular choline to P. syringae on leaves. Our data support a model in which this choline pool is derived from the phospholipid phosphatidylcholine through plant-encoded phospholipases that release choline into the intercellular spaces of plant tissues, such as for membrane lipid recycling. The consequent extracellular release of choline compounds enables their interception and exploitation by plant-associated microbes, and thus provides a selective advantage for microbes such as P. syringae that are adapted to maximally exploit choline. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

The induction of tomato leucine aminopeptidase genes (LapA) after Pseudomonas syringae pv. tomato infection is primarily a wound response triggered by coronatine.

PubMed

Pautot, V; Holzer, F M; Chaufaux, J; Walling, L L

2001-02-01

Tomato plants constitutively express a neutral leucine aminopeptidase (LAP-N) and an acidic LAP (LAP-A) during floral development and in leaves in response to insect infestation, wounding, and Pseudomonas syringae pv. tomato infection. To assess the physiological roles of LAP-A, a LapA-antisense construct (35S:asLapA1) was introduced into tomato. The 35S:asLapA1 plants had greatly reduced or showed undetectable levels of LAP-A and LAP-N proteins in healthy and wounded leaves and during floral development. Despite the loss of these aminopeptidases, no global changes in protein profiles were noted. The 35S:asLapA1 plants also exhibited no significant alteration in floral development and did not impact the growth and development of Manduca sexta and P. syringae pv. tomato growth rates during compatible or incompatible infections. To investigate the mechanism underlying the strong induction of LapA upon P. syringae pv. tomato infection, LapA expression was monitored after infection with coronatine-producing and -deficient P. syringae pv. tomato strains. LapA RNA and activity were detected only with the coronatine-producing P. syringae pv. tomato strain. Coronatine treatment of excised shoots caused increases in RNAs for jasmonic acid (JA)-regulated wound-response genes (LapA and pin2) but did not influence expression of a JA-regulated pathogenesis-related protein gene (PR-1). These results indicated that coronatine mimicked the wound response but was insufficient to activate JA-regulated PR genes.

Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

PubMed Central

Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

2013-01-01

Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library Jstacs, and is freely available as a web-application and a command line program. PMID:23526890

Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis

PubMed Central

Großkinsky, Dominik K.; Tafner, Richard; Moreno, María V.; Stenglein, Sebastian A.; García de Salamone, Inés E.; Nelson, Louise M.; Novák, Ondřej; Strnad, Miroslav; van der Graaff, Eric; Roitsch, Thomas

2016-01-01

Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience. PMID:26984671

Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis.

PubMed

Großkinsky, Dominik K; Tafner, Richard; Moreno, María V; Stenglein, Sebastian A; García de Salamone, Inés E; Nelson, Louise M; Novák, Ondřej; Strnad, Miroslav; van der Graaff, Eric; Roitsch, Thomas

2016-03-17

Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience.

New strategies for genetic engineering Pseudomonas syringae using recombination

USDA-ARS?s Scientific Manuscript database

Here we report that DNA oligonucleotides (oligos) introduced directly into bacteria by electroporation can recombine with the bacterial chromosome. This phenomenon was identified in Pseudomonas syringae and we subsequently found that Escherichia coli, Salmonella typhimurium and Shigella flexneri are...

Characterization of the Transcriptional Activators SalA and SyrF, Which Are Required for Syringomycin and Syringopeptin Production by Pseudomonas syringae pv. syringae

PubMed Central

Wang, Nian; Lu, Shi-En; Records, Angela R.; Gross, Dennis C.

2006-01-01

Production of the phytotoxins syringomycin and syringopeptin by Pseudomonas syringae pv. syringae is controlled by the regulatory genes salA and syrF. Analysis with 70-mer oligonucleotide microarrays established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to a maltose-binding protein (MBP) in Escherichia coli and P. syringae pv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by maltose affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment of the syrB1 promoter region containing the syr-syp box. Purified MBP-SalA caused a shift of a 324-bp band containing the putative syrF promoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of β-glucuronidase activities of the sypA::uidA and syrB1::uidA reporters by 59% to 74%. The effect of SalA on the expression of the syr-syp genes is mediated by SyrF, which activates the syr-syp genes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes. PMID:16621822

High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE.

PubMed

Fan, Jun; Crooks, Casey; Lamb, Chris

2008-01-01

Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F(2) population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance.

PubMed

Gruner, Katrin; Zeier, Tatyana; Aretz, Christina; Zeier, Jürgen

2018-04-16

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR-defective mlo2 mutants were still competent in systemically increasing the levels of the SAR-activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR-related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA- or Pip-inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Pseudomonas savastanoi pv. savastanoi: some like it knot.

PubMed

Ramos, Cayo; Matas, Isabel M; Bardaji, Leire; Aragón, Isabel M; Murillo, Jesús

2012-12-01

Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century witnessed a large number of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively prevented the pathogen from benefitting from the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A number of studies in recent years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other nonpathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, which encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi-olive interaction and to obtain insights into why some pathovars like it necrotic and why some like it knot. Pseudomonas syringae pv. savastanoi. Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi. Gram-negative rods, 0.4-0.8 × 1.0-3.0 μm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25-30 °C; oxidase negative, arginine dihydrolase negative; elicits the hypersensitive response on tobacco; most isolates are fluorescent and levan negative, although some isolates are nonfluorescent and levan positive. P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive have been reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii are differentiated from pathovar savastanoi mostly in their host range, and were not formally recognized until 1996. Literature before about 1996 generally names strains of the three pathovars as P. syringae ssp. savastanoi or P. savastanoi ssp. savastanoi, contributing to confusion on the host range and biological properties. Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated with nonpathogenic bacteria, both epiphytically and endophytically, and have been demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans, which could result in increased bacterial populations and disease symptoms. Based on preventive measures, mostly sanitary and cultural practices. Integrated control programmes benefit from regular applications of copper formulations, which should be maintained for at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen. http://www.pseudomonas-syringae.org/; http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Bacterial Leaf Spot of Parsley: Characterization of a New Disease

USDA-ARS?s Scientific Manuscript database

Since 2002, a severe leaf spot disease on parsley has occurred throughout central coastal California and particularly in Monterey County. Two different bacterial pathogens (Pseudomonas syringae pv. apii, and P. syringae pv. coriandricola) have been associated these outbreaks on parsley. Our research...

Foliar Diseases of Apiaceae Crops in Coastal California

USDA-ARS?s Scientific Manuscript database

The number of outbreaks of leaf spot, blight and streak diseases on celery, cilantro, fennel and parsley has been increasing throughout central coastal California and particularly in Monterey County since 2002. Two different bacterial pathogens (Pseudomonas syringae pv. apii, and P. syringae pv. cor...

[Fatty acids composition of cellular lipids of the collected and newly isolated Pseudomonas lupini strains].

PubMed

Hvozdiak, R I; Dankevych, L A; Votselko, S K; Holubets', O V

2005-01-01

Fatty acid composition of cellular lipids of 23 Pseudomonas lupini strains (Beltjukova et Koroljova 1968) has been investigated. Cellular fatty acids which contained from C10 to C19 carbon atoms have been identified. Basic fatty acid of those Pseudomonas cells are hexadecanoic, hexadecenoic and octadecanoic acids. The 3-hydroxydecanoic (C10:0 3OH), 3-hydroxydodecanoic (C12:0 3OH), 2-hydroxydodecanoic (C12:0 2OH) and cyclopropane fatty acids which contain 17 and 19 carbon atoms have been detected in cellular lipids. The cellular fatty acids spectra of 22 P. lupini strains are similar to cellular fatty acids spectrum of the type strain Pseudomonas syringae pv. syringae 8511. Pathogenic isolate 2, which fatty acid content of cell lipids significantly differ from lipids of cell fatty acids from P. lupini strains and cell lipids of fatty acids of typical strains Pseudomonas syringae pv. syringae 8511 and Pseudomonas savastanoi pv. phaseolicola 9066 is the exception.

Entomopathogenicity to Two Hemipteran Insects Is Common but Variable across Epiphytic Pseudomonas syringae Strains.

PubMed

Smee, Melanie R; Baltrus, David A; Hendry, Tory A

2017-01-01

Strains of the well-studied plant pathogen Pseudomonas syringae show large differences in their ability to colonize plants epiphytically and to inflict damage to hosts. Additionally, P. syringae can infect some sap-sucking insects and at least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. The likelihood of agricultural pest insects coming into contact with transient populations of P. syringae while feeding on plants is high, yet the ecological implications of these interactions are currently not well understood as virulence has not been tested across a wide range of strains. To investigate virulence differences across strains we exposed the sweet potato whitefly, Bemisia tabaci , and the pea aphid, Acyrthosiphon pisum , both of which are cosmopolitan agricultural pests, to 12 P. syringae strains. We used oral inoculations with bacteria suspended in artificial diet in order to assay virulence while controlling for other variables such as differences in epiphytic growth ability. Generally, patterns of pathogenicity remain consistent across the two species of hemipteran insects, with bacterial strains from phylogroup II, or genomospecies 1, causing the highest rate of mortality with up to 86% of individuals dead after 72 h post infection. The rate of mortality is highly variable across strains, some significantly different from negative control treatments and others showing no discernable difference. Interestingly, one of the most pathogenic strains to both aphids and whiteflies (Cit7) is thought to be non-pathogenic on plants. We also found Cit7 to establish the highest epiphytic population after 48 h on fava beans. Between the nine P. syringae strains tested for epiphytic ability there is also much variation, but epiphytic ability was positively correlated with pathogenicity to insects, suggesting that the two traits may be linked and that strains likely to be found on plants may often be entomopathogenic. Our study highlights that there may be a use for epiphytic bacteria in the biological control of insect crop pests. It also suggests that interactions with epiphytic bacteria could be evolutionary and ecological drivers for hemipteran insects.

The truncated NLR protein TIR-NBS13 is a MOS6/IMPORTIN-α3 interaction partner required for plant immunity.

PubMed

Roth, Charlotte; Lüdke, Daniel; Klenke, Melanie; Quathamer, Annalena; Valerius, Oliver; Braus, Gerhard H; Wiermer, Marcel

2017-12-01

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

PubMed

Urbanus, Malene L; Quaile, Andrew T; Stogios, Peter J; Morar, Mariya; Rao, Chitong; Di Leo, Rosa; Evdokimova, Elena; Lam, Mandy; Oatway, Christina; Cuff, Marianne E; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw P; Taipale, Mikko; Savchenko, Alexei; Ensminger, Alexander W

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

PubMed Central

Catz, Sergio Daniel

2013-01-01

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

The Biology and Biological Activity of Pseudomonas syringae pv. tagetis

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae pv. tagetis (Pst) is a disease of plants in the family Asteraceae. A distinctive characteristic of this bacterial pathogen is the symptom of apical chlorosis in infected plants, caused by the phytotoxin tagetitoxin. Strains of Pst have been isolated from several plant species ...

E622, a miniature, virulence-associated mobile element.

PubMed

Stavrinides, John; Kirzinger, Morgan W B; Beasley, Federico C; Guttman, David S

2012-01-01

Miniature inverted terminal repeat elements (MITEs) are nonautonomous mobile elements that have a significant impact on bacterial evolution. Here we characterize E622, a 611-bp virulence-associated MITE from Pseudomonas syringae, which contains no coding region but has almost perfect 168-bp inverted repeats. Using an antibiotic coupling assay, we show that E622 is transposable and can mobilize an antibiotic resistance gene contained between its borders. Its predicted parent element, designated TnE622, has a typical transposon structure with a three-gene operon, consisting of resolvase, integrase, and exeA-like genes, which is bounded by the same terminal inverted repeats as E622. A broader genome level survey of the E622/TnE622 inverted repeats identified homologs in Pseudomonas, Salmonella, Shewanella, Erwinia, Pantoea, and the cyanobacteria Nostoc and Cyanothece, many of which appear to encompass known virulence genes, including genes encoding toxins, enzymes, and type III secreted effectors. Its association with niche-specific genetic determinants, along with its persistence and evolutionary diversification, indicates that this mobile element family has played a prominent role in the evolution of many agriculturally and clinically relevant pathogenic bacteria.

Wide screening of phage-displayed libraries identifies immune targets in planta.

PubMed

Rioja, Cristina; Van Wees, Saskia C; Charlton, Keith A; Pieterse, Corné M J; Lorenzo, Oscar; García-Sánchez, Susana

2013-01-01

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Features of air masses associated with the deposition of Pseudomonas syringae and Botrytis cinerea by rain and snowfall

PubMed Central

Monteil, Caroline L; Bardin, Marc; Morris, Cindy E

2014-01-01

Clarifying the role of precipitation in microbial dissemination is essential for elucidating the processes involved in disease emergence and spread. The ecology of Pseudomonas syringae and its presence throughout the water cycle makes it an excellent model to address this issue. In this study, 90 samples of freshly fallen rain and snow collected from 2005–2011 in France were analyzed for microbiological composition. The conditions favorable for dissemination of P. syringae by this precipitation were investigated by (i) estimating the physical properties and backward trajectories of the air masses associated with each precipitation event and by (ii) characterizing precipitation chemistry, and genetic and phenotypic structures of populations. A parallel study with the fungus Botrytis cinerea was also performed for comparison. Results showed that (i) the relationship of P. syringae to precipitation as a dissemination vector is not the same for snowfall and rainfall, whereas it is the same for B. cinerea and (ii) the occurrence of P. syringae in precipitation can be linked to electrical conductivity and pH of water, the trajectory of the air mass associated with the precipitation and certain physical conditions of the air mass (i.e. temperature, solar radiation exposure, distance traveled), whereas these predictions are different for B. cinerea. These results are pertinent to understanding microbial survival, emission sources and atmospheric processes and how they influence microbial dissemination. PMID:24722630

Features of air masses associated with the deposition of Pseudomonas syringae and Botrytis cinerea by rain and snowfall.

PubMed

Monteil, Caroline L; Bardin, Marc; Morris, Cindy E

2014-11-01

Clarifying the role of precipitation in microbial dissemination is essential for elucidating the processes involved in disease emergence and spread. The ecology of Pseudomonas syringae and its presence throughout the water cycle makes it an excellent model to address this issue. In this study, 90 samples of freshly fallen rain and snow collected from 2005-2011 in France were analyzed for microbiological composition. The conditions favorable for dissemination of P. syringae by this precipitation were investigated by (i) estimating the physical properties and backward trajectories of the air masses associated with each precipitation event and by (ii) characterizing precipitation chemistry, and genetic and phenotypic structures of populations. A parallel study with the fungus Botrytis cinerea was also performed for comparison. Results showed that (i) the relationship of P. syringae to precipitation as a dissemination vector is not the same for snowfall and rainfall, whereas it is the same for B. cinerea and (ii) the occurrence of P. syringae in precipitation can be linked to electrical conductivity and pH of water, the trajectory of the air mass associated with the precipitation and certain physical conditions of the air mass (i.e. temperature, solar radiation exposure, distance traveled), whereas these predictions are different for B. cinerea. These results are pertinent to understanding microbial survival, emission sources and atmospheric processes and how they influence microbial dissemination.

Pseudomonas syringae pv. actinidiae Draft Genomes Comparison Reveal Strain-Specific Features Involved in Adaptation and Virulence to Actinidia Species

PubMed Central

Marcelletti, Simone; Ferrante, Patrizia; Petriccione, Milena; Firrao, Giuseppe; Scortichini, Marco

2011-01-01

A recent re-emerging bacterial canker disease incited by Pseudomonas syringae pv. actinidiae (Psa) is causing severe economic losses to Actinidia chinensis and A. deliciosa cultivations in southern Europe, New Zealand, Chile and South Korea. Little is known about the genetic features of this pathovar. We generated genome-wide Illumina sequence data from two Psa strains causing outbreaks of bacterial canker on the A. deliciosa cv. Hayward in Japan (J-Psa, type-strain of the pathovar) and in Italy (I-Psa) in 1984 and 1992, respectively as well as from a Psa strain (I2-Psa) isolated at the beginning of the recent epidemic on A. chinensis cv. Hort16A in Italy. All strains were isolated from typical leaf spot symptoms. The phylogenetic relationships revealed that Psa is more closely related to P. s. pv. theae than to P. avellanae within genomospecies 8. Comparative genomic analyses revealed both relevant intrapathovar variations and putative pathovar-specific genomic regions in Psa. The genomic sequences of J-Psa and I-Psa were very similar. Conversely, the I2-Psa genome encodes four additional effector protein genes, lacks a 50 kb plasmid and the phaseolotoxin gene cluster, argK-tox but has acquired a 160 kb plasmid and putative prophage sequences. Several lines of evidence from the analysis of the genome sequences support the hypothesis that this strain did not evolve from the Psa population that caused the epidemics in 1984–1992 in Japan and Italy but rather is the product of a recent independent evolution of the pathovar actinidiae for infecting Actinidia spp. All Psa strains share the genetic potential for copper resistance, antibiotic detoxification, high affinity iron acquisition and detoxification of nitric oxide of plant origin. Similar to other sequenced phytopathogenic pseudomonads associated with woody plant species, the Psa strains isolated from leaves also display a set of genes involved in the catabolism of plant-derived aromatic compounds. PMID:22132095

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

Virulence of Pseudomonas syringae pv. tomato DC3000 is modulated through the Catabolite Repression Control protein Crc

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae (P.s.) infects diverse plant species and several P.s. pathovars have been used in the study of molecular events that occur during plant-microbe interactions. Although the relationship between bacterial metabolism, nutrient acquisition and virulence has attracted increasing atten...

Phytophthora suppressor of RNA silencing 2 is a conserved RxLR effector that promotes infection in soybean and Arabidopsis thaliana.

PubMed

Xiong, Qin; Ye, Wenwu; Choi, Duseok; Wong, James; Qiao, Yongli; Tao, Kai; Wang, Yuanchao; Ma, Wenbo

2014-12-01

The genus Phytophthora consists of notorious and emerging pathogens of economically important crops. Each Phytophthora genome encodes several hundreds of cytoplasmic effectors, which are believed to manipulate plant immune response inside the host cells. However, the majority of Phytophthora effectors remain functionally uncharacterized. We recently discovered two effectors from the soybean stem and root rot pathogen Phytophthora sojae with the activity to suppress RNA silencing in plants. These effectors are designated Phytophthora suppressor of RNA silencing (PSRs). Here, we report that the P. sojae PSR2 (PsPSR2) belongs to a conserved and widespread effector family in Phytophthora. A PsPSR2-like effector produced by P. infestans (PiPSR2) can also suppress RNA silencing in plants and promote Phytophthora infection, suggesting that the PSR2 family effectors have conserved functions in plant hosts. Using Agrobacterium rhizogenes-mediated hairy roots induction, we demonstrated that the expression of PsPSR2 rendered hypersusceptibility of soybean to P. sojae. Enhanced susceptibility was also observed in PsPSR2-expressing Arabidopsis thaliana plants during Phytophthora but not bacterial infection. These experiments provide strong evidence that PSR2 is a conserved Phytophthora effector family that performs important virulence functions specifically during Phytophthora infection of various plant hosts.

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification.

USDA-ARS?s Scientific Manuscript database

Since 2002, severe leaf spotting on parsley (Petroselinum crispum L.) has occurred in Monterey County, California. One of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from seven distinct outbreaks and twice from the same outbreak (2002 and 2009). Frag...

EFFECT OF PHENOTYPIC PLASTICITY ON EPIPHYTIC SURVIVAL AND COLONIZATION BY PSEUDOMONAS SYRINGAE

EPA Science Inventory

The bacterial epiphyte Pseudomonas syringas MF714R was cultured on agar or in broth or collected form colonized leaves; it was then inoculated onto greenhouse-grown bean plants incubated in a growth chamber at low relative humidity or in the field or onto field-grown bean plants....

A Mathematical model to investigate quorum sensing regulation and its heterogenecity in pseudomonas syringae on leaves

USDA-ARS?s Scientific Manuscript database

The bacterium Pseudomonas syringae is a plant-pathogen, which through quorum sensing (QS), controls virulence. In this paper, by means of mathematical modeling, we investigate QS of this bacterium when living on leaf surfaces. We extend an existing stochastic model for the formation of Pseudomonas s...

The conserved hypothetical protein PSPTO_3957 is essential for virulence in the plant pathogen Pseudomonas syringae pv. tomato DC3000

USDA-ARS?s Scientific Manuscript database

The plant pathogen Pseudomonas syringae accounts for substantial crop losses and is considered an important agricultural issue. Although many genes involved in interactions of this pathogen with hosts have been identified and characterized, little is known about processes involving bacterial metabol...

Show us your spots! Researchers need samples of bacterial leaf spots on celery, cilantro, parsley, and other crops.

USDA-ARS?s Scientific Manuscript database

Since 2002, a severe leaf spot disease on parsley has occurred throughout central coastal California and particularly in Monterey County. Three different bacterial pathogens (Pseudomonas syringae pv. apii, P. syringae pv. coriandricola and an organism very closely related to P. viridiflava) have bee...

Space-based multifunctional end effector systems functional requirements and proposed designs

NASA Technical Reports Server (NTRS)

Mishkin, A. H.; Jau, B. M.

1988-01-01

The end effector is an essential element of teleoperator and telerobot systems to be employed in space in the next decade. The report defines functional requirements for end effector systems to perform operations that are currently only feasible through Extra-Vehicular Activity (EVA). Specific tasks and functions that the end effectors must be capable of performing are delineated. Required capabilities for forces and torques, clearances, compliance, and sensing are described, using current EVA requirements as guidelines where feasible. The implications of these functional requirements on the elements of potential end effector systems are discussed. The systems issues that must be considered in the design of space-based manipulator systems are identified; including impacts on subsystems tightly coupled to the end effector, i.e., control station, information processing, manipulator arm, tool and equipment stowage. Possible end effector designs are divided into three categories: single degree-of-freedom end effectors, multiple degree of freedom end effectors, and anthropomorphic hands. Specific design alternatives are suggested and analyzed within the individual categories. Two evaluations are performed: the first considers how well the individual end effectors could substitute for EVA; the second compares how manipulator systems composed of the top performers from the first evaluation would improve the space shuttle Remote Manipulator System (RMS) capabilities. The analysis concludes that the anthropomorphic hand is best-suited for EVA tasks. A left- and right-handed anthropomorphic manipulator arm configuration is suggested as appropriate to be affixed to the RMS, but could also be used as part of the Smart Front End for the Orbital Maneuvering Vehicle (OMV). The technical feasibility of the anthropomorphic hand and its control are demonstrated. An evolutionary development approach is proposed and approximate scheduling provided for implementing the suggested manipulator systems in time for space stations operations in the early 1990s.

Thermo-Regulation of Genes Mediating Motility and Plant Interactions in Pseudomonas syringae

PubMed Central

Hockett, Kevin L.; Burch, Adrien Y.; Lindow, Steven E.

2013-01-01

Pseudomonas syringae is an important phyllosphere colonist that utilizes flagellum-mediated motility both as a means to explore leaf surfaces, as well as to invade into leaf interiors, where it survives as a pathogen. We found that multiple forms of flagellum-mediated motility are thermo-suppressed, including swarming and swimming motility. Suppression of swarming motility occurs between 28° and 30°C, which coincides with the optimal growth temperature of P. syringae. Both fliC (encoding flagellin) and syfA (encoding a non-ribosomal peptide synthetase involved in syringafactin biosynthesis) were suppressed with increasing temperature. RNA-seq revealed 1440 genes of the P. syringae genome are temperature sensitive in expression. Genes involved in polysaccharide synthesis and regulation, phage and IS elements, type VI secretion, chemosensing and chemotaxis, translation, flagellar synthesis and motility, and phytotoxin synthesis and transport were generally repressed at 30°C, while genes involved in transcriptional regulation, quaternary ammonium compound metabolism and transport, chaperone/heat shock proteins, and hypothetical genes were generally induced at 30°C. Deletion of flgM, a key regulator in the transition from class III to class IV gene expression, led to elevated and constitutive expression of fliC regardless of temperature, but did not affect thermo-regulation of syfA. This work highlights the importance of temperature in the biology of P. syringae, as many genes encoding traits important for plant-microbe interactions were thermo-regulated. PMID:23527276

Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

PubMed

Frampton, Rebekah A; Acedo, Elena Lopez; Young, Vivienne L; Chen, Danni; Tong, Brian; Taylor, Corinda; Easingwood, Richard A; Pitman, Andrew R; Kleffmann, Torsten; Bostina, Mihnea; Fineran, Peter C

2015-06-24

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

PubMed Central

Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

PubMed Central

Charpentier, Xavier; Gabay, Joëlle E.; Reyes, Moraima; Zhu, Jing W.; Weiss, Arthur; Shuman, Howard A.

2009-01-01

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions. PMID:19578436

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

PubMed Central

Shames, Stephanie R.; Liu, Luying; Havey, James C.; Schofield, Whitman B.; Goodman, Andrew L.; Roy, Craig R.

2017-01-01

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis. PMID:29133401

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

PubMed

Whalen, M C; Innes, R W; Bent, A F; Staskawicz, B J

1991-01-01

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.

High immunosuppressive burden in advanced hepatocellular carcinoma patients: Can effector functions be restored?

PubMed

Lugade, Amit A; Kalathil, Suresh; Miller, Austin; Iyer, Renuka; Thanavala, Yasmin

2013-07-01

The accumulation of immunosuppressive cells and exhausted effector T cells highlight an important immune dysfunction in advanced stage hepatocellular carcinoma (HCC) patients. These cells significantly hamper the efficacy immunotherapies and facilitate HCC progression. We have recently demonstrated that the multipronged depletion of immunosuppressive cells potentially restores effector T-cell function in HCC.

Characterization of the bacterial stem blight pathogen of alfalfa, Pseudomonas syringae pv. syringae ALF3

USDA-ARS?s Scientific Manuscript database

Bacterial stem blight of alfalfa occurs sporadically in the central and western U.S. Yield losses of up to 50% of the first harvest can occur with some cultivars. Developing resistant cultivars is hampered by lack of information on the pathogen and a standard test for evaluating plant germplasm. Bac...

A rapid seedling resistance assay identifies wild tomato lines that are resistant to Psuedomonas syringe pv. tomato race 1

USDA-ARS?s Scientific Manuscript database

Bacterial speck caused by Pseudomonas syringae has historically been controlled by the Pto/Prf gene cluster. Emerging strains like P. syringae pv. tomato race 1 overcome resistance conferred by Pto/Prf, and can cause serious crop loss under appropriate environmental conditions. We developed a rapid ...

Characterization of Pseudomonas pathovars isolated from rosaceous fruit trees in East Algeria.

PubMed

Harzallah, D; Sadallah, S; Larous, L

2004-01-01

A survey of bacterial diseases due to Pseudomonas on rosaceous fruit trees was conducted. In forty two orchards located in the Constantine region ( East Algeria). Pseudomonas isolates were identified on the bases of their cultural and biochemical characteristics . A total of fifty nine phytopathogenic bacteria were isolated from diseased pome and stone fruit trees. Thirty one strains comparable to Pseudomonas syringae pv. syringae were isolated from cherry (Prunus avium L.), plum (P. domestica L.), apricot (P. armeniaca L.), almond (P. dulcis L.) and pear trees (Pirus communis L.); sixteen strains comparable to Pseudomonas syringae pv. morsprunorum were obtained from samples of cherry and plum. Twelve strains of Pseudomonas viridiflava were isolated from cherry, apricot and peach (Prunus persica L.).

Identification of legionella effectors using bioinformatic approaches.

PubMed

Segal, Gil

2013-01-01

Legionella pneumophila the causative agent of Legionnaires' disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which translocates about 300 effector proteins into host cells during infection. Many of these effectors were first identified as effector candidates by several bioinformatic approaches, and these predicted effectors were later examined experimentally for translocation and a large number of which were validated as effector proteins. Here, I summarized the bioinformatic approaches that were used to identify these effectors.

Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

USDA-ARS?s Scientific Manuscript database

Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

Sequence and Role in Virulence of the Three Plasmid Complement of the Model Tumor-Inducing Bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

PubMed Central

Bardaji, Leire; Pérez-Martínez, Isabel; Rodríguez-Moreno, Luis; Rodríguez-Palenzuela, Pablo; Sundin, George W.; Ramos, Cayo; Murillo, Jesús

2011-01-01

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin genes. PMID:22022435

The Xanthomonas campestris pv. vesicatoria Type-3 Effector XopB Inhibits Plant Defence Responses by Interfering with ROS Production

PubMed Central

Priller, Johannes Peter Roman; Reid, Stephen; Konein, Patrick; Dietrich, Petra

2016-01-01

The bacterial pathogen Xanthomonas campestris pv. vesicatoria 85–10 (Xcv) translocates about 30 type-3 effector proteins (T3Es) into pepper plants (Capsicum annuum) to suppress plant immune responses. Among them is XopB which interferes with PTI, ETI and sugar-mediated defence responses, but the underlying molecular mechanisms and direct targets are unknown so far. Here, we examined the XopB-mediated suppression of plant defence responses in more detail. Infection of susceptible pepper plants with Xcv lacking xopB resulted in delayed symptom development compared to Xcv wild type infection concomitant with an increased formation of salicylic acid (SA) and expression of pathogenesis-related (PR) genes. Expression of xopB in Arabidopsis thaliana promoted the growth of the virulent Pseudomonas syringae pv. tomato (Pst) DC3000 strain. This was paralleled by a decreased SA-pool and a lower induction of SA-dependent PR gene expression. The expression pattern of early flg22-responsive marker genes indicated that MAPK signalling was not altered in the presence of XopB. However, XopB inhibited the flg22-triggered burst of reactive oxygen species (ROS). Consequently, the transcript accumulation of AtOXI1, a ROS-dependent marker gene, was reduced in xopB-expressing Arabidopsis plants as well as callose deposition. The lower ROS production correlated with a low level of basal and flg22-triggered expression of apoplastic peroxidases and the NADPH oxidase RBOHD. Conversely, deletion of xopB in Xcv caused a higher production of ROS in leaves of susceptible pepper plants. Together our results demonstrate that XopB modulates ROS responses and might thereby compromise plant defence. PMID:27398933

The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henty-Ridilla, Jessica L.; Shimono, Masaki; Li, Jiejie

2013-04-04

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence thatmore » the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.« less

QueTAL: a suite of tools to classify and compare TAL effectors functionally and phylogenetically

PubMed Central

Pérez-Quintero, Alvaro L.; Lamy, Léo; Gordon, Jonathan L.; Escalon, Aline; Cunnac, Sébastien; Szurek, Boris; Gagnevin, Lionel

2015-01-01

Transcription Activator-Like (TAL) effectors from Xanthomonas plant pathogenic bacteria can bind to the promoter region of plant genes and induce their expression. DNA-binding specificity is governed by a central domain made of nearly identical repeats, each determining the recognition of one base pair via two amino acid residues (a.k.a. Repeat Variable Di-residue, or RVD). Knowing how TAL effectors differ from each other within and between strains would be useful to infer functional and evolutionary relationships, but their repetitive nature precludes reliable use of traditional alignment methods. The suite QueTAL was therefore developed to offer tailored tools for comparison of TAL effector genes. The program DisTAL considers each repeat as a unit, transforms a TAL effector sequence into a sequence of coded repeats and makes pair-wise alignments between these coded sequences to construct trees. The program FuncTAL is aimed at finding TAL effectors with similar DNA-binding capabilities. It calculates correlations between position weight matrices of potential target DNA sequence predicted from the RVD sequence, and builds trees based on these correlations. The programs accurately represented phylogenetic and functional relationships between TAL effectors using either simulated or literature-curated data. When using the programs on a large set of TAL effector sequences, the DisTAL tree largely reflected the expected species phylogeny. In contrast, FuncTAL showed that TAL effectors with similar binding capabilities can be found between phylogenetically distant taxa. This suite will help users to rapidly analyse any TAL effector genes of interest and compare them to other available TAL genes and should improve our understanding of TAL effectors evolution. It is available at http://bioinfo-web.mpl.ird.fr/cgi-bin2/quetal/quetal.cgi. PMID:26284082

Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana

PubMed Central

Han, Shaojie; Wang, Yan; Zheng, Xiyin; Jia, Qi; Zhao, Jinping; Bai, Fan; Hong, Yiguo; Liu, Yule

2015-01-01

Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity. PMID:25829441

Ubiquitin Ligases and Deubiquitinating Enzymes in CD4+ T Cell Effector Fate Choice and Function.

PubMed

Layman, Awo A K; Oliver, Paula M

2016-05-15

The human body is exposed to potentially pathogenic microorganisms at barrier sites such as the skin, lungs, and gastrointestinal tract. To mount an effective response against these pathogens, the immune system must recruit the right cells with effector responses that are appropriate for the task at hand. Several types of CD4(+) T cells can be recruited, including Th cells (Th1, Th2, and Th17), T follicular helper cells, and regulatory T cells. These cells help to maintain normal immune homeostasis in the face of constantly changing microbes in the environment. Because these cells differentiate from a common progenitor, the composition of their intracellular milieu of proteins changes to appropriately guide their effector function. One underappreciated process that impacts the levels and functions of effector fate-determining factors is ubiquitylation. This review details our current understanding of how ubiquitylation regulates CD4(+) T cell effector identity and function. Copyright © 2016 by The American Association of Immunologists, Inc.

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

PubMed Central

Schroeder, Gunnar N.

2018-01-01

The defective in organelle trafficking/intracellular multiplication (Dot/Icm) Type IVb secretion system (T4SS) is the essential virulence factor for the intracellular life style and pathogenicity of Legionella species. Screens demonstrated that an individual L. pneumophila strain can use the Dot/Icm T4SS to translocate an unprecedented number of more than 300 proteins into host cells, where these, so called Icm/Dot-translocated substrates (IDTS) or effectors, manipulate host cell functions to the benefit of the bacteria. Bioinformatic analysis of the pan-genus genome predicts at least 608 orthologous groups of putative effectors. Deciphering the function of these effectors is key to understanding Legionella pathogenesis; however, the analysis is challenging. Substantial functional redundancy renders classical, phenotypic screening of single gene deletion mutants mostly ineffective. Here, I review experimental approaches that were successfully used to identify, validate and functionally characterize T4SS effectors and highlight new methods, which promise to facilitate unlocking the secrets of Legionella's extraordinary weapons arsenal. PMID:29354599

Mutations in y-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid '-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome h...

Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity

PubMed Central

Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C.; Gilroy, Eleanor M.; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R. J.; Brunner, Frédéric

2014-01-01

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans. PMID:24763622

The Ca2+ induced two-component system, CvsSR regulates the Type III secretion system and the extracytoplasmic function sigma-factor AlgU in Pseudomonas syringae pv. tomato DC3000

USDA-ARS?s Scientific Manuscript database

Two-component systems (TCSs) of bacteria regulate many different aspects of the bacterial life cycle including pathogenesis. Most TCSs remain uncharacterized with no information about the signal(s) or regulatory targets and/or role in bacterial pathogenesis. Here, we characterize a TCS in the plant-...

Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides.

PubMed

Salomon, Dor; Guo, Yirui; Kinch, Lisa N; Grishin, Nick V; Gardner, Kevin H; Orth, Kim

2013-01-01

Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells.

CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

PubMed Central

Yao, Shuyu; Huang, Dan; Chen, Crystal Y.; Halliday, Lisa; Wang, Richard C.; Chen, Zheng W.

2014-01-01

The possibility that CD4+ T cells can act as “innate-like” cells to contain very-early M. tuberculosis (Mtb) dissemination and function as master helpers to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes during development of adaptive immunity against primary tuberculosis(TB) has not been demonstrated. We showed that pulmonary Mtb infection of CD4-depleted macaques surprisingly led to very-early extrathoracic Mtb dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during Mtb infection revealed the ability of CD8+ T cells to compensate and rapidly differentiate to Th17-like/Th1-like, and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. While CD3-negative non-T lymphocytes in presence of CD4+ T cells developed predominant Th22-like and NK-like (perforin production) responses to Mtb infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3-negative lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint of more severe TB, but presented pulmonary IL-4+ T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22+ and perforin+ cells despite presence of IL-17+ and IL-4+ cells. These results implicate previously-unknown “innate-like” ability of CD4+ T cells to contain extrathoracic Mtb dissemination at very early stage. Data also suggest that CD4+ T cells are required to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes and to prevent rapid TB progression during Mtb infection of nonhuman primates. PMID:24489088

Bacterial virulence effectors and their activities.

PubMed

Hann, Dagmar R; Gimenez-Ibanez, Selena; Rathjen, John P

2010-08-01

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes. Copyright 2010 Elsevier Ltd. All rights reserved.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

PubMed Central

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility

PubMed Central

Hutin, Mathilde; Pérez-Quintero, Alvaro L.; Lopez, Camilo; Szurek, Boris

2015-01-01

Many plant-pathogenic xanthomonads rely on Transcription Activator-Like (TAL) effectors to colonize their host. This particular family of type III effectors functions as specific plant transcription factors via a programmable DNA-binding domain. Upon binding to the promoters of plant disease susceptibility genes in a sequence-specific manner, the expression of these host genes is induced. However, plants have evolved specific strategies to counter the action of TAL effectors and confer resistance. One mechanism is to avoid the binding of TAL effectors by mutations of their DNA binding sites, resulting in resistance by loss-of-susceptibility. This article reviews our current knowledge of the susceptibility hubs targeted by Xanthomonas TAL effectors, possible evolutionary scenarios for plants to combat the pathogen with loss-of-function alleles, and how this knowledge can be used overall to develop new pathogen-informed breeding strategies and improve crop resistance. PMID:26236326

VIP1 and Its Homologs Are Not Required for Agrobacterium-Mediated Transformation, but Play a Role in Botrytis and Salt Stress Responses

PubMed Central

Lapham, Rachelle; Lee, Lan-Ying; Tsugama, Daisuke; Lee, Sanghun; Mengiste, Tesfaye; Gelvin, Stanton B.

2018-01-01

The bZIP transcription factor VIP1 interacts with the Agrobacterium virulence protein VirE2, but the role of VIP1 in Agrobacterium-mediated transformation remains controversial. Previously tested vip1-1 mutant plants produce a truncated protein containing the crucial bZIP DNA-binding domain. We generated the CRISPR/Cas mutant vip1-2 that lacks this domain. The transformation susceptibility of vip1-2 and wild-type plants is similar. Because of potential functional redundancy among VIP1 homologs, we tested transgenic lines expressing VIP1 fused to a SRDX repression domain. All VIP1-SRDX transgenic lines showed wild-type levels of transformation, indicating that neither VIP1 nor its homologs are required for Agrobacterium-mediated transformation. Because VIP1 is involved in innate immune response signaling, we tested the susceptibility of vip1 mutant and VIP1-SRDX plants to Pseudomonas syringae and Botrytis cinerea. vip1 mutant and VIP1-SRDX plants show increased susceptibility to B. cinerea but not to P. syringae infection, suggesting a role for VIP1 in B. cinerea, but not in P. syringae, defense signaling. B. cinerea susceptibility is dependent on abscisic acid (ABA) which is also important for abiotic stress responses. The germination of vip1 mutant and VIP1-SRDX seeds is sensitive to exogenous ABA, suggesting a role for VIP1 in response to ABA. vip1 mutant and VIP1-SRDX plants show increased tolerance to growth in salt, indicating a role for VIP1 in response to salt stress. PMID:29946325

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

PubMed

Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Effector prediction in host-pathogen interaction based on a Markov model of a ubiquitous EPIYA motif

PubMed Central

2010-01-01

Background Effector secretion is a common strategy of pathogen in mediating host-pathogen interaction. Eight EPIYA-motif containing effectors have recently been discovered in six pathogens. Once these effectors enter host cells through type III/IV secretion systems (T3SS/T4SS), tyrosine in the EPIYA motif is phosphorylated, which triggers effectors binding other proteins to manipulate host-cell functions. The objectives of this study are to evaluate the distribution pattern of EPIYA motif in broad biological species, to predict potential effectors with EPIYA motif, and to suggest roles and biological functions of potential effectors in host-pathogen interactions. Results A hidden Markov model (HMM) of five amino acids was built for the EPIYA-motif based on the eight known effectors. Using this HMM to search the non-redundant protein database containing 9,216,047 sequences, we obtained 107,231 sequences with at least one EPIYA motif occurrence and 3115 sequences with multiple repeats of the EPIYA motif. Although the EPIYA motif exists among broad species, it is significantly over-represented in some particular groups of species. For those proteins containing at least four copies of EPIYA motif, most of them are from intracellular bacteria, extracellular bacteria with T3SS or T4SS or intracellular protozoan parasites. By combining the EPIYA motif and the adjacent SH2 binding motifs (KK, R4, Tarp and Tir), we built HMMs of nine amino acids and predicted many potential effectors in bacteria and protista by the HMMs. Some potential effectors for pathogens (such as Lawsonia intracellularis, Plasmodium falciparum and Leishmania major) are suggested. Conclusions Our study indicates that the EPIYA motif may be a ubiquitous functional site for effectors that play an important pathogenicity role in mediating host-pathogen interactions. We suggest that some intracellular protozoan parasites could secrete EPIYA-motif containing effectors through secretion systems similar to the T3SS/T4SS in bacteria. Our predicted effectors provide useful hypotheses for further studies. PMID:21143776

TAL effectors and the executor R genes

PubMed Central

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized—recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance. PMID:26347759

TAL effectors and the executor R genes.

PubMed

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized-recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase.

PubMed

Morris, V L; Jackson, D P; Grattan, M; Ainsworth, T; Cuppels, D A

1995-04-01

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant.

Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase.

PubMed Central

Morris, V L; Jackson, D P; Grattan, M; Ainsworth, T; Cuppels, D A

1995-01-01

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant. PMID:7896694

An assay for entry of secreted fungal effectors into plant cells.

PubMed

Lo Presti, Libera; Zechmann, Bernd; Kumlehn, Jochen; Liang, Liang; Lanver, Daniel; Tanaka, Shigeyuki; Bock, Ralph; Kahmann, Regine

2017-01-01

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity.

PubMed

Ma, Ka-Wai; Ma, Wenbo

2016-12-01

Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

NASA Astrophysics Data System (ADS)

Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

2003-06-01

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

PubMed Central

2016-01-01

SUMMARY Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. PMID:27784797

Effector biology of plant-associated organisms: concepts and perspectives.

PubMed

Win, J; Chaparro-Garcia, A; Belhaj, K; Saunders, D G O; Yoshida, K; Dong, S; Schornack, S; Zipfel, C; Robatzek, S; Hogenhout, S A; Kamoun, S

2012-01-01

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

DOE PAGES

Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; ...

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

Putative terminator and/or effector functions of Arf GAPs in the trafficking of clathrin-coated vesicles.

PubMed

Kon, Shunsuke; Funaki, Tomo; Satake, Masanobu

2011-05-01

The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.

Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis

PubMed Central

Zhang, Chong; Xie, Qiguang; Anderson, Ryan G.; Ng, Gina; Seitz, Nicholas C.; Peterson, Thomas; McClung, C. Robertson; McDowell, John M.; Kong, Dongdong; Kwak, June M.; Lu, Hua

2013-01-01

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity. PMID:23754942

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection.

PubMed

Jaouannet, Maëlle; Rosso, Marie-Noëlle

2013-09-01

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

PubMed

Lee, Chang-Han; Romain, Gabrielle; Yan, Wupeng; Watanabe, Makiko; Charab, Wissam; Todorova, Biliana; Lee, Jiwon; Triplett, Kendra; Donkor, Moses; Lungu, Oana I; Lux, Anja; Marshall, Nicholas; Lindorfer, Margaret A; Goff, Odile Richard-Le; Balbino, Bianca; Kang, Tae Hyun; Tanno, Hidetaka; Delidakis, George; Alford, Corrine; Taylor, Ronald P; Nimmerjahn, Falk; Varadarajan, Navin; Bruhns, Pierre; Zhang, Yan Jessie; Georgiou, George

2017-08-01

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

PubMed Central

Cianfanelli, Francesca R.; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J.

2016-01-01

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery. PMID:27352036

Transcription Factor Networks Directing the Development, Function, and Evolution of Innate Lymphoid Effectors

PubMed Central

Kang, Joonsoo; Malhotra, Nidhi

2015-01-01

Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity. PMID:25650177

Identification and functional analysis of secreted effectors from phytoparasitic nematodes.

PubMed

Rehman, Sajid; Gupta, Vijai K; Goyal, Aakash K

2016-03-21

Plant parasitic nematodes develop an intimate and long-term feeding relationship with their host plants. They induce a multi-nucleate feeding site close to the vascular bundle in the roots of their host plant and remain sessile for the rest of their life. Nematode secretions, produced in the oesophageal glands and secreted through a hollow stylet into the host plant cytoplasm, are believed to play key role in pathogenesis. To combat these persistent pathogens, the identity and functional analysis of secreted effectors can serve as a key to devise durable control measures. In this review, we will recapitulate the knowledge over the identification and functional characterization of secreted nematode effector repertoire from phytoparasitic nematodes. Despite considerable efforts, the identity of genes encoding nematode secreted proteins has long been severely hampered because of their microscopic size, long generation time and obligate biotrophic nature. The methodologies such as bioinformatics, protein structure modeling, in situ hybridization microscopy, and protein-protein interaction have been used to identify and to attribute functions to the effectors. In addition, RNA interference (RNAi) has been instrumental to decipher the role of the genes encoding secreted effectors necessary for parasitism and genes attributed to normal development. Recent comparative and functional genomic approaches have accelerated the identification of effectors from phytoparasitic nematodes and offers opportunities to control these pathogens. Plant parasitic nematodes pose a serious threat to global food security of various economically important crops. There is a wealth of genomic and transcriptomic information available on plant parasitic nematodes and comparative genomics has identified many effectors. Bioengineering crops with dsRNA of phytonematode genes can disrupt the life cycle of parasitic nematodes and therefore holds great promise to develop resistant crops against plant-parasitic nematodes.

Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora.

PubMed

Siamer, Sabrina; Gaubert, Stéphane; Boureau, Tristan; Brisset, Marie-Noëlle; Barny, Marie-Anne

2013-05-01

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Functional versus effector-specific organization of the human posterior parietal cortex: revisited

PubMed Central

Leone, Frank T. M.; Medendorp, W. Pieter

2016-01-01

It has been proposed that the posterior parietal cortex (PPC) is characterized by an effector-specific organization. However, strikingly similar functional MRI (fMRI) activation patterns have been found in the PPC for hand and foot movements. Because the fMRI signal is related to average neuronal activity, similar activation levels may result either from effector-unspecific neurons or from intermingled subsets of effector-specific neurons within a voxel. We distinguished between these possibilities using fMRI repetition suppression (RS). Participants made delayed, goal-directed eye, hand, and foot movements to visual targets. In each trial, the instructed effector was identical or different to that of the previous trial. RS effects indicated an attenuation of the fMRI signal in repeat trials. The caudal PPC was active during the delay but did not show RS, suggesting that its planning activity was effector independent. Hand and foot-specific RS effects were evident in the anterior superior parietal lobule (SPL), extending to the premotor cortex, with limb overlap in the anterior SPL. Connectivity analysis suggested information flow between the caudal PPC to limb-specific anterior SPL regions and between the limb-unspecific anterior SPL toward limb-specific motor regions. These results underline that both function and effector specificity should be integrated into a concept of PPC action representation not only on a regional but also on a fine-grained, subvoxel level. PMID:27466132

Gene duplication and fragment recombination drive functional diversification of a superfamily of cytoplasmic effectors in Phytophthora sojae.

PubMed

Shen, Danyu; Liu, Tingli; Ye, Wenwu; Liu, Li; Liu, Peihan; Wu, Yuren; Wang, Yuanchao; Dou, Daolong

2013-01-01

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Antagonism of Lactic Acid Bacteria against Phytopathogenic Bacteria

PubMed Central

Visser, Ronèl; Holzapfel, Wilhelm H.; Bezuidenhout, Johannes J.; Kotzé, Johannes M.

1986-01-01

A variety of lactic acid bacteria, isolated from plant surfaces and plant-associated products, were found to be antagonistic to test strains of the phytopathogens Xanthomonas campestris, Erwinia carotovora, and Pseudomonas syringae. Effective “in vitro” inhibition was found both on agar plates and in broth cultures. In pot trials, treatment of bean plants with a Lactobacillus plantarum strain before inoculation with P. syringae caused a significant reduction of the disease incidence. Images PMID:16347150

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, Ravindra; Usui, Kota; Livingstone, Ruth A.

Ice-nucleating organisms play important roles in the environment. With their ability to induce ice formation at temperatures just below the ice melting point, bacteria such as Pseudomonas syringae attack plants through frost damage using specialized ice-nucleating proteins. Besides the impact on agriculture and microbial ecology, airborne P. syringae can affect atmospheric glaciation processes, with consequences for cloud evolution, precipitation, and climate. Biogenic ice nucleation is also relevant for artificial snow production and for biomimetic materials for controlled interfacial freezing. We use interface-specific sum frequency generation (SFG) spectroscopy to show that hydrogen bonding at the water-bacteria contact imposes structural ordering onmore » the adjacent water network. Experimental SFG data and molecular dynamics simulations demonstrate that ice active sites within P. syringae feature unique hydrophilic-hydrophobic patterns to enhance ice nucleation. Finally, the freezing transition is further facilitated by the highly effective removal of latent heat from the nucleation site, as apparent from time-resolved SFG spectroscopy.« less

Polyamines Attenuate Ethylene-Mediated Defense Responses to Abrogate Resistance to Botrytis cinerea in Tomato1[C][W][OA

PubMed Central

Nambeesan, Savithri; AbuQamar, Synan; Laluk, Kristin; Mattoo, Autar K.; Mickelbart, Michael V.; Ferruzzi, Mario G.; Mengiste, Tesfaye; Handa, Avtar K.

2012-01-01

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense. PMID:22128140

Impact of end effector technology on telemanipulation performance

NASA Technical Reports Server (NTRS)

Bejczy, A. K.; Szakaly, Z.; Ohm, T.

1990-01-01

Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

Space Station end effector strategy study

NASA Technical Reports Server (NTRS)

Katzberg, Stephen J.; Jensen, Robert L.; Willshire, Kelli F.; Satterthwaite, Robert E.

1987-01-01

The results of a study are presented for terminology definition, identification of functional requirements, technolgy assessment, and proposed end effector development strategies for the Space Station Program. The study is composed of a survey of available or under-developed end effector technology, identification of requirements from baselined Space Station documents, a comparative assessment of the match between technology and requirements, and recommended strategies for end effector development for the Space Station Program.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

PubMed

Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

PubMed Central

Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

2017-01-01

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

Glycine Betaine Catabolism Contributes to Pseudomonas syringae Tolerance to Hyperosmotic Stress by Relieving Betaine-Mediated Suppression of Compatible Solute Synthesis

PubMed Central

Li, Shanshan; Yu, Xilan

2013-01-01

Many bacteria can accumulate glycine betaine for osmoprotection and catabolize it as a growth substrate, but how they regulate these opposing roles is poorly understood. In Pseudomonas syringae B728a, expression of the betaine catabolism genes was reduced by an osmotic upshift to an intermediate stress level, consistent with betaine accumulation, but was increased by an upshift to a high stress level, as confirmed by an accompanying increase in degradation of radiolabeled betaine. Deletion of the gbcAB betaine catabolism genes reduced osmotolerance at a high osmolarity, and this reduction was due to the relief of betaine-mediated suppression of compatible solute synthesis. This conclusion was supported by the findings that, at high osmolarity, the ΔgbcAB mutant accumulated high betaine levels and low endogenous solutes and exhibited reduced expression of the solute synthesis genes. Moreover, the ΔgbcAB mutant and a mutant deficient in the synthesis of the compatible solutes NAGGN and trehalose exhibited similar reductions in osmotolerance and also in fitness on bean leaves. Activation of betaine catabolism at high osmotic stress resulted, in part, from induction of gbdR, which encodes the transcriptional activator GbdR. Betaine catabolism was subject to partial repression by succinate under hyperosmotic stress conditions, in contrast to strong repression in the absence of stress, suggesting that betaine functions both in nutrition and as an intracellular signal modulating solute synthesis under hyperosmotic stress conditions. Collectively, these results begin to provide a detailed mechanistic understanding of how P. syringae transitions from reliance on exogenously derived betaine to the use of endogenous solutes during adaptation to hyperosmotic conditions. PMID:23524610

Survival, growth, and localization of epiphytic fitness mutants of pseudomonas syringae on leaves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beattie, G.A.; Lindow, S.E.

Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated into and into plant leaves. For example, while non showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular space of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parentalmore » strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sizes protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possible several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes. 52 refs., 6 figs., 1 tab.« less

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing.

PubMed

Kowalczyk, Agnieszka; Eyice, Özge; Schäfer, Hendrik; Price, Oliver R; Finnegan, Christopher J; van Egmond, Roger A; Shaw, Liz J; Barrett, Glyn; Bending, Gary D

2015-10-01

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [(13)C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The YopJ superfamily of type III efforts in plant-associated bacteria

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defense responses or innate immunity. However, some effector proteins may be recognized by...

Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis.

PubMed

Lu, Hua; Zhang, Chong; Albrecht, Ute; Shimizu, Rena; Wang, Guanfeng; Bowman, Kim D

2013-01-01

Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of Arabidopsis and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The Arabidopsis gene NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of NDR1 from citrus, CsNDR1. Overexpression of CsNDR1 complemented susceptibility conferred by the Arabidopsis ndr1-1 mutant to Pseudomonas syringae strains and also led to enhanced resistance to an oomycete pathogen Hyaloperonospora arabidopsidis. Such heightened resistance is associated with increased SA production and expression of the defense marker gene PATHOGENESIS RELATED 1 (PR1). In addition, we found that expression of PR1 and accumulation of SA were induced to modest levels in citrus infected with Candidatus Liberibacter asiaticus, the bacterial pathogen associated with HLB disease. Thus, our data suggest that CsNDR1 is a functional ortholog of Arabidopsis NDR1. Since Ca. L. asiaticus infection only activates modest levels of defense responses in citrus, we propose that genetically increasing SA/NDR1-mediated pathways could potentially lead to enhanced resistance against HLB, citrus canker, and other destructive diseases challenging global citrus production.

Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis

PubMed Central

Lu, Hua; Zhang, Chong; Albrecht, Ute; Shimizu, Rena; Wang, Guanfeng; Bowman, Kim D.

2013-01-01

Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of Arabidopsis and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The Arabidopsis gene NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of NDR1 from citrus, CsNDR1. Overexpression of CsNDR1 complemented susceptibility conferred by the Arabidopsis ndr1-1 mutant to Pseudomonas syringae strains and also led to enhanced resistance to an oomycete pathogen Hyaloperonospora arabidopsidis. Such heightened resistance is associated with increased SA production and expression of the defense marker gene PATHOGENESIS RELATED 1 (PR1). In addition, we found that expression of PR1 and accumulation of SA were induced to modest levels in citrus infected with Candidatus Liberibacter asiaticus, the bacterial pathogen associated with HLB disease. Thus, our data suggest that CsNDR1 is a functional ortholog of Arabidopsis NDR1. Since Ca. L. asiaticus infection only activates modest levels of defense responses in citrus, we propose that genetically increasing SA/NDR1-mediated pathways could potentially lead to enhanced resistance against HLB, citrus canker, and other destructive diseases challenging global citrus production. PMID:23761797

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

PubMed

Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Frickel, Eva-Maria; Ploegh, Hidde L

2016-11-01

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. © 2016 The Authors.

A Proposal for a Genome Similarity-Based Taxonomy for Plant-Pathogenic Bacteria that Is Sufficiently Precise to Reflect Phylogeny, Host Range, and Outbreak Affiliation Applied to Pseudomonas syringae sensu lato as a Proof of Concept.

PubMed

Vinatzer, Boris A; Weisberg, Alexandra J; Monteil, Caroline L; Elmarakeby, Haitham A; Sheppard, Samuel K; Heath, Lenwood S

2017-01-01

Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of Pseudomonas syringae sensu lato, LINs were compared with a P. syringae genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of P. syringae pathovar actinidiae, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

Emerging concepts in effector biology of plant-associated organisms.

PubMed

Hogenhout, Saskia A; Van der Hoorn, Renier A L; Terauchi, Ryohei; Kamoun, Sophien

2009-02-01

Plant-associated organisms secrete proteins and other molecules to modulate plant defense circuitry and enable colonization of plant tissue. Understanding the molecular function of these secreted molecules, collectively known as effectors, became widely accepted as essential for a mechanistic understanding of the processes underlying plant colonization. This review summarizes recent findings in the field of effector biology and highlights the common concepts that have emerged from the study of cellular plant pathogen effectors.

Phytophthora parasitica Effector PpRxLR2 Suppresses Nicotiana benthamiana Immunity.

PubMed

Dalio, R J D; Maximo, H J; Oliveira, T S; Dias, R O; Breton, M C; Felizatti, H; Machado, M

2018-04-01

Phytophthora species secrete several classes of effector proteins during interaction with their hosts. These proteins can have multiple functions including modulation of host physiology and immunity. The RxLR effectors have the ability to enter plant cells using the plant machinery. Some of these effectors have been characterized as immunity suppressors; however, very little is known about their functions in the interaction between Phytophthora parasitica and its hosts. Using a bioinformatics pipeline, we have identified 172 candidate RxLR effectors (CREs) in the isolate IAC 01_95 of P. parasitica. Of these 172 CREs, 93 were found to be also present in eight other genomes of P. parasitica, isolated from different hosts and continents. After transcriptomics and gene expression analysis, we have found five CREs to be up-regulated in in-vitro and in-planta samples. Subsequently, we selected three CREs for functional characterization in the model plant Nicotiana benthamiana. We show that PpRxLR2 is able to completely suppress INF-1-induced cell death, whereas PpRxLR3 and PpRxLR5 moderately suppressed N. benthamiana immunity in a less-extensive manner. Moreover, we confirmed the effector-triggered susceptibility activity of these proteins after transient transformation and infection of N. benthamiana plants. All three CREs enhanced virulence of P. parasitica during the interaction with N. benthamiana. These effectors, in particular PpRxLR2, can be targeted for the development of biotechnology-based control strategies of P. parasitica diseases.

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Expanded functions for a family of plant intracellular immune receptors beyond specific recognition of pathogen effectors

PubMed Central

Bonardi, Vera; Tang, Saijun; Stallmann, Anna; Roberts, Melinda; Cherkis, Karen; Dangl, Jeffery L.

2011-01-01

Plants and animals deploy intracellular immune receptors that perceive specific pathogen effector proteins and microbial products delivered into the host cell. We demonstrate that the ADR1 family of Arabidopsis nucleotide-binding leucine-rich repeat (NB-LRR) receptors regulates accumulation of the defense hormone salicylic acid during three different types of immune response: (i) ADRs are required as “helper NB-LRRs” to transduce signals downstream of specific NB-LRR receptor activation during effector-triggered immunity; (ii) ADRs are required for basal defense against virulent pathogens; and (iii) ADRs regulate microbial-associated molecular pattern-dependent salicylic acid accumulation induced by infection with a disarmed pathogen. Remarkably, these functions do not require an intact P-loop motif for at least one ADR1 family member. Our results suggest that some NB-LRR proteins can serve additional functions beyond canonical, P-loop–dependent activation by specific virulence effectors, extending analogies between intracellular innate immune receptor function from plants and animals. PMID:21911370

Legionella and Coxiella effectors: strength in diversity and activity.

PubMed

Qiu, Jiazhang; Luo, Zhao-Qing

2017-10-01

Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular pathogens that use the Dot/Icm type IV secretion system to translocate effectors into host cells. These effectors are essential for the establishment of membrane-bound compartments known as replication vacuoles, which enable the survival and replication of bacteria inside host cells. The effectors interfere with diverse signalling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, to promote pathogen survival. In this Review, we explore Dot/Icm effectors from L. pneumophila and C. burnetii as key virulence factors, and we examine the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence.

Effects of Botrytis cinerea and Pseudomonas syringae infection on the antioxidant profile of Mesembryanthemum crystallinum C3/CAM intermediate plant.

PubMed

Libik-Konieczny, Marta; Surówka, Ewa; Kuźniak, Elżbieta; Nosek, Michał; Miszalski, Zbigniew

2011-07-01

Mesembryathemum crystallinum plants performing C(3) or CAM (crassulacean acid metabolism) appear to be highly resistant to Botrytis cinerea as well as to Pseudomonas syringae. Fungal hyphae growth was restricted to 48h post-inoculation (hpi) in both metabolic types and morphology of hyphae differed between those growing in C(3) and CAM plants. Growth of bacteria was inhibited significantly 24 hpi in both C(3) and CAM plants. B. cinerea and P. syringae infection led to an increase in the concentration of H(2)O(2) in C(3) plants 3 hpi, while a decrease in H(2)O(2) content was observed in CAM performing plants. The concentration of H(2)O(2) returned to the control level 24 and 48 hpi. Changes in H(2)O(2) content corresponded with the activity of guaiacol peroxidase (POD), mostly 3 hpi. We noted that its activity decreased significantly in C(3) plants and increased in CAM plants in response to inoculation with both pathogens. On the contrary, changes in the activity of CAT did not correlate with H(2)O(2) level. It increased significantly after interaction of C(3) plants with B. cinerea or P. syringae, but in CAM performing plants, the activity of this enzyme was unchanged. Inoculation with B. cinerea or P. syringae led to an increase in the total SOD activity in C(3) plants while CAM plants did not exhibit changes in the total SOD activity after interaction with both pathogens. In conclusion, the pathogen-induced changes in H(2)O(2) content and in SOD, POD and CAT activities in M. crystallinum leaves, were related to the photosynthetic metabolism type of the stressed plants rather than to the lifestyle of the invading pathogen. Copyright © 2011 Elsevier GmbH. All rights reserved.

Temporal Occurrence and Niche Preferences of Phytophthora spp. Causing Brown Rot of Citrus in the Central Valley of California.

PubMed

Hao, Wei; Miles, Timothy D; Martin, Frank N; Browne, Gregory T; Förster, Helga; Adaskaveg, James E

2018-03-01

Brown rot of citrus fruit is caused by several species of Phytophthora and is currently of serious concern for the California citrus industry. Two species, Phytophthora syringae and P. hibernalis, are quarantine pathogens in China, a major export market for California citrus. To maintain trade and estimate the risk of exporting a quarantine pathogen, the distribution and frequency of Phytophthora spp. causing brown rot of orange in major growing areas of California was investigated. Symptomatic fruit were collected from navel (winter to late spring) and Valencia (late spring to summer) orange orchards from 2013 to 2015. Species identification of isolates was based on morphological characteristics, random amplified polymorphic DNA banding patterns, and sequencing of the internal transcribed spacer and the partial cox2/spacer/cox1 regions from axenic cultures, or directly on DNA from fruit tissue using a multiplex TaqMan quantitative polymerase chain reaction assay. In winter samplings, the incidence of P. syringae based on the number of fruit with Phytophthora spp. detection ranged from 73.6 to 96.1% for the two counties surveyed. The remaining isolates were identified as P. citrophthora. In late spring or summer, only P. citrophthora was recovered. P. hibernalis and P. nicotianae were not detected in any fruit with brown rot symptoms. These results indicate that P. syringae is currently an important brown rot pathogen of citrus fruit in California during the cooler seasons of the year. In winter 2016 and 2017, P. syringae was recovered by pear baiting at a high incidence from leaf litter and from a small number of rhizosphere soil or root samples but not from living leaves on the tree. In contrast, P. citrophthora was rarely found in leaf litter but was commonly detected in the rhizosphere. Thus, leaf litter is a major inoculum source for P. syringae and this species occupies a distinct ecological niche.

Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme

PubMed Central

Lucca, Liliana E.; Lerner, Benjamin A.; Gunel, Murat; Raddassi, Khadir; Coric, Vlad; Hafler, David A.; Love, J. Christopher

2017-01-01

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM), the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25—CD127+Foxp3—effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1—CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1—CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. PMID:28880903

A bio-inspired approach for the design of a multifunctional robotic end-effector customized for automated maintenance of a reconfigurable vibrating screen.

PubMed

Makinde, O A; Mpofu, K; Vrabic, R; Ramatsetse, B I

2017-01-01

The development of a robotic-driven maintenance solution capable of automatically maintaining reconfigurable vibrating screen (RVS) machine when utilized in dangerous and hazardous underground mining environment has called for the design of a multifunctional robotic end-effector capable of carrying out all the maintenance tasks on the RVS machine. In view of this, the paper presents a bio-inspired approach which unfolds the design of a novel multifunctional robotic end-effector embedded with mechanical and control mechanisms capable of automatically maintaining the RVS machine. To achieve this, therblig and morphological methodologies (which classifies the motions as well as the actions required by the robotic end-effector in carrying out RVS machine maintenance tasks), obtained from a detailed analogy of how human being (i.e. a machine maintenance manager) will carry out different maintenance tasks on the RVS machine, were used to obtain the maintenance objective functions or goals of the multifunctional robotic end-effector as well as the maintenance activity constraints of the RVS machine that must be adhered to by the multifunctional robotic end-effector during the machine maintenance. The results of the therblig and morphological analyses of five (5) different maintenance tasks capture and classify one hundred and thirty-four (134) repetitive motions and fifty-four (54) functions required in automating the maintenance tasks of the RVS machine. Based on these findings, a worm-gear mechanism embedded with fingers extruded with a hexagonal shaped heads capable of carrying out the "gripping and ungrasping" and "loosening and bolting" functions of the robotic end-effector and an electric cylinder actuator module capable of carrying out "unpinning and hammering" functions of the robotic end-effector were integrated together to produce the customized multifunctional robotic end-effector capable of automatically maintaining the RVS machine. The axial forces ([Formula: see text] and [Formula: see text]), normal forces ([Formula: see text]) and total load [Formula: see text] acting on the teeth of the worm-gear module of the multifunctional robotic end-effector during the gripping of worn-out or new RVS machine subsystems, which are 978.547, 1245.06 and 1016.406 N, respectively, were satisfactory. The nominal bending and torsional stresses acting on the shoulder of the socket module of the multifunctional robotic end-effector during the loosing and tightening of bolts, which are 1450.72 and 179.523 MPa, respectively, were satisfactory. The hammering and unpinning forces utilized by the electric cylinder actuator module of the multifunctional robotic end-effector during the unpinning and hammering of screen panel pins out of and into the screen panels were satisfactory.

Kinematic functions for the 7 DOF robotics research arm

NASA Technical Reports Server (NTRS)

Kreutz, K.; Long, M.; Seraji, Homayoun

1989-01-01

The Robotics Research Model K-1207 manipulator is a redundant 7R serial link arm with offsets at all joints. To uniquely determine joint angles for a given end-effector configuration, the redundancy is parameterized by a scalar variable which corresponds to the angle between the manipulator elbow plane and the vertical plane. The forward kinematic mappings from joint-space to end-effector configuration and elbow angle, and the augmented Jacobian matrix which gives end-effector and elbow angle rates as a function of joint rates, are also derived.

Electroporation of Functional Bacterial Effectors into Mammalian Cells

DOE PAGES

Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

2015-01-19

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less

Deciphering Interplay between Salmonella Invasion Effectors

PubMed Central

Koronakis, Vassilis

2008-01-01

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction. PMID:18389058

Regulation of Effector Treg Cells in Murine Lupus.

PubMed

Chandrasekaran, Uma; Yi, Woelsung; Gupta, Sanjay; Weng, Chien-Huan; Giannopoulou, Eugenia; Chinenov, Yurii; Jessberger, Rolf; Weaver, Casey T; Bhagat, Govind; Pernis, Alessandra B

2016-06-01

Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could limit disease development. © 2016, American College of Rheumatology.

Ice-nucleating bacteria control the order and dynamics of interfacial water

DOE PAGES

Pandey, Ravindra; Usui, Kota; Livingstone, Ruth A.; ...

2016-04-22

Ice-nucleating organisms play important roles in the environment. With their ability to induce ice formation at temperatures just below the ice melting point, bacteria such as Pseudomonas syringae attack plants through frost damage using specialized ice-nucleating proteins. Besides the impact on agriculture and microbial ecology, airborne P. syringae can affect atmospheric glaciation processes, with consequences for cloud evolution, precipitation, and climate. Biogenic ice nucleation is also relevant for artificial snow production and for biomimetic materials for controlled interfacial freezing. We use interface-specific sum frequency generation (SFG) spectroscopy to show that hydrogen bonding at the water-bacteria contact imposes structural ordering onmore » the adjacent water network. Experimental SFG data and molecular dynamics simulations demonstrate that ice active sites within P. syringae feature unique hydrophilic-hydrophobic patterns to enhance ice nucleation. Finally, the freezing transition is further facilitated by the highly effective removal of latent heat from the nucleation site, as apparent from time-resolved SFG spectroscopy.« less

The life history of Pseudomonas syringae: linking agriculture to earth system processes.

PubMed

Morris, Cindy E; Monteil, Caroline L; Berge, Odile

2013-01-01

The description of the ecology of Pseudomonas syringae is moving away from that of a ubiquitous epiphytic plant pathogen to one of a multifaceted bacterium sans frontières in fresh water and other ecosystems linked to the water cycle. Discovery of the aquatic facet of its ecology has led to a vision of its life history that integrates spatial and temporal scales spanning billions of years and traversing catchment basins, continents, and the planet and that confronts the implication of roles that are potentially conflicting for agriculture (as a plant pathogen and as an actor in processes leading to rain and snowfall). This new ecological perspective has also yielded insight into epidemiological phenomena linked to disease emergence. Overall, it sets the stage for the integration of more comprehensive contexts of ecology and evolutionary history into comparative genomic analyses to elucidate how P. syringae subverts the attack and defense responses of the cohabitants of the diverse environments it occupies.

Clarification of Taxonomic Status within the Pseudomonas syringae Species Group Based on a Phylogenomic Analysis.

PubMed

Gomila, Margarita; Busquets, Antonio; Mulet, Magdalena; García-Valdés, Elena; Lalucat, Jorge

2017-01-01

The Pseudomonas syringae phylogenetic group comprises 15 recognized bacterial species and more than 60 pathovars. The classification and identification of strains is relevant for practical reasons but also for understanding the epidemiology and ecology of this group of plant pathogenic bacteria. Genome-based taxonomic analyses have been introduced recently to clarify the taxonomy of the whole genus. A set of 139 draft and complete genome sequences of strains belonging to all species of the P. syringae group available in public databases were analyzed, together with the genomes of closely related species used as outgroups. Comparative genomics based on the genome sequences of the species type strains in the group allowed the delineation of phylogenomic species and demonstrated that a high proportion of strains included in the study are misclassified. Furthermore, representatives of at least 7 putative novel species were detected. It was also confirmed that P. ficuserectae, P. meliae , and P. savastanoi are later synonyms of P. amygdali and that " P. coronafaciens " should be revived as a nomenspecies.

Ice-nucleating bacteria control the order and dynamics of interfacial water

PubMed Central

Pandey, Ravindra; Usui, Kota; Livingstone, Ruth A.; Fischer, Sean A.; Pfaendtner, Jim; Backus, Ellen H. G.; Nagata, Yuki; Fröhlich-Nowoisky, Janine; Schmüser, Lars; Mauri, Sergio; Scheel, Jan F.; Knopf, Daniel A.; Pöschl, Ulrich; Bonn, Mischa; Weidner, Tobias

2016-01-01

Ice-nucleating organisms play important roles in the environment. With their ability to induce ice formation at temperatures just below the ice melting point, bacteria such as Pseudomonas syringae attack plants through frost damage using specialized ice-nucleating proteins. Besides the impact on agriculture and microbial ecology, airborne P. syringae can affect atmospheric glaciation processes, with consequences for cloud evolution, precipitation, and climate. Biogenic ice nucleation is also relevant for artificial snow production and for biomimetic materials for controlled interfacial freezing. We use interface-specific sum frequency generation (SFG) spectroscopy to show that hydrogen bonding at the water-bacteria contact imposes structural ordering on the adjacent water network. Experimental SFG data and molecular dynamics simulations demonstrate that ice-active sites within P. syringae feature unique hydrophilic-hydrophobic patterns to enhance ice nucleation. The freezing transition is further facilitated by the highly effective removal of latent heat from the nucleation site, as apparent from time-resolved SFG spectroscopy. PMID:27152346

Rapid determination of floral aroma compounds of lilac blossom by fast gas chromatography combined with surface acoustic wave sensor.

PubMed

Oh, Se Yeon; Shin, Hyun Du; Kim, Sung Jean; Hong, Jongki

2008-03-07

A novel analytical method using fast gas chromatography combined with surface acoustic wave sensor (GC/SAW) has been developed for the detection of volatile aroma compounds emanated from lilac blossom (Syringa species: Syringa vulgaris variginata and Syringa dilatata). GC/SAW could detect and quantify various fragrance emitted from lilac blossom, enabling to provide fragrance pattern analysis results. The fragrance pattern analysis could easily characterize the delicate differences in aromas caused by the substantial difference of chemical composition according to different color and shape of petals. Moreover, the method validation of GC/SAW was performed for the purpose of volatile floral actual aroma analysis, achieving a high reproducibility and excellent sensitivity. From the validation results, GC/SAW could serve as an alternative analytical technique for the analysis of volatile floral actual aroma of lilac. In addition, headspace solid-phase microextraction (HS-SPME) GC-MS was employed to further confirm the identification of fragrances emitted from lilac blossom and compared to GC/SAW.

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

PubMed Central

Zhou, Yan; Dong, Na; Hu, Liyan; Shao, Feng

2013-01-01

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells. PMID:23469023

Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach.

PubMed

Zhou, Bangjun; Zeng, Lirong

2017-01-01

Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets. Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana . Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae . Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s. We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and plant immunity-associated ROS production.

The plant host pathogen interface: cell wall and membrane dynamics of pathogen-induced responses.

PubMed

Day, Brad; Graham, Terry

2007-10-01

Perception of pathogens by their hosts is the outcome of a highly coordinated and sophisticated surveillance network, tightly regulated by both host and pathogen elicitors, effectors, and signaling processes. In this article, we focus on two relatively well-studied host-pathogens systems, one involving a bacterial-plant interaction (Pseudomonas syringae-Arabidopsis) and the other involving an oomycete-plant interaction (Phytophthora sojae-soybean). We discuss the status of current research related to events occurring at the host-pathogen interface in these two systems, and how these events influence the organization and activation of resistance responses in the respective hosts. This recent research has revealed that in addition to the previously identified resistance machinery (R-proteins, molecular chaperones, etc.), the dynamics of the cell wall, membrane trafficking, and the actin cytoskeleton are intimately associated with the activation of resistance in plants. Specifically, in Arabidopsis, a possible connection between the actin machinery and R-protein- mediated induction of disease resistance is described. In the case of the P. sojae-soybean interaction, we describe the fact that a classical basal resistance elicitor, the cell wall glucan elicitor from the pathogen, can directly activate host hypersensitive cell death, which is apparently modulated in a race-specific manner by the presence of R genes in the host.

Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae.

PubMed Central

Kinscherf, T G; Coleman, R H; Barta, T M; Willis, D K

1991-01-01

Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the beta-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked Tn5 insertions and retained their resistance to the active toxin form, tabtoxnine-beta-lactam (T beta L). The wild-type DNA corresponding to the mutated region was cloned and found to restore the Tn5 mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10(-3)/CFU) in BR2. The Tox- deletion mutants also lost resistance to tabtoxinine-beta-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox- derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease. Images PMID:1648077

Mitochondrial Ca2+ and membrane potential, an alternative pathway for Interleukin 6 to regulate CD4 cell effector function

PubMed Central

Yang, Rui; Lirussi, Dario; Thornton, Tina M; Jelley-Gibbs, Dawn M; Diehl, Sean A; Case, Laure K; Madesh, Muniswamy; Taatjes, Douglas J; Teuscher, Cory; Haynes, Laura; Rincón, Mercedes

2015-01-01

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca2+ late during activation of CD4 cells. Increased levels of mitochondrial Ca2+ in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca2+ is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases. DOI: http://dx.doi.org/10.7554/eLife.06376.001 PMID:25974216

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells

PubMed Central

Carrette, Florent; Henriquez, Monique L.; Fujita, Yu

2018-01-01

T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus–specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7−/− effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7−/− CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells. PMID:29491007

Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells.

PubMed

Quentin, Michaëel; Abad, Pierre; Favery, Bruno

2013-01-01

Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells (FCs). Effectors synthesized in the esophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized FCs requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defense responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalized within FC nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesized roles in the unique feeding behavior of these pests.

Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens.

PubMed

Vleeshouwers, Vivianne G A A; Oliver, Richard P

2014-03-01

One of most important challenges in plant breeding is improving resistance to the plethora of pathogens that threaten our crops. The ever-growing world population, changing pathogen populations, and fungicide resistance issues have increased the urgency of this task. In addition to a vital inflow of novel resistance sources into breeding programs, the functional characterization and deployment of resistance also needs improvement. Therefore, plant breeders need to adopt new strategies and techniques. In modern resistance breeding, effectors are emerging as tools to accelerate and improve the identification, functional characterization, and deployment of resistance genes. Since genome-wide catalogues of effectors have become available for various pathogens, including biotrophs as well as necrotrophs, effector-assisted breeding has been shown to be successful for various crops. "Effectoromics" has contributed to classical resistance breeding as well as for genetically modified approaches. Here, we present an overview of how effector-assisted breeding and deployment is being exploited for various pathosystems.

Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function

USDA-ARS?s Scientific Manuscript database

The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization via activation of Arp2/3 and plays a role in the dynamics of the immunological synapse. How these events influence subsequent gene expression and effector function is unclear. We studied the role of WASp in CD4+ T cell effe...

Neural control of the kidney: functionally specific renal sympathetic nerve fibers.

PubMed

DiBona, G F

2000-11-01

The sympathetic nervous system provides differentiated regulation of the functions of various organs. This differentiated regulation occurs via mechanisms that operate at multiple sites within the classic reflex arc: peripherally at the level of afferent input stimuli to various reflex pathways, centrally at the level of interconnections between various central neuron pools, and peripherally at the level of efferent fibers targeted to various effectors within the organ. In the kidney, increased renal sympathetic nerve activity regulates the functions of the intrarenal effectors: the tubules, the blood vessels, and the juxtaglomerular granular cells. This enables a physiologically appropriate coordination between the circulatory, filtration, reabsorptive, excretory, and renin secretory contributions to overall renal function. Anatomically, each of these effectors has a dual pattern of innervation consisting of a specific and selective innervation by unmyelinated slowly conducting C-type renal sympathetic nerve fibers in addition to an innervation that is shared among all the effectors. This arrangement permits the maximum flexibility in the coordination of physiologically appropriate responses of the tubules, the blood vessels, and the juxtaglomerular granular cells to a variety of homeostatic requirements.

Functionally specific renal sympathetic nerve fibers: role in cardiovascular regulation.

PubMed

DiBona, G F

2001-06-01

The sympathetic nervous system provides differentiated regulation of the functions of various organs. This differentiated regulation occurs through mechanisms that operate at multiple sites within the classic reflex arc: peripherally at the level of afferent input stimuli to various reflex pathways, centrally at the level of interconnections between various central neuron pools, and peripherally at the level of efferent fibers targeted to various effectors within the organ. In the kidney, increased renal sympathetic nerve activity regulates the functions of the intrarenal effectors: the tubules, the blood vessels, and the juxtaglomerular granular cells. This enables a physiologically appropriate coordination between the circulatory, filtration, reabsorptive, excretory, and renin secretory contributions to overall renal function. Anatomically, each of these effectors has a dual pattern of innervation consisting of a specific and selective innervation by unmyelinated slowly conducting C-type renal sympathetic nerve fibers and an innervation that is shared among all the effectors. This arrangement facilitates maximum flexibility in the coordination of the tubules, the blood vessels, and the juxtaglomerular granular cells so as to produce physiologically appropriate responses to a variety of homeostatic requirements.

Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity1[OPEN

PubMed Central

Kim, Denis; Schreiber, Stefan; Zeier, Tatyana; Schuck, Stefan; Reichel-Deland, Vanessa

2017-01-01

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/μ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae. Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction. PMID:28330936

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins.

PubMed

Hewezi, Tarek

2015-10-01

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. © 2015 American Society of Plant Biologists. All Rights Reserved.

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

PubMed Central

Selin, Carrie; de Kievit, Teresa R.; Belmonte, Mark F.; Fernando, W. G. Dilantha

2016-01-01

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. PMID:27199930

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

PubMed Central

Liu, Yunxiao; Lan, Xia; Song, Shiren; Yin, Ling; Dry, Ian B.; Qu, Junjie; Xiang, Jiang; Lu, Jiang

2018-01-01

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria. PMID:29706971

Bacterial avirulence genes.

PubMed

Leach, J E; White, F F

1996-01-01

Although more than 30 bacterial avirulence genes have been cloned and characterized, the function of the gene products in the elictitation of resistance is unknown in all cases but one. The product of avrD from Pseudomonas syringae pv. glycinea likely functions indirectly to elicit resistance in soybean, that is, evidence suggests the gene product is an enzyme involved in elicitor production. In most if not all cases, bacterial avirulence gene function is dependent on interactions with the hypersensitive response and pathogenicity (hrp) genes. Many hrp genes are similar to genes involved in delivery of pathogenicity factors in mammalian bacterial pathogens. Thus, analogies between mammalian and plant pathogens may provide needed clues to elucidate how virulence gene products control induction of resistance.

Modulation of hemoglobin dynamics by an allosteric effector

DOE PAGES

Lal, Jyotsana; Maccarini, Marco; Fouquet, Peter; ...

2016-12-15

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb andmore » HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo (NSE) measurements accompanied by wideangle x-ray scattering (WAXS) to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. Furthermore, these observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.« less

SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual function protein that facilitates infection while protecting from wheat-produced chitinases

USDA-ARS?s Scientific Manuscript database

All fungal plant pathogens produce effectors to manipulate the plant immune system to colonize and gain nutrients from the plant cell. Much is known about how fungal pathogens classified as biotrophs use effectors to interact with their hosts and how the host responds, however, less is known about ...

A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat

USDA-ARS?s Scientific Manuscript database

It has now been established that the wheat pathogen Stagonospora nodorum causes disease on wheat in an inverse gene-for-gene manner through the interaction of pathogen effector proteins and corresponding dominant susceptibility host genes. One such effector, SnToxA, interacts with the Tsn1 gene to c...

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

Abscisic Acid-Cytokinin Antagonism Modulates Resistance Against Pseudomonas syringae in Tobacco.

PubMed

Großkinsky, Dominik K; van der Graaff, Eric; Roitsch, Thomas

2014-12-01

Phytohormones are known as essential regulators of plant defenses, with ethylene, jasmonic acid, and salicylic acid as the central immunity backbone, while other phytohormones have been demonstrated to interact with this. Only recently, a function of the classic phytohormone cytokinin in plant immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco, antagonistic interaction of these phytohormones in plant immunity was identified. Kinetin reduced abscisic acid levels in tobacco, while increased abscisic acid levels by exogenous application or inhibition of abscisic acid catabolism by diniconazole neutralized kinetin-induced resistance. Based on these results, we conclude that reduction of abscisic acid levels by enhanced abscisic acid catabolism strongly contributes to cytokinin-mediated resistance effects. Thus, the identified cytokinin-abscisic acid antagonism is a novel regulatory mechanism in plant immunity.

Coronatine Facilitates Pseudomonas syringae Infection of Arabidopsis Leaves at Night.

PubMed

Panchal, Shweta; Roy, Debanjana; Chitrakar, Reejana; Price, Lenore; Breitbach, Zachary S; Armstrong, Daniel W; Melotto, Maeli

2016-01-01

In many land plants, the stomatal pore opens during the day and closes during the night. Thus, periods of darkness could be effective in decreasing pathogen penetration into leaves through stomata, the primary sites for infection by many pathogens. Pseudomonas syringae pv. tomato (Pst) DC3000 produces coronatine (COR) and opens stomata, raising an intriguing question as to whether this is a virulence strategy to facilitate bacterial infection at night. In fact, we found that (a) biological concentration of COR is effective in opening dark-closed stomata of Arabidopsis thaliana leaves, (b) the COR defective mutant Pst DC3118 is less effective in infecting Arabidopsis in the dark than under light and this difference in infection is reduced with the wild type bacterium Pst DC3000, and (c) cma, a COR biosynthesis gene, is induced only when the bacterium is in contact with the leaf surface independent of the light conditions. These findings suggest that Pst DC3000 activates virulence factors at the pre-invasive phase of its life cycle to infect plants even when environmental conditions (such as darkness) favor stomatal immunity. This functional attribute of COR may provide epidemiological advantages for COR-producing bacteria on the leaf surface.

Coronatine Facilitates Pseudomonas syringae Infection of Arabidopsis Leaves at Night

PubMed Central

Panchal, Shweta; Roy, Debanjana; Chitrakar, Reejana; Price, Lenore; Breitbach, Zachary S.; Armstrong, Daniel W.; Melotto, Maeli

2016-01-01

In many land plants, the stomatal pore opens during the day and closes during the night. Thus, periods of darkness could be effective in decreasing pathogen penetration into leaves through stomata, the primary sites for infection by many pathogens. Pseudomonas syringae pv. tomato (Pst) DC3000 produces coronatine (COR) and opens stomata, raising an intriguing question as to whether this is a virulence strategy to facilitate bacterial infection at night. In fact, we found that (a) biological concentration of COR is effective in opening dark-closed stomata of Arabidopsis thaliana leaves, (b) the COR defective mutant Pst DC3118 is less effective in infecting Arabidopsis in the dark than under light and this difference in infection is reduced with the wild type bacterium Pst DC3000, and (c) cma, a COR biosynthesis gene, is induced only when the bacterium is in contact with the leaf surface independent of the light conditions. These findings suggest that Pst DC3000 activates virulence factors at the pre-invasive phase of its life cycle to infect plants even when environmental conditions (such as darkness) favor stomatal immunity. This functional attribute of COR may provide epidemiological advantages for COR-producing bacteria on the leaf surface. PMID:27446113

Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Germane, Katherine L.; Spiller, Benjamin W.

2011-09-20

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

PubMed

Suzuki, Godai; Wang, Yang; Kubo, Karen; Hirata, Eri; Ohnuki, Shinsuke; Ohya, Yoshikazu

2018-02-20

The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Nuclear processes associated with plant immunity and pathogen susceptibility

PubMed Central

Motion, Graham B.; Amaro, Tiago M.M.M.; Kulagina, Natalja

2015-01-01

Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant–microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants. PMID:25846755

Nuclear processes associated with plant immunity and pathogen susceptibility.

PubMed

Motion, Graham B; Amaro, Tiago M M M; Kulagina, Natalja; Huitema, Edgar

2015-07-01

Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant-microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants. © The Author 2015. Published by Oxford University Press.

AtMIN7 mediated disease resistance to Pseudomonas syringae in Arabidopsis

DOEpatents

He, Sheng Yang [Okemos, MI; Nomura, Kinya [East Lansing, MI

2011-07-26

The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein AtMIN7 mediated protection is enhanced and/or there is a decrease in activity of an AtMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1. Reagents of the present invention provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

New insights into Blimp-1 in T lymphocytes: a divergent regulator of cell destiny and effector function.

PubMed

Fu, Shin-Huei; Yeh, Li-Tzu; Chu, Chin-Chen; Yen, B Lin-Ju; Sytwu, Huey-Kang

2017-07-21

B lymphocyte-induced maturation protein-1 (Blimp-1) serves as a master regulator of the development and function of antibody-producing B cells. Given that its function in T lymphocytes has been identified within the past decade, we review recent findings with emphasis on its role in coordinated control of gene expression during the development, differentiation, and function of T cells. Expression of Blimp-1 is mainly confined to activated T cells and is essential for the production of interleukin (IL)-10 by a subset of forkhead box (Fox)p3 + regulatory T cells with an effector phenotype. Blimp-1 is also required to induce cell elimination in the thymus and critically modulates peripheral T cell activation and proliferation. In addition, Blimp-1 promotes T helper (Th) 2 lineage commitment and limits Th1, Th17 and follicular helper T cell differentiation. Furthermore, Blimp-1 coordinates with other transcription factors to regulate expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. In CD8 + T cells, Blimp-1 expression is distinct in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation when they encounter antigens. Moreover, Blimp-1 plays a fundamental role in coordinating cytokine receptor signaling networks and transcriptional programs to regulate diverse aspects of the formation and function of effector and memory CD8 + T cells and their exhaustion. Blimp-1 also functions as a gatekeeper of T cell activation and suppression to prevent or dampen autoimmune disease, antiviral responses and antitumor immunity. In this review, we discuss the emerging roles of Blimp-1 in the complex regulation of gene networks that regulate the destiny and effector function of T cells and provide a Blimp-1-dominated transcriptional framework for T lymphocyte homeostasis.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

PubMed

Genga, Ryan M; Kearns, Nicola A; Maehr, René

2016-05-15

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai

Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment. Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with differentmore » specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.« less

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

PubMed

Sanders, Anne M; Stehle, John R; Blanks, Michael J; Riedlinger, Gregory; Kim-Shapiro, Jung W; Monjazeb, Arta M; Adams, Jonathan M; Willingham, Mark C; Cui, Zheng

2010-03-31

Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

PubMed

Mukhtar, M Shahid; Carvunis, Anne-Ruxandra; Dreze, Matija; Epple, Petra; Steinbrenner, Jens; Moore, Jonathan; Tasan, Murat; Galli, Mary; Hao, Tong; Nishimura, Marc T; Pevzner, Samuel J; Donovan, Susan E; Ghamsari, Lila; Santhanam, Balaji; Romero, Viviana; Poulin, Matthew M; Gebreab, Fana; Gutierrez, Bryan J; Tam, Stanley; Monachello, Dario; Boxem, Mike; Harbort, Christopher J; McDonald, Nathan; Gai, Lantian; Chen, Huaming; He, Yijian; Vandenhaute, Jean; Roth, Frederick P; Hill, David E; Ecker, Joseph R; Vidal, Marc; Beynon, Jim; Braun, Pascal; Dangl, Jeffery L

2011-07-29

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.

A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Julie Anne Roden, Branids Belt, Jason Barzel Ross, Thomas Tachibana, Joe Vargas, Mary Beth Mudgett

2004-11-23

The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resultedmore » in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.« less

Extensive Field Survey, Laboratory and Greenhouse Studies Reveal Complex Nature of Pseudomonas syringae-Associated Hazelnut Decline in Central Italy

PubMed Central

Lamichhane, Jay Ram; Bartoli, Claudia; Varvaro, Leonardo

2016-01-01

Pseudomonas avellanae (Pav) has been reported as the causal agent of bacterial decline and bacterial canker of hazelnut in Italy and Greece, respectively. Both hazelnut diseases were reported to be similar in terms of symptoms, severity and persistence. In this study, we found that both symptomatic and asymptomatic trees in the field were colonized by Pav. Multilocus Sequence Typing (MLST) analysis showed that Pav strains isolated during this study in Italy belong to the P. syringae phylogroup 1 and they are closely related to Pav strains previously isolated in Greece from hazelnut bacterial canker. On the other hand, strains isolated in earlier studies from hazelnut decline in Italy belong to both phylogroup 1 and 2 of P. syringae. Both phylogroup 1 strains of P. syringae from Greece and Italy are different than strains isolated in this study in terms of their capacity to excrete fluorescent pigments on different media. Despite the same plant genotype and cropping practices adopted, the incidence of hazelnut decline ranged from nearly 0 to 91% across our study sites. No disease developed on plants inoculated with Pav through wounding while leaf scar inoculations produced only mild disease symptoms. Based on our results and the previously reported correlation between pedo-climatic conditions and hazelnut decline, we conclude that hazelnut decline in central Italy could be incited by a combination of predisposing (adverse pedo-climatic conditions) and contributing factors (Pav). Because this is a true decline different from “bacterial canker” described in Greece, we refer to it as hazelnut decline (HD). PMID:26840951

Extensive Field Survey, Laboratory and Greenhouse Studies Reveal Complex Nature of Pseudomonas syringae-Associated Hazelnut Decline in Central Italy.

PubMed

Lamichhane, Jay Ram; Bartoli, Claudia; Varvaro, Leonardo

2016-01-01

Pseudomonas avellanae (Pav) has been reported as the causal agent of bacterial decline and bacterial canker of hazelnut in Italy and Greece, respectively. Both hazelnut diseases were reported to be similar in terms of symptoms, severity and persistence. In this study, we found that both symptomatic and asymptomatic trees in the field were colonized by Pav. Multilocus Sequence Typing (MLST) analysis showed that Pav strains isolated during this study in Italy belong to the P. syringae phylogroup 1 and they are closely related to Pav strains previously isolated in Greece from hazelnut bacterial canker. On the other hand, strains isolated in earlier studies from hazelnut decline in Italy belong to both phylogroup 1 and 2 of P. syringae. Both phylogroup 1 strains of P. syringae from Greece and Italy are different than strains isolated in this study in terms of their capacity to excrete fluorescent pigments on different media. Despite the same plant genotype and cropping practices adopted, the incidence of hazelnut decline ranged from nearly 0 to 91% across our study sites. No disease developed on plants inoculated with Pav through wounding while leaf scar inoculations produced only mild disease symptoms. Based on our results and the previously reported correlation between pedo-climatic conditions and hazelnut decline, we conclude that hazelnut decline in central Italy could be incited by a combination of predisposing (adverse pedo-climatic conditions) and contributing factors (Pav). Because this is a true decline different from "bacterial canker" described in Greece, we refer to it as hazelnut decline (HD).

Improvement of the Efficacy of Linear Undecapeptides against Plant-Pathogenic Bacteria by Incorporation of d-Amino Acids ▿

PubMed Central

Güell, Imma; Cabrefiga, Jordi; Badosa, Esther; Ferre, Rafael; Talleda, Montserrat; Bardají, Eduard; Planas, Marta; Feliu, Lidia; Montesinos, Emilio

2011-01-01

A set of 31 undecapeptides, incorporating 1 to 11 d-amino acids and derived from the antimicrobial peptide BP100 (KKLFKKILKYL-NH2), was designed and synthesized. This set was evaluated for inhibition of growth of the plant-pathogenic bacteria Erwinia amylovora, Pseudomonas syringae pv. syringae, and Xanthomonas axonopodis pv. vesicatoria, hemolysis, and protease degradation. Two derivatives were as active as BP100, and 10 peptides displayed improved activity, with the all-d isomer being the most active. Twenty-six peptides were less hemolytic than BP100, and all peptides were more stable against protease degradation. Plant extracts inhibited the activity of BP100 as well as that of the d-isomers. Ten derivatives incorporating one d-amino acid each were tested in an infectivity inhibition assay with the three plant-pathogenic bacteria by using detached pear and pepper leaves and pear fruits. All 10 peptides studied were active against E. amylovora, 6 displayed activity against P. syringae pv. syringae, and 2 displayed activity against X. axonopodis pv. vesicatoria. Peptides BP143 (KKLFKKILKYL-NH2) and BP145 (KKLFKKILKYL-NH2), containing one d-amino acid at positions 4 and 2 (underlined), respectively, were evaluated in whole-plant assays for the control of bacterial blight of pepper and pear and fire blight of pear. Peptide BP143 was as effective as streptomycin in the three pathosystems, was more effective than BP100 against bacterial blight of pepper and pear, and equally effective against fire blight of pear. PMID:21335383

The effector candidate repertoire of the arbuscular mycorrhizal fungus Rhizophagus clarus.

PubMed

Sędzielewska Toro, Kinga; Brachmann, Andreas

2016-02-09

Arbuscular mycorrhizal fungi (AMF) form an ecologically important symbiosis with more than two thirds of studied land plants. Recent studies of plant-pathogen interactions showed that effector proteins play a key role in host colonization by controlling the plant immune system. We hypothesise that also for symbiotic-plant interactions the secreted effectome of the fungus is a major component of communication and the conservation level of effector proteins between AMF species may be indicative whether they play a fundamental role. In this study, we used a bioinformatics pipeline to predict and compare the effector candidate repertoire of the two AMF species, Rhizophagus irregularis and Rhizophagus clarus. Our in silico pipeline revealed a list of 220 R. irregularis candidate effector genes that create a valuable information source to elucidate the mechanism of plant infection and colonization by fungi during AMF symbiotic interaction. While most of the candidate effectors show no homologies to known domains or proteins, the candidates with homologies point to potential roles in signal transduction, cell wall modification or transcription regulation. A remarkable aspect of our work is presence of a large portion of the effector proteins involved in symbiosis, which are not unique to each fungi or plant species, but shared along the Glomeromycota phylum. For 95% of R. irregularis candidates we found homologs in a R. clarus genome draft generated by Illumina high-throughput sequencing. Interestingly, 9% of the predicted effectors are at least as conserved between the two Rhizophagus species as proteins with housekeeping functions (similarity > 90%). Therefore, we state that this group of highly conserved effector proteins between AMF species may play a fundamental role during fungus-plant interaction. We hypothesise that in symbiotic interactions the secreted effectome of the fungus might be an important component of communication. Identification and functional characterization of the primary AMF effectors that regulate symbiotic development will help in understanding the mechanisms of fungus-plant interaction.

Characterization of CTL Recognized Epitopes on Human Breast Tumors

DTIC Science & Technology

1996-09-01

maturation and effector function of cellular immune cytotoxic effectors such as CTL (11). (c) The epitopes defined on tumor Ag are self-peptides of...have been reported to be expressed in breast and ovarian cancer cells (18), and they apparently function by maintaining the undifferentiated state...Body of the Report The purpose of the present work continues to be the characterization of the functional significance of the CTL epitopes as potential

The Genome Biology of Effector Gene Evolution in Filamentous Plant Pathogens.

PubMed

Sánchez-Vallet, Andrea; Fouché, Simone; Fudal, Isabelle; Hartmann, Fanny E; Soyer, Jessica L; Tellier, Aurélien; Croll, Daniel

2018-05-16

Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Clarification of Taxonomic Status within the Pseudomonas syringae Species Group Based on a Phylogenomic Analysis

PubMed Central

Gomila, Margarita; Busquets, Antonio; Mulet, Magdalena; García-Valdés, Elena; Lalucat, Jorge

2017-01-01

The Pseudomonas syringae phylogenetic group comprises 15 recognized bacterial species and more than 60 pathovars. The classification and identification of strains is relevant for practical reasons but also for understanding the epidemiology and ecology of this group of plant pathogenic bacteria. Genome-based taxonomic analyses have been introduced recently to clarify the taxonomy of the whole genus. A set of 139 draft and complete genome sequences of strains belonging to all species of the P. syringae group available in public databases were analyzed, together with the genomes of closely related species used as outgroups. Comparative genomics based on the genome sequences of the species type strains in the group allowed the delineation of phylogenomic species and demonstrated that a high proportion of strains included in the study are misclassified. Furthermore, representatives of at least 7 putative novel species were detected. It was also confirmed that P. ficuserectae, P. meliae, and P. savastanoi are later synonyms of P. amygdali and that “P. coronafaciens” should be revived as a nomenspecies. PMID:29270162

JMJ27, an Arabidopsis H3K9 histone demethylase, modulates defense against Pseudomonas syringae and flowering time.

PubMed

Dutta, Aditya; Choudhary, Pratibha; Caruana, Julie; Raina, Ramesh

2017-09-01

Histone methylation is known to dynamically regulate diverse developmental and physiological processes. Histone methyl marks are written by methyltransferases and erased by demethylases, and result in modification of chromatin structure to repress or activate transcription. However, little is known about how histone methylation may regulate defense mechanisms and flowering time in plants. Here we report characterization of JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), an Arabidopsis JHDM2 (JmjC domain-containing histone demethylase 2) family protein, which modulates defense against pathogens and flowering time. JMJ27 is a nuclear protein containing a zinc-finger motif and a catalytic JmjC domain with conserved Fe(II) and α-ketoglutarate binding sites, and displays H3K9me1/2 demethylase activity both in vitro and in vivo. JMJ27 is induced in response to virulent Pseudomonas syringae pathogens and is required for resistance against these pathogens. JMJ27 is a negative modulator of WRKY25 (a repressor of defense) and a positive modulator of several pathogenesis-related (PR) proteins. Additionally, loss of JMJ27 function leads to early flowering. JMJ27 negatively modulates the major flowering regulator CONSTANS (CO) and positively modulates FLOWERING LOCUS C (FLC). Taken together, our results indicate that JMJ27 functions as a histone demethylase to modulate both physiological (defense) and developmental (flowering time) processes in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

PubMed Central

Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

1999-01-01

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection.

PubMed

Ederli, Luisa; Madeo, Laura; Calderini, Ornella; Gehring, Chris; Moretti, Chiaraluce; Buonaurio, Roberto; Paolocci, Francesco; Pasqualini, Stefania

2011-10-15

In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O(3)). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O(3) and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O(3) sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H(2)O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen. Copyright © 2011 Elsevier GmbH. All rights reserved.

Elongator Plays a Positive Role in Exogenous NAD-Induced Defense Responses in Arabidopsis.

PubMed

An, Chuanfu; Ding, Yezhang; Zhang, Xudong; Wang, Chenggang; Mou, Zhonglin

2016-05-01

Extracellular NAD is emerging as an important signal molecule in animal cells, but its role in plants has not been well-established. Although it has been shown that exogenous NAD(+) activates defense responses in Arabidopsis, components in the exogenous NAD(+)-activated defense pathway remain to be fully discovered. In a genetic screen for mutants insensitive to exogenous NAD(+) (ien), we isolated a mutant named ien2. Map-based cloning revealed that IEN2 encodes ELONGATA3 (ELO3)/AtELP3, a subunit of the Arabidopsis Elongator complex, which functions in multiple biological processes, including histone modification, DNA (de)methylation, and transfer RNA modification. Mutations in the ELO3/AtELP3 gene compromise exogenous NAD(+)-induced expression of pathogenesis-related (PR) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326, and transgenic expression of the coding region of ELO3/AtELP3 in elo3/Atelp3 restores NAD(+) responsiveness to the mutant plants, demonstrating that ELO3/AtELP3 is required for exogenous NAD(+)-induced defense responses. Furthermore, mutations in genes encoding the other five Arabidopsis Elongator subunits (ELO2/AtELP1, AtELP2, ELO1/AtELP4, AtELP5, and AtELP6) also compromise exogenous NAD(+)-induced PR gene expression and resistance to P. syringae pv. maculicola ES4326. These results indicate that the Elongator complex functions as a whole in exogenous NAD(+)-activated defense signaling in Arabidopsis.

Endosome-Associated CRT1 Functions Early in Resistance Gene–Mediated Defense Signaling in Arabidopsis and Tobacco[W

PubMed Central

Kang, Hong-Gu; Oh, Chang-Sik; Sato, Masanao; Katagiri, Fumiaki; Glazebrook, Jane; Takahashi, Hideki; Kachroo, Pradeep; Martin, Gregory B.; Klessig, Daniel F.

2010-01-01

Resistance gene–mediated immunity confers protection against pathogen infection in a wide range of plants. A genetic screen for Arabidopsis thaliana mutants compromised for recognition of turnip crinkle virus previously identified CRT1, a member of the GHKL ATPase/kinase superfamily. Here, we demonstrate that CRT1 interacts with various resistance proteins from different structural classes, and this interaction is disrupted when these resistance proteins are activated. The Arabidopsis mutant crt1-2 crh1-1, which lacks CRT1 and its closest homolog, displayed compromised resistance to avirulent Pseudomonas syringae and Hyaloperonospora arabidopsidis. Additionally, resistance-associated hypersensitive cell death was suppressed in Nicotiana benthamiana silenced for expression of CRT1 homolog(s). Thus, CRT1 appears to be a general factor for resistance gene–mediated immunity. Since elevation of cytosolic calcium triggered by avirulent P. syringae was compromised in crt1-2 crh1-1 plants, but cell death triggered by Nt MEK2DD was unaffected in CRT1-silenced N. benthamiana, CRT1 likely functions at an early step in this pathway. Genome-wide transcriptome analysis led to identification of CRT1-Associated genes, many of which are associated with transport processes, responses to (a)biotic stress, and the endomembrane system. Confocal microscopy and subcellular fractionation revealed that CRT1 localizes to endosome-like vesicles, suggesting a key process in resistance protein activation/signaling occurs in this subcellular compartment. PMID:20332379

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

PubMed

Gupta, Bhawna; Iancu, Emanuela M; Gannon, Philippe O; Wieckowski, Sébastien; Baitsch, Lukas; Speiser, Daniel E; Rufer, Nathalie

2012-07-01

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

PubMed

Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

2010-07-23

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues. Copyright 2010 Elsevier Inc. All rights reserved.

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

PubMed Central

Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

2015-01-01

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

PubMed Central

Lippé, Roger; Miaczynska, Marta; Rybin, Vladimir; Runge, Anja; Zerial, Marino

2001-01-01

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases. PMID:11452015

Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

PubMed Central

Boesch, Austin W.; Osei-Owusu, Nana Yaw; Crowley, Andrew R.; Chu, Thach H.; Chan, Ying N.; Weiner, Joshua A.; Bharadwaj, Pranay; Hards, Rufus; Adamo, Mark E.; Gerber, Scott A.; Cocklin, Sarah L.; Schmitz, Joern E.; Miles, Adam R.; Eckman, Joshua W.; Belli, Aaron J.; Reimann, Keith A.; Ackerman, Margaret E.

2016-01-01

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies. PMID:28018355

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

PubMed Central

Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

HOPM1 mediated disease resistance to Pseudomonas syringae in Arabidopsis

DOEpatents

He, Sheng Yang [Okemos, MI; Nomura, Kinya [East Lansing, MI

2011-11-15

The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein HopM1.sub.1-300 mediated protection is enhanced, such as increased protection to Pseudomonas syringae pv. tomato DC3000 HopM1 and/or there is an increase in activity of an ATMIN associated plant protection protein, such as ATMIN7. Reagents of the present invention further provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.

Advanced Aerodynamic Design of Passive Porosity Control Effectors

NASA Technical Reports Server (NTRS)

Hunter, Craig A.; Viken, Sally A.; Wood, Richard M.; Bauer, Steven X. S.

2001-01-01

This paper describes aerodynamic design work aimed at developing a passive porosity control effector system for a generic tailless fighter aircraft. As part of this work, a computational design tool was developed and used to layout passive porosity effector systems for longitudinal and lateral-directional control at a low-speed, high angle of attack condition. Aerodynamic analysis was conducted using the NASA Langley computational fluid dynamics code USM3D, in conjunction with a newly formulated surface boundary condition for passive porosity. Results indicate that passive porosity effectors can provide maneuver control increments that equal and exceed those of conventional aerodynamic effectors for low-speed, high-alpha flight, with control levels that are a linear function of porous area. This work demonstrates the tremendous potential of passive porosity to yield simple control effector systems that have no external moving parts and will preserve an aircraft's fixed outer mold line.

Evolutionary Plasticity of AmrZ Regulation in Pseudomonas

PubMed Central

Dougherty, Kevin; Diaz, Beatriz; Murillo, Rachel

2018-01-01

ABSTRACT amrZ encodes a master regulator protein conserved across pseudomonads, which can be either a positive or negative regulator of swimming motility depending on the species examined. To better understand plasticity in the regulatory function of AmrZ, we characterized the mode of regulation for this protein for two different motility-related phenotypes in Pseudomonas stutzeri. As in Pseudomonas syringae, AmrZ functions as a positive regulator of swimming motility within P. stutzeri, which suggests that the functions of this protein with regard to swimming motility have switched at least twice across pseudomonads. Shifts in mode of regulation cannot be explained by changes in AmrZ sequence alone. We further show that AmrZ acts as a positive regulator of colony spreading within this strain and that this regulation is at least partially independent of swimming motility. Closer investigation of mechanistic shifts in dual-function regulators like AmrZ could provide unique insights into how transcriptional pathways are rewired between closely related species. IMPORTANCE Microbes often display finely tuned patterns of gene regulation across different environments, with major regulatory changes controlled by a small group of “master” regulators within each cell. AmrZ is a master regulator of gene expression across pseudomonads and can be either a positive or negative regulator for a variety of pathways depending on the strain and genomic context. Here, we demonstrate that the phenotypic outcomes of regulation of swimming motility by AmrZ have switched at least twice independently in pseudomonads, so that AmrZ promotes increased swimming motility in P. stutzeri and P. syringae but represses this phenotype in Pseudomonas fluorescens and Pseudomonas aeruginosa. Since examples of switches in regulatory mode are relatively rare, further investigation into the mechanisms underlying shifts in regulator function for AmrZ could provide unique insights into the evolution of bacterial regulatory proteins. PMID:29669886

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

PubMed

Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae.

PubMed

Verdier, Valérie; Triplett, Lindsay R; Hummel, Aaron W; Corral, Rene; Cernadas, R Andres; Schmidt, Clarice L; Bogdanove, Adam J; Leach, Jan E

2012-12-01

Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

PubMed

Zhang, Lei; Davies, Laura J; Elling, Axel A

2015-01-01

Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Functional classification of memory CD8(+) T cells by CX3CR1 expression.

PubMed

Böttcher, Jan P; Beyer, Marc; Meissner, Felix; Abdullah, Zeinab; Sander, Jil; Höchst, Bastian; Eickhoff, Sarah; Rieckmann, Jan C; Russo, Caroline; Bauer, Tanja; Flecken, Tobias; Giesen, Dominik; Engel, Daniel; Jung, Steffen; Busch, Dirk H; Protzer, Ulrike; Thimme, Robert; Mann, Matthias; Kurts, Christian; Schultze, Joachim L; Kastenmüller, Wolfgang; Knolle, Percy A

2015-09-25

Localization of memory CD8(+) T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX3CR1 distinguishes memory CD8(+) T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX3CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8(+) T cells with effector function. We find CD62L(hi)CX3CR1(+) memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX3CR1(+) memory CD8(+) T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3CR1-based functional classification will help to resolve the principles of protective CD8(+) T-cell memory.

Transcription Factor Foxo1 Is a Negative Regulator of NK Cell Maturation and Function

PubMed Central

Deng, Youcai; Kerdiles, Yann; Chu, Jianhong; Yuan, Shunzong; Wang, Youwei; Chen, Xilin; Mao, Hsiaoyin; Zhang, Lingling; Zhang, Jianying; Hughes, Tiffany; Deng, Yafei; Zhang, Qi; Wang, Fangjie; Zou, Xianghong; Liu, Chang-Gong; Freud, Aharon G.; Li, Xiaohui; Caligiuri, Michael A; Vivier, Eric; Yu, Jianhua

2015-01-01

SUMMARY Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes through upregulating CD62L expression, and impaired late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21+/− mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions. PMID:25769609

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P.

PubMed

Zhang, Xiaoxiao; Farah, Nadya; Rolston, Laura; Ericsson, Daniel J; Catanzariti, Ann-Maree; Bernoux, Maud; Ve, Thomas; Bendak, Katerina; Chen, Chunhong; Mackay, Joel P; Lawrence, Gregory J; Hardham, Adrienne; Ellis, Jeffrey G; Williams, Simon J; Dodds, Peter N; Jones, David A; Kobe, Bostjan

2018-05-01

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Coolwater culmination: Sensitive high-resolution ion microprobe (SHRIMP) U-Pb and isotopic evidence for continental delamination in the Syringa Embayment, Salmon River suture, Idaho

USGS Publications Warehouse

Lund, K.; Aleinikoff, J.N.; Yacob, E.Y.; Unruh, D.M.; Fanning, C.M.

2008-01-01

During dextral oblique translation along Laurentia in western Idaho, the Blue Mountains superterrane underwent clockwise rotation and impinged into the Syringa embayment at the northern end of the Salmon River suture. Along the suture, the superterrane is juxtaposed directly against western Laurentia, making this central Cordilleran accretionary-margin segment unusually attenuated. In the embayment, limited orthogonal contraction produced a crustal wedge of oceanic rocks that delaminated Laurentian crust. The wedge is exposed through Laurentian crust in the Coolwater culmination as documented by mapping and by sensitive high-resolution ion microprobe U-Pb, Sri, and ??Nd data for gneisses that lie inboard of the suture. The predominant country rock is Mesoproterozoic paragneiss overlying Laurentian basement. An overlying Neoproterozoic (or younger) paragneiss belt in the Syringa embayment establishes the form of the Cordilleran miogeocline and that the embayment is a relict of Rodinia rifting. An underlying Cretaceous paragneiss was derived from arc terranes and suture-zone orogenic welt but also from Laurentia. The Cretaceous paragneiss and an 86-Ma orthogneiss that intruded it formed the wedge of oceanic rocks that were inserted into the Laurentian margin between 98 and 73 Ma, splitting supracrustal Laurentian rocks from their basement. Crustal thickening, melting and intrusion within the wedge, and folding to form the Coolwater culmination continued until 61 Ma. The embayment formed a restraining bend at the end of the dextral transpressional suture. Clockwise rotation of the impinging superterrane and overthrusting of Laurentia that produced the crustal wedge in the Coolwater culmination are predicted by oblique collision into the Syringa embayment. Copyright 2008 by the American Geophysical Union.

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajasekaran, Mohan B; Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS; Mitchell, Sue A

2010-07-30

Research highlights: {yields} Bioinformatic analysis reveals EfeM is a metallopeptidase with conserved HXXE motif. {yields} Mass spectrometry confirms EfeM consists of 251 residues, molecular weight 27,772Da. {yields} SRCD spectroscopy shows an {alpha}-helical secondary structure. {yields} Single crystals of EfeM are orthorhombic and diffract to 1.6A resolution. {yields} Space group is P22{sub 1}2{sub 1} with cell dimensions a = 46.74, b = 95.17 and c = 152.61 A. -- Abstract: The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signalmore » peptide of 34 amino acid residues and a C-terminal 'Peptidase{sub M}75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr{sub 3}370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M{sub W} 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly {alpha}-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6 A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.« less

Modern Technologies for Creating Synthetic Antibodies for Clinical application

PubMed Central

Lebedenko, E. N.

2009-01-01

The modular structure and versatility of antibodies enables one to modify natural immunoglobulins in different ways for various clinical applications. Rational design and molecular engineering make it possible to directionally modify the molecular size, affinity, specificity, and immunogenicity and effector functions of an antibody, as well as to combine them with other functional agents. This review focuses on up-to-date methods of antibody engineering for diagnosing and treating various diseases, particularly on new technologies meant to refine the effector functions of therapeutic antibodies. PMID:22649585

Functional Proteomics to Identify Moderators of CD8+ T Cell Function in Melanoma

DTIC Science & Technology

2015-05-01

identified 17 phage that selectively bind TIL rather than effector cells. However, none of these phage influenced CD8+ TIL expansion or function in vitro...Using a novel NextGeneration sequencing approach, we have further defined another 1,000,000 phage that selectively bind TIL , of which 100,000 are unique...Using the original approach outlined in the application, we identified a total of 17 unique phage that selectively bind CD8+ TIL but not effector or

The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

PubMed

Siggers, Keri A; Lesser, Cammie F

2008-07-17

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.

Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

PubMed

Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

2013-04-01

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.

Tailored immune responses: novel effector helper T cell subsets in protective immunity.

PubMed

Kara, Ervin E; Comerford, Iain; Fenix, Kevin A; Bastow, Cameron R; Gregor, Carly E; McKenzie, Duncan R; McColl, Shaun R

2014-02-01

Differentiation of naïve CD4⁺ cells into functionally distinct effector helper T cell subsets, characterised by distinct "cytokine signatures," is a cardinal strategy employed by the mammalian immune system to efficiently deal with the rapidly evolving array of pathogenic microorganisms encountered by the host. Since the T(H)1/T(H)2 paradigm was first described by Mosmann and Coffman, research in the field of helper T cell biology has grown exponentially with seven functionally unique subsets having now been described. In this review, recent insights into the molecular mechanisms that govern differentiation and function of effector helper T cell subsets will be discussed in the context of microbial infections, with a focus on how these different helper T cell subsets orchestrate immune responses tailored to combat the nature of the pathogenic threat encountered.

Effector CD8+ T cell IFN-γ production and cytotoxicity are enhanced by mild hyperthermia

PubMed Central

Mace, Thomas A.; Zhong, Lingwen; Kokolus, Kathleen M.; Repasky, Elizabeth A.

2012-01-01

Purpose Clinical trials combining hyperthermia with radiation and/or chemotherapy for cancer treatment have resulted in improved overall survival and control of local recurrences. The contribution of thermally enhanced anti-immune function in these effects is of considerable interest, but not understood; studies on the fundamental effects of elevated temperature on immune effector cells are needed. The goal of this study is to investigate the potential of mild hyperthermia to impact tumor antigen-specific (Ag) effector CD8+ T cell functions. Method Pmel-1 Ag-specific CD8+ T cells were exposed to mild hyperthermia and tested for changes in IFN-γ production and cytotoxicity. Additionally, overall plasma membrane organization and the phosphorylation of signaling proteins were also investigated following heat treatment. Results Exposing effector Pmel-1 specific CD8+ T cells to mild hyperthermia (39.5°C) resulted in significantly enhanced Ag-specific IFN-γ production and tumor target cell killing compared to that seen using lower temperatures (33 and 37°C). Further, inhibition of protein synthesis during hyperthermia did not reduce subsequent Ag-induced IFN-γ production by CD8+ T cells. Correlated with these effects, we observed a distinct clustering of GM1+ lipid microdomains at the plasma membrane and enhanced phosphorylation of LAT and PKCθ which may be related to an observed enhancement of Ag-specific effector CD8+ T cell IFN-γ gene transcription following mild hyperthermia. However, mitogen–mediated production of IFN-γ, which bypasses T cell receptor activation with antigen, was not enhanced. Conclusions Antigen-dependent effector T cell activity is enhanced following mild hyperthermia. These effects could potentially occur in patients being treated with thermal therapies. These data also provide support for the use of thermal therapy as an adjuvant for immunotherapies to improve CD8+ effector cell function. PMID:22235780

Arabidopsis ACCELERATED CELL DEATH2 Modulates Programmed Cell DeathW⃞

PubMed Central

Yao, Nan; Greenberg, Jean T.

2006-01-01

The Arabidopsis thaliana chloroplast protein ACCELERATED CELL DEATH2 (ACD2) modulates the amount of programmed cell death (PCD) triggered by Pseudomonas syringae and protoporphyrin IX (PPIX) treatment. In vitro, ACD2 can reduce red chlorophyll catabolite, a chlorophyll derivative. We find that ACD2 shields root protoplasts that lack chlorophyll from light- and PPIX-induced PCD. Thus, chlorophyll catabolism is not obligatory for ACD2 anti-PCD function. Upon P. syringae infection, ACD2 levels and localization change in cells undergoing PCD and in their close neighbors. Thus, ACD2 shifts from being largely in chloroplasts to partitioning to chloroplasts, mitochondria, and, to a small extent, cytosol. ACD2 protects cells from PCD that requires the early mitochondrial oxidative burst. Later, the chloroplasts of dying cells generate NO, which only slightly affects cell viability. Finally, the mitochondria in dying cells have dramatically altered movements and cellular distribution. Overproduction of both ACD2 (localized to mitochondria and chloroplasts) and ascorbate peroxidase (localized to chloroplasts) greatly reduces P. syringae–induced PCD, suggesting a pro-PCD role for mitochondrial and chloroplast events. During infection, ACD2 may bind to and/or reduce PCD-inducing porphyrin-related molecules in mitochondria and possibly chloroplasts that generate reactive oxygen species, cause altered organelle behavior, and activate a cascade of PCD-inducing events. PMID:16387834

LOV-domain photoreceptor, encoded in a genomic island, attenuates the virulence of Pseudomonas syringae in light-exposed Arabidopsis leaves.

PubMed

Moriconi, Victoria; Sellaro, Romina; Ayub, Nicolás; Soto, Gabriela; Rugnone, Matías; Shah, Rashmi; Pathak, Gopal P; Gärtner, Wolfgang; Casal, Jorge J

2013-10-01

In Arabidopsis thaliana, light signals modulate the defences against bacteria. Here we show that light perceived by the LOV domain-regulated two-component system (Pst-Lov) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) modulates virulence against A. thaliana. Bioinformatic analysis and the existence of an episomal circular intermediate indicate that the locus encoding Pst-Lov is present in an active genomic island acquired by horizontal transfer. Strains mutated at Pst-Lov showed enhanced growth on minimal medium and in leaves of A. thaliana exposed to light, but not in leaves incubated in darkness or buried in the soil. Pst-Lov repressed the expression of principal and alternative sigma factor genes and their downstream targets linked to bacterial growth, virulence and quorum sensing, in a strictly light-dependent manner. We propose that the function of Pst-Lov is to distinguish between soil (dark) and leaf (light) environments, attenuating the damage caused to host tissues while releasing growth out of the host. Therefore, in addition to its direct actions via photosynthesis and plant sensory receptors, light may affect plants indirectly via the sensory receptors of bacterial pathogens. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function

PubMed Central

Tomalka, Amanda G.; Stopford, Charles M.; Lee, Pei-Chung; Rietsch, Arne

2012-01-01

Summary Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the P. aeruginosa translocator protein PopD as a model to identify its export signals. The amino-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone-binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. PMID:23121689

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

PubMed

Etzl, Stefan; Lindner, Robert; Nelson, Matthew D; Winkler, Andreas

2018-06-08

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties. © 2018 Etzl et al.

Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease

PubMed Central

McLane, Laura M.; Steblyanko, Maria; Anikeeva, Nadia; Ablanedo-Terrazas, Yuria; Demers, Korey; Eller, Michael A.; Streeck, Hendrik; Jansson, Marianne; Sönnerborg, Anders; Canaday, David H.; Naji, Ali; Wherry, E. John; Robb, Merlin L.; Reyes-Teran, Gustavo; Sykulev, Yuri; Betts, Michael R.

2018-01-01

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues. PMID:29652923

Role of the glucocorticoid-induced TNFR-related protein (GITR)-GITR ligand pathway in innate and adaptive immunity.

PubMed

Azuma, Miyuki

2010-01-01

Glucocorticoid-induced TNF receptor-related protein (GITR) is expressed in regulatory T cells at high levels, but is also inducible in conventional effector T cells after activation. Initial studies using an agonistic anti- GITR mAb mislead this line of research with respect to the contribution of GITR stimulation on the function of regulatory T cells. In fact, GITR acts as a costimulatory receptor for both effector and regulatory T cells by enhancing effector and regulatory functions, respectively. Unlike other costimulatory ligands, GITR ligand (GITRL) expression on mature myeloid dendritic cells (DCs) is extremely limited and the GITR-GITRL pathway does not contribute markedly to direct interactions with T cells and antigen-presenting cells in the secondary lymphoid tissues. Rather, GITRL is constitutively expressed on parenchymal tissue cells and interacts with GITR expressed on tissue-infiltrating macrophages and DCs, or effector and regulatory T cells. Interactions with GITR and GITRL at local inflammatory sites induce site-specific production of cytokines and chemokines, resulting in control activation of tissue-infiltrating effector or regulatory cells and their migration. This review summarizes recent reports on the GITR-GITRL pathway, which controls both innate and adaptive immune responses.

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity.

PubMed

Medina, Cesar Augusto; Reyes, Paola Andrea; Trujillo, Cesar Augusto; Gonzalez, Juan Luis; Bejarano, David Alejandro; Montenegro, Nathaly Andrea; Jacobs, Jonathan M; Joe, Anna; Restrepo, Silvia; Alfano, James R; Bernal, Adriana

2018-03-01

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Genetic Diversity of Coastal Bottlenose Dolphins Revealed by Structurally and Functionally Diverse Hemoglobins

PubMed Central

Remington, Nicole; Stevens, Robert D.; Wells, Randall S.; Hohn, Aleta; Dhungana, Suraj; Taboy, Celine H.; Crumbliss, Alvin L.; Henkens, Robert; Bonaventura, Celia

2007-01-01

Studies of structure-function relationships in the respiratory proteins of marine mammals revealed unexpected variations in the number and types of hemoglobins (Hbs) present in coastal bottlenose dolphins, Tursiops truncatus. We obtained blood samples from free-ranging coastal bottlenose dolphins as a component of capture-release studies. We found that the oxygen-binding functions of bottlenose dolphin blood are poised between effector-saturated and unsaturated levels, enabling exercise-dependent shifts in oxygen transfer functions. Isolated bottlenose dolphin Hbs showed elevated pH sensitivities (Bohr effects) and appreciably lower oxygen affinities than adult human Hb in the absence of allosteric effectors. These properties may be an adaptive modification that enhance oxygen delivery during diving episodes when oxygen tensions and effector levels are low. The Hbs of individual dolphins showed similar oxygen affinities, responses to effectors, and expression of heme-heme interaction in oxygen binding, but differed in their redox potentials and rates of autoxidation. The heterogeneity suggested by these functional variations in Hbs of individual dolphins was born out by variations in the molecular weights and numbers of their α and β globin chains. Although coastal bottlenose dolphins were expected to have a single type of Hb, the mass differences observed revealed considerable genetic diversity. There were multiple Hb forms in some individuals and differences in Hb patterns among individuals within the same community. PMID:17604574

Genetic diversity of coastal bottlenose dolphins revealed by structurally and functionally diverse hemoglobins.

PubMed

Remington, Nicole; Stevens, Robert D; Wells, Randall S; Holn, Aleta; Dhungana, Suraj; Taboy, Celine H; Crumbliss, Alvin L; Henkens, Robert; Bonaventura, Celia

2007-08-15

Studies of structure-function relationships in the respiratory proteins of marine mammals revealed unexpected variations in the number and types of hemoglobins (Hbs) present in coastal bottlenose dolphins, Tursiops truncatus. We obtained blood samples from free-ranging coastal bottlenose dolphins as a component of capture-release studies. We found that the oxygen-binding functions of bottlenose dolphin blood are poised between effector-saturated and unsaturated levels, enabling exercise-dependent shifts in oxygen transfer functions. Isolated bottlenose dolphin Hbs showed elevated pH sensitivities (Bohr effects) and appreciably lower oxygen affinities than adult human Hb in the absence of allosteric effectors. These properties may be an adaptive modification that enhances oxygen delivery during diving episodes when oxygen tensions and effector levels are low. The Hbs of individual dolphins showed similar oxygen affinities, responses to effectors, and expression of heme-heme interaction in oxygen binding, but differed in their redox potentials and rates of autoxidation. The heterogeneity suggested by these functional variations in Hbs of individual dolphins was born out by variations in the molecular weights and numbers of their alpha and beta globin chains. Although coastal bottlenose dolphins were expected to have a single type of Hb, the mass differences observed revealed considerable genetic diversity. There were multiple Hb forms in some individuals and differences in Hb patterns among individuals within the same community.

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene-for-gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus 'Evereste'

USDA-ARS?s Scientific Manuscript database

The Gram-negative bacterium Erwinia amylovora (Burrill) Winslow. et al., causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that functions to translocate effector proteins into plant cells that collectively function to suppress host defenses and ena...

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

PubMed

Ve, Thomas; Williams, Simon J; Catanzariti, Ann-Maree; Rafiqi, Maryam; Rahman, Motiur; Ellis, Jeffrey G; Hardham, Adrienne R; Jones, David A; Anderson, Peter A; Dodds, Peter N; Kobe, Bostjan

2013-10-22

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

Distinct regions of the Phytophthora essential effector Avh238 determine its function in cell death activation and plant immunity suppression.

PubMed

Yang, Bo; Wang, Qunqing; Jing, Maofeng; Guo, Baodian; Wu, Jiawei; Wang, Haonan; Wang, Yang; Lin, Long; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Wang, Yuanchao

2017-04-01

Phytophthora pathogens secrete effectors to manipulate host innate immunity, thus facilitating infection. Among the RXLR effectors highly induced during Phytophthora sojae infection, Avh238 not only contributes to pathogen virulence but also triggers plant cell death. However, the detailed molecular basis of Avh238 functions remains largely unknown. We mapped the regions responsible for Avh238 functions in pathogen virulence and plant cell death induction using a strategy that combines investigation of natural variation and large-scale mutagenesis assays. The correlation between cellular localization and Avh238 functions was also evaluated. We found that the 79 th residue (histidine or leucine) of Avh238 determined its cell death-inducing activity, and that the 53 amino acids in its C-terminal region are responsible for promoting Phytophthora infection. Transient expression of Avh238 in Nicotiana benthamiana revealed that nuclear localization is essential for triggering cell death, while Avh238-mediated suppression of INF1-triggered cell death requires cytoplasmic localization. Our results demonstrate that a representative example of an essential Phytophthora RXLR effector can evolve to escape recognition by the host by mutating one nucleotide site, and can also retain plant immunosuppressive activity to enhance pathogen virulence in planta. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

N-Acyl-Homoserine Lactone Primes Plants for Cell Wall Reinforcement and Induces Resistance to Bacterial Pathogens via the Salicylic Acid/Oxylipin Pathway[C][W][OPEN

PubMed Central

Schenk, Sebastian T.; Hernández-Reyes, Casandra; Samans, Birgit; Stein, Elke; Neumann, Christina; Schikora, Marek; Reichelt, Michael; Mithöfer, Axel; Becker, Annette; Kogel, Karl-Heinz; Schikora, Adam

2014-01-01

The ability of plants to monitor their surroundings, for instance the perception of bacteria, is of crucial importance. The perception of microorganism-derived molecules and their effector proteins is the best understood of these monitoring processes. In addition, plants perceive bacterial quorum sensing (QS) molecules used for cell-to-cell communication between bacteria. Here, we propose a mechanism for how N-acyl-homoserine lactones (AHLs), a group of QS molecules, influence host defense and fortify resistance in Arabidopsis thaliana against bacterial pathogens. N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for enhanced callose deposition, accumulation of phenolic compounds, and lignification of cell walls. Moreover, increased levels of oxylipins and salicylic acid favored closure of stomata in response to Pseudomonas syringae infection. The AHL-induced resistance seems to differ from the systemic acquired and the induced systemic resistances, providing new insight into inter-kingdom communication. Consistent with the observation that short-chain AHLs, unlike oxo-C14-HSL, promote plant growth, treatments with C6-HSL, oxo-C10-HSL, or oxo-C14-HSL resulted in different transcriptional profiles in Arabidopsis. Understanding the priming induced by bacterial QS molecules augments our knowledge of plant reactions to bacteria and suggests strategies for using beneficial bacteria in plant protection. PMID:24963057

Characterization of siderophore produced by Pseudomonas syringae BAF.1 and its inhibitory effects on spore germination and mycelium morphology of Fusarium oxysporum.

PubMed

Yu, Sumei; Teng, Chunying; Liang, Jinsong; Song, Tao; Dong, Liying; Bai, Xin; Jin, Yu; Qu, Juanjuan

2017-11-01

In this study, an antagonistic bacterium against Fusarium oxysporum was identified and designated as Pseudomonas syringae strain BAF.1 on the basis of 16S rDNA sequence analysis and physiological-biochemical characteristics. It produced catechol-species siderophore at a molecular weight of 488.59 Da and a maximum amount of 55.27 μg/ml with glucose as a carbon source and asparagine as a nitrogen source at a C/N ratio of 10:1, 30°C and pH 7. The siderophore exhibited prominent antagonistic activity against Fusarium oxysporum with a maximum inhibition rate of 95.24% and had also suppressive effects on other kinds of 11 phytopathogenic fungi in the absence of FeCl 3 ·6H 2 O. Spore germination was completely inhibited by 50 μl of the siderophorecontaining solution, and the ultrastructures of mycelia and spores were also considerably suppressed by siderophore treatment as established by electron microscopy observation. These results indicate that the siderophore produced by Pseudomonas syringae BAF.1 could be potentially used for biocontrol of pathogenic Fusarium oxysporum.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants

PubMed Central

Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants. PMID:29033963

A Plethora of Virulence Strategies Hidden Behind Nuclear Targeting of Microbial Effectors

PubMed Central

Rivas, Susana; Genin, Stéphane

2011-01-01

Plant immune responses depend on the ability to couple rapid recognition of the invading microbe to an efficient response. During evolution, plant pathogens have acquired the ability to deliver effector molecules inside host cells in order to manipulate cellular and molecular processes and establish pathogenicity. Following translocation into plant cells, microbial effectors may be addressed to different subcellular compartments. Intriguingly, a significant number of effector proteins from different pathogenic microorganisms, including viruses, oomycetes, fungi, nematodes, and bacteria, is targeted to the nucleus of host cells. In agreement with this observation, increasing evidence highlights the crucial role played by nuclear dynamics, and nucleocytoplasmic protein trafficking during a great variety of analyzed plant–pathogen interactions. Once in the nucleus, effector proteins are able to manipulate host transcription or directly subvert essential host components to promote virulence. Along these lines, it has been suggested that some effectors may affect histone packing and, thereby, chromatin configuration. In addition, microbial effectors may either directly activate transcription or target host transcription factors to alter their regular molecular functions. Alternatively, nuclear translocation of effectors may affect subcellular localization of their cognate resistance proteins in a process that is essential for resistance protein-mediated plant immunity. Here, we review recent progress in our field on the identification of microbial effectors that are targeted to the nucleus of host plant cells. In addition, we discuss different virulence strategies deployed by microbes, which have been uncovered through examination of the mechanisms that guide nuclear localization of effector proteins. PMID:22639625

Integration of two RAB5 groups during endosomal transport in plants

PubMed Central

Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

2018-01-01

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

Manipulation of intestinal epithelial cell function by the cell contact-dependent type III secretion systems of Vibrio parahaemolyticus

PubMed Central

O'Boyle, Nicky; Boyd, Aoife

2013-01-01

Vibrio parahaemolyticus elicits gastroenteritis by deploying Type III Secretion Systems (TTSS) to deliver effector proteins into epithelial cells of the human intestinal tract. The bacteria must adhere to the human cells to allow colonization and operation of the TTSS translocation apparatus bridging the bacterium and the host cell. This article first reviews recent advances in identifying the molecules responsible for intercellular adherence. V. parahaemolyticus possesses two TTSS, each of which delivers an exclusive set of effectors and mediates unique effects on the host cell. TTSS effectors primarily target and alter the activation status of host cell signaling proteins, thereby bringing about changes in the regulation of cellular behavior. TTSS1 is responsible for the cytotoxicity of V. parahaemolyticus, while TTSS2 is necessary for the enterotoxicity of the pathogen. Recent publications have elucidated the function of several TTSS effectors and their importance in the virulence of the bacterium. This review will explore the ability of the TTSS to manipulate activities of human intestinal cells and how this modification of cell function favors bacterial colonization and persistence of V. parahaemolyticus in the host. PMID:24455490

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases*

PubMed Central

Westfall, Corey S.; Xu, Ang; Jez, Joseph M.

2014-01-01

Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1–3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme. PMID:25160622

Machine learning methods enable predictive modeling of antibody feature:function relationships in RV144 vaccinees.

PubMed

Choi, Ickwon; Chung, Amy W; Suscovich, Todd J; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Kaewkungwal, Jaranit; O'Connell, Robert J; Francis, Donald; Robb, Merlin L; Michael, Nelson L; Kim, Jerome H; Alter, Galit; Ackerman, Margaret E; Bailey-Kellogg, Chris

2015-04-01

The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.

Pivotal advance: CTLA-4+ T cells exhibit normal antiviral functions during acute viral infection.

PubMed

Raué, Hans-Peter; Slifka, Mark K

2007-05-01

Previous studies have shown that T cells, which are genetically deficient in CTLA-4/CD152 expression, will proliferate uncontrollably, resulting in lethal autoimmune disease. This and other evidence indicate that CTLA-4 plays a critical role in the negative regulation of effector T cell function. In contrast to expectations, BrdU incorporation experiments demonstrated that CTLA-4 expression was associated with normal or even enhanced in vivo proliferation of virus-specific CD4+ and CD8+ T cells following acute lymphocytic choriomeningitis virus or vaccinia virus infection. When compared with CTLA-4- T cells directly ex vivo, CTLA-4+ T cells also exhibited normal antiviral effector functions following stimulation with peptide-coated cells, virus-infected cells, plate-bound anti-CD3/anti-CTLA-4, or the cytokines IL-12 and IL-18. Together, this indicates that CTLA-4 does not directly inhibit antiviral T cell expansion or T cell effector functions, at least not under the normal physiological conditions associated with either of these two acute viral infections.

Machine Learning Methods Enable Predictive Modeling of Antibody Feature:Function Relationships in RV144 Vaccinees

PubMed Central

Choi, Ickwon; Chung, Amy W.; Suscovich, Todd J.; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Kaewkungwal, Jaranit; O'Connell, Robert J.; Francis, Donald; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Alter, Galit; Ackerman, Margaret E.; Bailey-Kellogg, Chris

2015-01-01

The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates. PMID:25874406

A new approach to global control of redundant manipulators

NASA Technical Reports Server (NTRS)

Seraji, Homayoun

1989-01-01

A new and simple approach to configuration control of redundant manipulators is presented. In this approach, the redundancy is utilized to control the manipulator configuration directly in task space, where the task will be performed. A number of kinematic functions are defined to reflect the desirable configuration that will be achieved for a given end-effector position. The user-defined kinematic functions and the end-effector Cartesian coordinates are combined to form a set of task-related configuration variables as generalized coordinates for the manipulator. An adaptive scheme is then utilized to globally control the configuration variables so as to achieve tracking of some desired reference trajectories. This accomplishes the basic task of desired end-effector motion, while utilizing the redundancy to achieve any additional task through the desired time variation of the kinematic functions. The control law is simple and computationally very fast, and does not require the complex manipulator dynamic model.

CD4+ T helper 2 cells – microbial triggers, differentiation requirements and effector functions

PubMed Central

Okoye, Isobel S; Wilson, Mark S

2011-01-01

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate-like cells with the capacity to secrete large amounts of interleukin-5 (IL-5), IL-13 and IL-9 as well as IL-4-producing and antigen-processing basophils have (re)-emerged onto the type 2 scene. To what extent these new players influence αβ+ CD4+ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T-cell receptor ligation, co-stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned. PMID:22043920

Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

PubMed

Johnston, Joe; Gopalarathnam, Ashok

2012-09-01

A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the stall behavior more gentle. The benefits of using the effector could include care-free operations at high angles of attack during perching and maneuvering flight, especially in gusty conditions.

Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

PubMed

Rajput, Nasir Ahmed; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Zhang, Qimeng; Ru, Yanyan; Sun, Peng; Dou, Daolong

2015-12-01

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Intrinsic disorder in pathogen effectors: protein flexibility as an evolutionary hallmark in a molecular arms race.

PubMed

Marín, Macarena; Uversky, Vladimir N; Ott, Thomas

2013-09-01

Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants' innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein-protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors.

Contrasting Roles of the Apoplastic Aspartyl Protease APOPLASTIC, ENHANCED DISEASE SUSCEPTIBILITY1-DEPENDENT1 and LEGUME LECTIN-LIKE PROTEIN1 in Arabidopsis Systemic Acquired Resistance1,2[W

PubMed Central

Breitenbach, Heiko H.; Wenig, Marion; Wittek, Finni; Jordá, Lucia; Maldonado-Alconada, Ana M.; Sarioglu, Hakan; Colby, Thomas; Knappe, Claudia; Bichlmeier, Marlies; Pabst, Elisabeth; Mackey, David; Parker, Jane E.; Vlot, A. Corina

2014-01-01

Systemic acquired resistance (SAR) is an inducible immune response that depends on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Here, we show that Arabidopsis (Arabidopsis thaliana) EDS1 is required for both SAR signal generation in primary infected leaves and SAR signal perception in systemic uninfected tissues. In contrast to SAR signal generation, local resistance remains intact in eds1 mutant plants in response to Pseudomonas syringae delivering the effector protein AvrRpm1. We utilized the SAR-specific phenotype of the eds1 mutant to identify new SAR regulatory proteins in plants conditionally expressing AvrRpm1. Comparative proteomic analysis of apoplast-enriched extracts from AvrRpm1-expressing wild-type and eds1 mutant plants led to the identification of 12 APOPLASTIC, EDS1-DEPENDENT (AED) proteins. The genes encoding AED1, a predicted aspartyl protease, and another AED, LEGUME LECTIN-LIKE PROTEIN1 (LLP1), were induced locally and systemically during SAR signaling and locally by salicylic acid (SA) or its functional analog, benzo 1,2,3-thiadiazole-7-carbothioic acid S-methyl ester. Because conditional overaccumulation of AED1-hemagglutinin inhibited SA-induced resistance and SAR but not local resistance, the data suggest that AED1 is part of a homeostatic feedback mechanism regulating systemic immunity. In llp1 mutant plants, SAR was compromised, whereas the local resistance that is normally associated with EDS1 and SA as well as responses to exogenous SA appeared largely unaffected. Together, these data indicate that LLP1 promotes systemic rather than local immunity, possibly in parallel with SA. Our analysis reveals new positive and negative components of SAR and reinforces the notion that SAR represents a distinct phase of plant immunity beyond local resistance. PMID:24755512

The Arabidopsis immune regulator SRFR1 dampens defences against herbivory by Spodoptera exigua and parasitism by Heterodera schachtii.

PubMed

Nguyen, Phuong Dung T; Pike, Sharon; Wang, Jianying; Nepal Poudel, Arati; Heinz, Robert; Schultz, Jack C; Koo, Abraham J; Mitchum, Melissa G; Appel, Heidi M; Gassmann, Walter

2016-05-01

Plants have developed diverse mechanisms to fine tune defence responses to different types of enemy. Cross-regulation between signalling pathways may allow the prioritization of one response over another. Previously, we identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent effector-triggered immunity against the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4. The use of multiple stresses is a powerful tool to further define gene function. Here, we examined whether SRFR1 also impacts resistance to a herbivorous insect in leaves and to a cyst nematode in roots. Interestingly, srfr1-1 plants showed increased resistance to herbivory by the beet army worm Spodoptera exigua and to parasitism by the cyst nematode Heterodera schachtii compared with the corresponding wild-type Arabidopsis accession RLD. Using quantitative real-time PCR (qRT-PCR) to measure the transcript levels of salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathway genes, we found that enhanced resistance of srfr1-1 plants to S. exigua correlated with specific upregulation of the MYC2 branch of the JA pathway concurrent with suppression of the SA pathway. In contrast, the greater susceptibility of RLD was accompanied by simultaneously increased transcript levels of SA, JA and JA/ET signalling pathway genes. Surprisingly, mutation of either SRFR1 or EDS1 increased resistance to H. schachtii, indicating that the concurrent presence of both wild-type genes promotes susceptibility. This finding suggests a novel form of resistance in Arabidopsis to the biotrophic pathogen H. schachtii or a root-specific regulation of the SA pathway by EDS1, and places SRFR1 at an intersection between multiple defence pathways. © 2015 THE AUTHORS MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

PubMed

Cressman, William J; Beckett, Dorothy

2016-01-19

Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

PubMed

Cooper, Colin A; Zhang, Kun; Andres, Sara N; Fang, Yuan; Kaniuk, Natalia A; Hannemann, Mandy; Brumell, John H; Foster, Leonard J; Junop, Murray S; Coombes, Brian K

2010-02-05

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

Anionic lipids and the cytoskeletal proteins MreB and RodZ define the spatio-temporal distribution and function of membrane stress controller PspA in Escherichia coli.

PubMed

Jovanovic, Goran; Mehta, Parul; Ying, Liming; Buck, Martin

2014-11-01

All cell types must maintain the integrity of their membranes. The conserved bacterial membrane-associated protein PspA is a major effector acting upon extracytoplasmic stress and is implicated in protection of the inner membrane of pathogens, formation of biofilms and multi-drug-resistant persister cells. PspA and its homologues in Gram-positive bacteria and archaea protect the cell envelope whilst also supporting thylakoid biogenesis in cyanobacteria and higher plants. In enterobacteria, PspA is a dual function protein negatively regulating the Psp system in the absence of stress and acting as an effector of membrane integrity upon stress. We show that in Escherichia coli the low-order oligomeric PspA regulatory complex associates with cardiolipin-rich, curved polar inner membrane regions. There, cardiolipin and the flotillin 1 homologue YqiK support the PspBC sensors in transducing a membrane stress signal to the PspA-PspF inhibitory complex. After stress perception, PspA high-order oligomeric effector complexes initially assemble in polar membrane regions. Subsequently, the discrete spatial distribution and dynamics of PspA effector(s) in lateral membrane regions depend on the actin homologue MreB and the peptidoglycan machinery protein RodZ. The consequences of loss of cytoplasmic membrane anionic lipids, MreB, RodZ and/or YqiK suggest that the mode of action of the PspA effector is closely associated with cell envelope organization. © 2014 The Authors.

PSTha5a23, a candidate effector from the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici, is involved in plant defense suppression and rust pathogenicity.

PubMed

Cheng, Yulin; Wu, Kuan; Yao, Juanni; Li, Shumin; Wang, Xiaojie; Huang, Lili; Kang, Zhensheng

2017-05-01

During the infection of host plants, pathogens can deliver virulence-associated 'effector' proteins to promote plant susceptibility. However, little is known about effector function in the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici (Pst) that is an important fungal pathogen in wheat production worldwide. Here, they report their findings on an in planta highly induced candidate effector from Pst, PSTha5a23. The PSTha5a23 gene is unique to Pst and shows a low level of intra-species polymorphism. It has a functional N-terminal signal peptide and is translocated to the host cytoplasm after infection. Overexpression of PSTha5a23 in Nicotiana benthamiana was found to suppress the programmed cell death triggered by BAX, PAMP-INF1 and two resistance-related mitogen-activated protein kinases (MKK1 and NPK1). Overexpression of PSTha5a23 in wheat also suppressed pattern-triggered immunity (PTI)-associated callose deposition. In addition, silencing of PSTha5a23 did not change Pst virulence phenotypes; however, overexpression of PSTha5a23 significantly enhanced Pst virulence in wheat. These results indicate that the Pst candidate effector PSTha5a23 plays an important role in plant defense suppression and rust pathogenicity, and also highlight the utility of gene overexpression in plants as a tool for studying effectors from obligate biotrophic pathogens. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Functional heterogeneity of human effector CD8+ T cells.

PubMed

Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

The targeting of plant cellular systems by injected type III effector proteins.

PubMed

Lewis, Jennifer D; Guttman, David S; Desveaux, Darrell

2009-12-01

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector-host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.

The Rab-binding Profiles of Bacterial Virulence Factors during Infection*

PubMed Central

So, Ernest C.; Schroeder, Gunnar N.; Carson, Danielle; Mattheis, Corinna; Mousnier, Aurélie; Broncel, Malgorzata; Tate, Edward W.; Frankel, Gad

2016-01-01

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection. PMID:26755725

Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

USDA-ARS?s Scientific Manuscript database

Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins that are delivered to the apoplast, as well as...

Effector-triggered immunity: from pathogen perception to robust defense.

PubMed

Cui, Haitao; Tsuda, Kenichi; Parker, Jane E

2015-01-01

In plant innate immunity, individual cells have the capacity to sense and respond to pathogen attack. Intracellular recognition mechanisms have evolved to intercept perturbations by pathogen virulence factors (effectors) early in host infection and convert it to rapid defense. One key to resistance success is a polymorphic family of intracellular nucleotide-binding/leucine-rich-repeat (NLR) receptors that detect effector interference in different parts of the cell. Effector-activated NLRs connect, in various ways, to a conserved basal resistance network in order to transcriptionally boost defense programs. Effector-triggered immunity displays remarkable robustness against pathogen disturbance, in part by employing compensatory mechanisms within the defense network. Also, the mobility of some NLRs and coordination of resistance pathways across cell compartments provides flexibility to fine-tune immune outputs. Furthermore, a number of NLRs function close to the nuclear chromatin by balancing actions of defense-repressing and defense-activating transcription factors to program cells dynamically for effective disease resistance.

Robotic end-effector for rewaterproofing shuttle tiles

NASA Astrophysics Data System (ADS)

Manouchehri, Davoud; Hansen, Joseph M.; Wu, Cheng M.; Yamamoto, Brian S.; Graham, Todd

1992-11-01

This paper summarizes work by Rockwell International's Space Systems Division's Robotics Group at Downey, California. The work is part of a NASA-led team effort to automate Space Shuttle rewaterproofing in the Orbiter Processing Facility at the Kennedy Space Center and the ferry facility at the Ames-Dryden Flight Research Facility. Rockwell's effort focuses on the rewaterproofing end-effector, whose function is to inject hazardous dimethylethyloxysilane into thousands of ceramic tiles on the underside of the orbiter after each flight. The paper has five sections. First, it presents background on the present manual process. Second, end-effector requirements are presented, including safety and interface control. Third, a design is presented for the five end-effector systems: positioning, delivery, containment, data management, and command and control. Fourth, end-effector testing and integrating to the total system are described. Lastly, future applications for this technology are discussed.

Diverse secreted effectors are required for Salmonella persistence in a mouse infection model.

PubMed

Kidwai, Afshan S; Mushamiri, Ivy; Niemann, George S; Brown, Roslyn N; Adkins, Joshua N; Heffron, Fred

2013-01-01

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kidwai, Afshan S.; Mushamiri, Ivy T.; Niemann, George

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 moremore » strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.« less

Effector functions of memory CTLs can be affected by signals received during reactivation.

PubMed

Lv, Yingjun; Mattson, Elliot; Bhadurihauck, Anjuli; Garcia, Karla; Li, Lei; Xiao, Zhengguo

2017-08-01

Memory cytotoxic T lymphocytes (CTLs) are able to provide protections to the host against repeated insults from intracellular pathogens. However, it has not been completely understood how the effector functions of memory CTLs are induced upon antigen challenge, which is directly related to the efficacy of their protection. Third signal cytokines, such as IL-12 and type I interferon, have been suggested to be involved in the protective function of memory CTLs, but direct evidence is warranted. In this report, we found that memory CTLs need to be reactivated to exert effector functions. Infusion of a large population of quiescent memory CTLs did not lead to cancer control in tumor-bearing mice, whereas infusion of a reactivated memory CTL population did. This reactivation of memory CTLs requires cytokines such as IL-12 in addition to antigen but was less dependent upon costimulation and IL-2 compared to naive CTLs. Memory CTLs responded more quickly and with greater strength than their naive counterparts upon stimulation, which is associated with higher upregulation of important transcription factors such as T-bet and phosphorylated STAT4. In addition, memory CTLs underwent less expansion than naive CTLs upon pathogen challenge. In conclusion, effector functions of established memory CTLs may be affected by certain cytokines such as IL-12 and type I IFN. Thus, a pathogen's ability to induce cytokines could contribute to the efficacy of protection of an established memory CTL population.

Mechanism and function of type IV secretion during infection of the human host

PubMed Central

Gonzalez-Rivera, Christian; Bhatty, Minny; Christie, Peter J.

2015-01-01

Bacterial pathogens employ type IV secretion systems (T4SSs) for various purposes to aid in survival and proliferation in eukaryotic host. One large T4SS subfamily, the conjugation systems, confers a selective advantage to the invading pathogen in clinical settings through dissemination of antibiotic resistance genes and virulence traits. Besides their intrinsic importance as principle contributors to the emergence of multiply drug-resistant ‘superbugs’, detailed studies of these highly tractable systems have generated important new insights into the mode of action and architectures of paradigmatic T4SSs as a foundation for future efforts aimed at suppressing T4SS machine function. Over the past decade, extensive work on the second large T4SS subfamily, the effector translocators, has identified a myriad of mechanisms employed by pathogens to subvert, subdue, or bypass cellular processes and signaling pathways of the host cell. An overarching theme in the evolution of many effectors is that of molecular mimicry. These effectors carry domains similar to those of eukaryotic proteins and exert their effects through stealthy interdigitation of cellular pathways, often with the outcome not of inducing irreversible cell damage but rather of reversibly modulating cellular functions. This chapter summarizes the major developments for the actively studied pathogens with an emphasis on the structural and functional diversity of the T4SSs and the emerging common themes surrounding effector function in the human host. PMID:27337453

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

PubMed

Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

2016-02-01

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

Combover/CG10732, a Novel PCP Effector for Drosophila Wing Hair Formation

PubMed Central

Fagan, Jeremy K.; Dollar, Gretchen; Lu, Qiuheng; Barnett, Austen; Pechuan Jorge, Joaquin; Schlosser, Andreas; Pfleger, Cathie; Adler, Paul; Jenny, Andreas

2014-01-01

The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh. PMID:25207969

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

PubMed

Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

2005-03-29

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

Suppression of polymorphonuclear (PMN) and monocyte-mediated inhibition of Candida albicans growth by delta-9-tetrahydrocannabinol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Djeu, J.Y.; Parapanios, A.; Halkias, D.

This study was an in vitro attempt to identify the effector cells responsible for growth inhibition of the opportunistic fungus, candida albicans, and to determine if THC or another marijuana derivatives, 11-hydroxyTHC, would adversely affect their function. Using a 24h radiolabel assay, the authors found that growth inhibition of C. albicans was primarily mediated by PMN and monocytes that could be isolated normal human peripheral blood. Both effector cell types caused almost complete inhibition of Candida growth at effector/target ratio of 300/1 and inhibition was often still seen at 30/1-. Incubation of PMN, PBL, or monocytes for 1 hr atmore » 37C with THC or 11-hydroxyTHC caused a marked suppression of function in all 3 cell populations. Maximal suppression was obtained with 7.5-10..mu..g/ml of the drugs in medium containing 10% fetal bovine serum (FBS) or with 2-4..mu..g/ml in 1% FBS. These drug concentrations did not affect lymphoid cell viability or candida growth in the absence of lymphoid effector cells. Marijuana derivatives, therefore, are doubly dangerous in that opportunistic fungi such as C. albicans can grow in their presence while the effector cells that control fungal growth are readily inactivated.« less

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

PubMed Central

2017-01-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

PubMed

Caillaud, Marie-Cécile; Piquerez, Sophie J M; Fabro, Georgina; Steinbrenner, Jens; Ishaque, Naveed; Beynon, Jim; Jones, Jonathan D G

2012-01-01

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Genetically modified luminescent bacteria Ralostonia solanacerum, Pseudomonas syringae, Pseudomonas savastanoi, and wild type bacterium Vibrio fischeri in biosynthesis of gold nanoparticles from gold chloride trihydrate.

PubMed

Attaran, Neda; Eshghi, Hossein; Rahimizadeh, Mohammad; Mashreghi, Mansour; Bakavoli, Mehdi

2014-08-04

The effect of different genetically engineered bacteria, Pseudomonas syringae, Pseudomonas savastanoi, and Ralostonia solanacerum and also a natural marine bacterial species, Vibrio fischeri NRRL B-11177, is studied in producing gold nanoparticles. This is the first report about the biosynthesis of gold nanoparticles by natural and genetically engineered luminescent bacteria. These microorganisms reduced gold ions and produced fairly monodisperse nanoparticles. TEM analysis indicated that spherical nano gold particles in the different diameters and shapes were obtained at pH values of 6.64. In this biosynthesis protocol, the gold nanoparticles with desired shape and size can be prepared.

Representation of the speech effectors in the human motor cortex: somatotopy or overlap?

PubMed

Takai, Osamu; Brown, Steven; Liotti, Mario

2010-04-01

Somatotopy within the orofacial region of the human motor cortex has been a central concept in interpreting the results of neuroimaging and transcranial magnetic stimulation studies of normal and disordered speech. Yet, somatotopy has been challenged by studies showing overlap among the effectors within the homunculus. In order to address this dichotomy, we performed four voxel-based meta-analyses of 54 functional neuroimaging studies of non-speech tasks involving respiration, lip movement, tongue movement, and swallowing, respectively. While the centers of mass of the clusters supported the classic homuncular view of the motor cortex, there was significant variability in the locations of the activation-coordinates among studies, resulting in an overlapping arrangement. This "somatotopy with overlap" might reflect the intrinsic functional interconnectedness of the oral effectors for speech production.

Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response

PubMed Central

2014-01-01

Background Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA“s” and PthC“s” of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange. Results Based on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA“s” and PthC“s” in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription. Conclusions The identification of PthA“s” and PthC“s” targets, such as the LOB (LATERAL ORGAN BOUNDARY) and CCNBS genes that we report here, is key for the understanding of the canker symptoms development during host susceptibility, or the defenses of sweet orange against the canker bacteria. We have narrowed down candidate targets to a few, which pointed out the host metabolic pathways explored by the pathogens. PMID:24564253

Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response.

PubMed

Pereira, Andre L A; Carazzolle, Marcelo F; Abe, Valeria Y; de Oliveira, Maria L P; Domingues, Mariane N; Silva, Jaqueline C; Cernadas, Raul A; Benedetti, Celso E

2014-02-25

Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA"s" and PthC"s" of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange. Based on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA"s" and PthC"s" in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription. The identification of PthA"s" and PthC"s" targets, such as the LOB (lateral organ boundary) and CCNBS genes that we report here, is key for the understanding of the canker symptoms development during host susceptibility, or the defenses of sweet orange against the canker bacteria. We have narrowed down candidate targets to a few, which pointed out the host metabolic pathways explored by the pathogens.

Intestinal Effector T Cells in Health and Disease

PubMed Central

Maynard, Craig L.; Weaver, Casey T.

2011-01-01

Summary Crohn’s disease and ulcerative colitis are the two major forms of chronic relapsing inflammatory disorders of the human intestines collectively referred to as inflammatory bowel disease (IBD). Though a complex set of autoinflammatory disorders that can be precipitated by diverse genetic and environmental factors, a feature that appears common to IBD pathogenesis is a dysregulated effector T cell response to the commensal microbiota. Due to the heightened effector T cell activity in IBD, developmental and functional pathways that give rise to these cells are potential targets for therapeutic intervention. In this review, we highlight recent advances in our understanding of effector T cell biology in the context of intestinal immune regulation and speculate on their potential clinical significance. PMID:19766082

Development and testing of the rack insertion device

NASA Technical Reports Server (NTRS)

Strickland, G. Scott

1995-01-01

Installing and removing experiment racks in a Space Station Logistics Module will become a repetitive operation at Kennedy Space Center (KSC) in the near future. A Rack Insertion Device (RID) consisting of an Extendible Boom, End Effector, and Positioning Base is being developed for the task. This paper discusses the key elements of the RlD's function and design. Prototype test results for the RlD's Extendible Boom and End Effector are presented. Also discussed are future end effectors that will further enhance the RlD's Space Station processing capability.

Novel bacterial ADP-ribosylating toxins: structure and function

PubMed Central

Simon, Nathan C.; Aktories, Klaus; Barbieri, Joseph T.

2018-01-01

Preface Bacterial ADP-ribosyltransferase toxins (bARTTs) transfer ADP-ribose to eukaryotic proteins to promote bacterial pathogenesis. In this review we use prototype bARTTs, such as diphtheria and pertussis toxins, as references for the characterization of several new bARTTs from human, insect, and plant pathogens, which were identified recently through bioinformatic analyses. Several of these toxins, including Cholix toxin from Vibrio cholerae, SpyA from Streptococcus pyogenes, HopU1 from Pseudomonas syringae, and the Tcc toxins from Photorhabdus luminescens, ADP-ribosylate novel substrates and possess unique organizations, which distinguish them from the reference toxins. The characterization of these toxins extends our appreciation for the variety of structure-function properties possessed by bARTTs and their roles in bacterial pathogenesis. PMID:25023120

Regulatory T cells inhibit acute IFN-γ synthesis without blocking T-helper cell type 1 (Th1) differentiation via a compartmentalized requirement for IL-10

PubMed Central

Sojka, Dorothy K.; Fowell, Deborah J.

2011-01-01

CD4+CD25+Forkhead box P3 (Foxp3)+ regulatory T cells (Tregs) control immune responses to self and foreign antigens in secondary lymphoid organs and at tissue sites of inflammation. Tregs can modify the function of many immune cells and have been proposed to block early proliferation, differentiation, and effector function. Acute ablation of Tregs has revealed rapid cytokine production immediately after Treg removal, suggesting that Tregs may regulate effector function acutely rather than regulating the programming for immune function. We developed in vitro and in vivo models that enabled the direct test of Treg regulation of T-helper cell type 1 (Th1) differentiation. CD28 signaling is known to abrogate Treg suppression of IL-2 secretion and proliferation, but our studies show that Treg suppression of IFN-γ during Th1 priming proceeds despite enhanced CD28 signaling. Importantly, during Th1 differentiation, Tregs inhibited early IFN-γ transcription without disrupting expression of Th1-specific T-box transcription factor (Tbet) and Th1 programming. Acute shutoff of effector cytokine production by Tregs was selective for IFN-γ but not TNF-α and was independent of TGF-β and Epstein-Barr virus-induced gene 3. In vivo, Tregs potently controlled CD4 IFN-γ and CD4 effector cell expansion in the lymph node (four- to fivefold reduction) but not Th1 programming, independent of IL-10. Tregs additionally reduced CD4 IFN-γ in the inflamed dermis (twofold reduction) dependent on their production of IL-10. We propose a model for Treg inhibition of effector function based on acute cytokine regulation. Interestingly, Tregs used different regulatory mechanisms to regulate IFN-γ (IL-10–dependent or –independent) subject to the target T-cell stage of activation and its tissue location. PMID:22025707

Functions of tissue-resident eosinophils.

PubMed

Weller, Peter F; Spencer, Lisa A

2017-12-01

Eosinophils are a prominent cell type in particular host responses such as the response to helminth infection and allergic disease. Their effector functions have been attributed to their capacity to release cationic proteins stored in cytoplasmic granules by degranulation. However, eosinophils are now being recognized for more varied functions in previously underappreciated diverse tissue sites, based on the ability of eosinophils to release cytokines (often preformed) that mediate a broad range of activities into the local environment. In this Review, we consider evolving insights into the tissue distribution of eosinophils and their functional immunobiology, which enable eosinophils to secrete in a selective manner cytokines and other mediators that have diverse, 'non-effector' functions in health and disease.

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

PubMed

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

2015-06-03

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

T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression1

PubMed Central

Long, Meixiao; Slaiby, Aaron M.; Hagymasi, Adam T.; Mihalyo, Marianne A.; Lichtler, Alexander C.; Reiner, Steven L.; Adler, Adam J.

2010-01-01

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ -expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure. PMID:16393991

The Tomato Kinase Pti1 Contributes to Production of Reactive Oxygen Species in Response to Two Flagellin-Derived Peptides and Promotes Resistance to Pseudomonas syringae Infection.

PubMed

Schwizer, Simon; Kraus, Christine M; Dunham, Diane M; Zheng, Yi; Fernandez-Pozo, Noé; Pombo, Marina A; Fei, Zhangjun; Chakravarthy, Suma; Martin, Gregory B

2017-09-01

The Pti1 kinase was identified from a reverse genetic screen as contributing to pattern-triggered immunity (PTI) against Pseudomonas syringae pv. tomato (Pst). The tomato genome has two Pti1 genes, referred to as Pti1a and Pti1b. A hairpin-Pti1 (hpPti1) construct was developed and was used to generate two independent stable transgenic tomato lines that had reduced transcript abundance of both genes. In response to P. syringae pv. tomato inoculation, these hpPti1 plants developed more severe disease symptoms, supported higher bacterial populations, and had reduced transcript accumulation of PTI-associated genes, as compared with wild-type plants. In response to two flagellin-derived peptides, the hpPti1 plants produced lesser amounts of reactive oxygen species (ROS) but showed no difference in mitogen-activated protein kinase (MAPK). Synthetic Pti1a and Pti1b genes designed to avoid silencing were transiently expressed in the hpPti1 plants and restored the ability of the plants to produce wild-type levels of ROS. Our results identify a new component of PTI in tomato that, because it affects ROS production but not MAPK signaling, appears to act early in the immune response.

Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum, Pseudomonas syringae, and Myzus persicae.

PubMed

Ellis, Christine; Karafyllidis, Ioannis; Turner, John G

2002-10-01

In Arabidopsis spp., the jasmonate (JA) response pathway generally is required for defenses against necrotrophic pathogens and chewing insects, while the salicylic acid (SA) response pathway is generally required for specific, resistance (R) gene-mediated defenses against both biotrophic and necrotrophic pathogens. For example, SA-dependent defenses are required for resistance to the biotrophic fungal pathogen Erysiphe cichoracearum UCSC1 and the bacterial pathogen Pseudomonas syringae pv. maculicola, and also are expressed during response to the green peach aphid Myzus persicae. However, recent evidence indicates that the expression of JA-dependent defenses also may confer resistance to E. cichoracearum. To confirm and to extend this observation, we have compared the disease and pest resistance of wild-type Arabidopsis plants with that of the mutants coil, which is insensitive to JA, and cev1, which has constitutive JA signaling. Measurements of the colonization of these plants by E. cichoracearum, P. syringae pv. maculicola, and M. persicae indicated that activation of the JA signal pathway enhanced resistance, and was associated with the activation of JA-dependent defense genes and the suppression of SA-dependent defense genes. We conclude that JA and SA induce alternative defense pathways that can confer resistance to the same pathogens and pests.

Differential Coexpression Analysis Reveals Extensive Rewiring of Arabidopsis Gene Coexpression in Response to Pseudomonas syringae Infection

PubMed Central

Jiang, Zhenhong; Dong, Xiaobao; Li, Zhi-Gang; He, Fei; Zhang, Ziding

2016-01-01

Plant defense responses to pathogens involve massive transcriptional reprogramming. Recently, differential coexpression analysis has been developed to study the rewiring of gene networks through microarray data, which is becoming an important complement to traditional differential expression analysis. Using time-series microarray data of Arabidopsis thaliana infected with Pseudomonas syringae, we analyzed Arabidopsis defense responses to P. syringae through differential coexpression analysis. Overall, we found that differential coexpression was a common phenomenon of plant immunity. Genes that were frequently involved in differential coexpression tend to be related to plant immune responses. Importantly, many of those genes have similar average expression levels between normal plant growth and pathogen infection but have different coexpression partners. By integrating the Arabidopsis regulatory network into our analysis, we identified several transcription factors that may be regulators of differential coexpression during plant immune responses. We also observed extensive differential coexpression between genes within the same metabolic pathways. Several metabolic pathways, such as photosynthesis light reactions, exhibited significant changes in expression correlation between normal growth and pathogen infection. Taken together, differential coexpression analysis provides a new strategy for analyzing transcriptional data related to plant defense responses and new insights into the understanding of plant-pathogen interactions. PMID:27721457

Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins

USDA-ARS?s Scientific Manuscript database

Soybean cyst nematodes (Heterodera glycines) produce secreted effector proteins that function as peptide mimics of plant CLAVATA3 / ESR (CLE)-like peptides probably involved in the developmental reprogramming of root cells to form specialized feeding cells called syncytia. The site of action and me...

The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins.

PubMed

Ma, Lay-Sun; Wang, Lei; Trippel, Christine; Mendoza-Mendoza, Artemio; Ullmann, Steffen; Moretti, Marino; Carsten, Alexander; Kahnt, Jörg; Reissmann, Stefanie; Zechmann, Bernd; Bange, Gert; Kahmann, Regine

2018-04-27

To cause disease in maize, the biotrophic fungus Ustilago maydis secretes a large arsenal of effector proteins. Here, we functionally characterize the repetitive effector Rsp3 (repetitive secreted protein 3), which shows length polymorphisms in field isolates and is highly expressed during biotrophic stages. Rsp3 is required for virulence and anthocyanin accumulation. During biotrophic growth, Rsp3 decorates the hyphal surface and interacts with at least two secreted maize DUF26-domain family proteins (designated AFP1 and AFP2). AFP1 binds mannose and displays antifungal activity against the rsp3 mutant but not against a strain constitutively expressing rsp3. Maize plants silenced for AFP1 and AFP2 partially rescue the virulence defect of rsp3 mutants, suggesting that blocking the antifungal activity of AFP1 and AFP2 by the Rsp3 effector is an important virulence function. Rsp3 orthologs are present in all sequenced smut fungi, and the ortholog from Sporisorium reilianum can complement the rsp3 mutant of U. maydis, suggesting a novel widespread fungal protection mechanism.

Apoplastic peroxidases are required for salicylic acid-mediated defense against Pseudomonas syringae.

PubMed

Mammarella, Nicole D; Cheng, Zhenyu; Fu, Zheng Qing; Daudi, Arsalan; Bolwell, G Paul; Dong, Xinnian; Ausubel, Frederick M

2015-04-01

Reactive oxygen species (ROS) generated by NADPH oxidases or apoplastic peroxidases play an important role in the plant defense response. Diminished expression of at least two Arabidopsis thaliana peroxidase encoding genes, PRX33 (At3g49110) and PRX34 (At3g49120), as a consequence of anti-sense expression of a heterologous French bean peroxidase gene (asFBP1.1), were previously shown to result in reduced levels of ROS following pathogen attack, enhanced susceptibility to a variety of bacterial and fungal pathogens, and reduced levels of callose production and defense-related gene expression in response to the microbe associated molecular pattern (MAMP) molecules flg22 and elf26. These data demonstrated that the peroxidase-dependent oxidative burst plays an important role in the elicitation of pattern-triggered immunity (PTI). Further work reported in this paper, however, shows that asFBP1.1 antisense plants are not impaired in all PTI-associated responses. For example, some but not all flg22-elicited genes are induced to lower levels by flg22 in asFPB1.1, and callose deposition in asFPB1.1 is similar to wild-type following infiltration with a Pseudomonas syringae hrcC mutant or with non-host P. syringae pathovars. Moreover, asFPB1.1 plants did not exhibit any apparent defect in their ability to mount a hypersensitive response (HR). On the other hand, salicylic acid (SA)-mediated activation of PR1 was dramatically impaired in asFPB1.1 plants. In addition, P. syringae-elicited expression of many genes known to be SA-dependent was significantly reduced in asFBP1.1 plants. Consistent with this latter result, in asFBP1.1 plants the key regulator of SA-mediated responses, NPR1, showed both dramatically decreased total protein abundance and a failure to monomerize, which is required for its translocation into the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative Genome Analysis Provides Insights into the Evolution and Adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum

PubMed Central

Green, Sarah; Studholme, David J.; Laue, Bridget E.; Dorati, Federico; Lovell, Helen; Arnold, Dawn; Cottrell, Joan E.; Bridgett, Stephen; Blaxter, Mark; Huitema, Edgar; Thwaites, Richard; Sharp, Paul M.

2010-01-01

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree. PMID:20419105

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

PubMed

Green, Sarah; Studholme, David J; Laue, Bridget E; Dorati, Federico; Lovell, Helen; Arnold, Dawn; Cottrell, Joan E; Bridgett, Stephen; Blaxter, Mark; Huitema, Edgar; Thwaites, Richard; Sharp, Paul M; Jackson, Robert W; Kamoun, Sophien

2010-04-19

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

The receptor-like cytoplasmic kinase PCRK1 contributes to pattern-triggered immunity against Pseudomonas syringae in Arabidopsis thaliana.

PubMed

Sreekanta, Suma; Bethke, Gerit; Hatsugai, Noriyuki; Tsuda, Kenichi; Thao, Amanda; Wang, Lin; Katagiri, Fumiaki; Glazebrook, Jane

2015-07-01

In this paper we describe PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) of Arabidopsis thaliana, an RLCK that is important for defense against the pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326). We examined defense responses such as bacterial growth, production of reactive oxygen species (ROS) and callose deposition in pcrk1 mutant plants to determine the role of PCRK1 during pathogen infection. Expression of PCRK1 was induced following pathogen infection. Pathogen growth was significantly higher in pcrk1 mutant lines than in wild-type Col-0. Mutant pcrk1 plants showed reduced pattern-triggered immunity (PTI) against Pma ES4326 after pretreatment with peptides derived from flagellin (flg22), elongation factor-Tu (elf18), or an endogenous protein (pep1). Deposition of callose was reduced in pcrk1 plants, indicating a role of PCRK1 in activation of early immune responses. A PCRK1 transgene containing a mutation in a conserved lysine residue important for phosphorylation activity of kinases (K118E) failed to complement a pcrk1 mutant for the Pma ES4326 growth phenotype. Our study shows that PCRK1 plays an important role during PTI and that a conserved lysine residue in the putative kinase domain is important for PCRK1 function. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana

PubMed Central

Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

2013-01-01

We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response. PMID:23451199

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita

PubMed Central

Rutter, William B.; Hewezi, Tarek; Abubucker, Sahar; Maier, Tom R.; Huang, Guozhong; Mitreva, Makedonka; Hussey, Richard S.; Baum, Thomas J.

2014-01-01

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins, which M. incognita secretes into its host plants during infection, is an important step towards finding new ways to manage this pest. In this study we have identified the cDNAs for 18 putative effectors, i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants. These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically up-regulated during different stages of the nematode’s life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of Meloidogyne hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed, and reproduce on their host plants. Future studies investigating the roles these proteins play in planta will help mitigate the effects of this damaging pest. PMID:24875667

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

PubMed

Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

2017-03-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita.

PubMed

Rutter, William B; Hewezi, Tarek; Abubucker, Sahar; Maier, Tom R; Huang, Guozhong; Mitreva, Makedonka; Hussey, Richard S; Baum, Thomas J

2014-09-01

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematode's life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.

rab3 mediates cortical granule exocytosis in the sea urchin egg.

PubMed

Conner, S; Wessel, G M

1998-11-15

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking. Copyright 1998 Academic Press.

The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

PubMed

Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph

2017-01-03

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activated Tissue-Resident Mesenchymal Stromal Cells Regulate Natural Killer Cell Immune and Tissue-Regenerative Function.

PubMed

Petri, Robert Michael; Hackel, Alexander; Hahnel, Katrin; Dumitru, Claudia Alexandra; Bruderek, Kirsten; Flohe, Stefanie B; Paschen, Annette; Lang, Stephan; Brandau, Sven

2017-09-12

The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-β and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Rab-binding Profiles of Bacterial Virulence Factors during Infection.

PubMed

So, Ernest C; Schroeder, Gunnar N; Carson, Danielle; Mattheis, Corinna; Mousnier, Aurélie; Broncel, Malgorzata; Tate, Edward W; Frankel, Gad

2016-03-11

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Posttranscriptional Control of T Cell Effector Function by Aerobic Glycolysis

PubMed Central

Chang, Chih-Hao; Curtis, Jonathan D.; Maggi, Leonard B.; Faubert, Brandon; Villarino, Alejandro V.; O’Sullivan, David; Huang, Stanley Ching-Cheng; van der Windt, Gerritje J.W.; Blagih, Julianna; Qiu, Jing; Weber, Jason D.; Pearce, Edward J.; Jones, Russell G.; Pearce, Erika L.

2013-01-01

SUMMARY A “switch” from oxidative phosphorylation (OXPHOS) to aerobic glycolysis is a hallmark of T cell activation and is thought to be required to meet the metabolic demands of proliferation. However, why proliferating cells adopt this less efficient metabolism, especially in an oxygen-replete environment, remains incompletely understood. We show here that aerobic glycolysis is specifically required for effector function in T cells but that this pathway is not necessary for proliferation or survival. When activated T cells are provided with costimulation and growth factors but are blocked from engaging glycolysis, their ability to produce IFN-γ is markedly compromised. This defect is translational and is regulated by the binding of the glycolysis enzyme GAPDH to AU-rich elements within the 3′ UTR of IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through fluctuations in its expression, controls effector cytokine production. Thus, aerobic glycolysis is a metabolically regulated signaling mechanism needed to control cellular function. PMID:23746840

Regulation of disease-responsive genes mediated by epigenetic factors: interaction of Arabidopsis-Pseudomonas.

PubMed

De-La-Peña, Clelia; Rangel-Cano, Alicia; Alvarez-Venegas, Raúl

2012-05-01

Genes in eukaryotic organisms function within the context of chromatin, and the mechanisms that modulate the structure of chromatin are defined as epigenetic. In Arabidopsis, pathogen infection induces the expression of at least one histone deacetylase, suggesting that histone acetylation/deacetylation has an important role in the pathogenic response in plants. How/whether histone methylation affects gene response to pathogen infection is unknown. To gain a better understanding of the epigenetic mechanisms regulating the interaction between Pseudomonas syringae and Arabidopsis thaliana, we analysed three different Arabidopsis ash1-related (absent, small or homeotic discs 1) mutants. We found that the loss of function of ASHH2 and ASHR1 resulted in faster hypersensitive responses (HRs) to both mutant (hrpA) and pathogenic (DC3000) strains of P. syringae, whereas control (Col-0) and ashr3 mutants appeared to be more resistant to the infection after 2 days. Furthermore, we showed that, in the ashr3 background, the PR1 gene (PATHOGENESIS-RELATED GENE 1) displayed the highest expression levels on infection with DC3000, correlating with increased resistance against this pathogen. Our results show that, in both the ashr1 and ashh2 backgrounds, the histone H3 lysine 4 dimethylation (H3K4me2) levels decreased at the promoter region of PR1 on infection with the DC3000 strain, suggesting that an epigenetically regulated PR1 expression is involved in the plant defence. Our results suggest that histone methylation in response to pathogen infection may be a critical component in the signalling and defence processes occurring between plants and microbes. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

PubMed

Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

2017-11-30

The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

Effector CD8 T cells dedifferentiate into long-lived memory cells.

PubMed

Youngblood, Ben; Hale, J Scott; Kissick, Haydn T; Ahn, Eunseon; Xu, Xiaojin; Wieland, Andreas; Araki, Koichi; West, Erin E; Ghoneim, Hazem E; Fan, Yiping; Dogra, Pranay; Davis, Carl W; Konieczny, Bogumila T; Antia, Rustom; Cheng, Xiaodong; Ahmed, Rafi

2017-12-21

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

PubMed

Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies

PubMed Central

Richardson, Simone I.; Mabvakure, Batsirai; Mkhize, Nonhlanhla N.; Moore, Penny L.; Alter, Galit

2018-01-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies. PMID:29630668

Greasy tactics in the plant-pathogen molecular arms race.

PubMed

Boyle, Patrick C; Martin, Gregory B

2015-03-01

The modification of proteins by the attachment of fatty acids is a targeting tactic involved in mechanisms of both plant immunity and bacterial pathogenesis. The plant plasma membrane (PM) is a key battleground in the war against disease-causing microbes. This membrane is armed with an array of sensor proteins that function as a surveillance system to detect invading pathogens. Several of these sensor proteins are directed to the plasma membrane through the covalent addition of fatty acids, a process termed fatty acylation. Phytopathogens secrete effector proteins into the plant cell to subvert these surveillance mechanisms, rendering the host susceptible to infection. The targeting of effectors to specific locales within plant cells, particularly the internal face of the host PM, is critical for their virulence function. Several bacterial effectors hijack the host fatty acylation machinery to be modified and directed to this contested locale. To find and fight these fatty acylated effectors the plant leverages lipid-modified intracellular sensors. This review provides examples featuring how fatty acylation is a battle tactic used by both combatants in the molecular arms race between plants and pathogens. Also highlighted is the exploitation of a specific form of host-mediated fatty acid modification, which appears to be exclusively employed by phytopathogenic effector proteins. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structural and Functional Investigations of the Effector Protein LpiR1 from Legionella pneumophila.

PubMed

Beyrakhova, Ksenia A; van Straaten, Karin; Li, Lei; Boniecki, Michal T; Anderson, Deborah H; Cygler, Miroslaw

2016-07-22

Legionella pneumophila is a causative agent of a severe pneumonia, known as Legionnaires' disease. Legionella pathogenicity is mediated by specific virulence factors, called bacterial effectors, which are injected into the invaded host cell by the bacterial type IV secretion system. Bacterial effectors are involved in complex interactions with the components of the host cell immune and signaling pathways, which eventually lead to bacterial survival and replication inside the mammalian cell. Structural and functional studies of bacterial effectors are, therefore, crucial for elucidating the mechanisms of Legionella virulence. Here we describe the crystal structure of the LpiR1 (Lpg0634) effector protein and investigate the effects of its overexpression in mammalian cells. LpiR1 is an α-helical protein that consists of two similar domains aligned in an antiparallel fashion. The hydrophilic cleft between the domains might serve as a binding site for a potential host cell interaction partner. LpiR1 binds the phosphate group at a conserved site and is stabilized by Mn(2+), Ca(2+), or Mg(2+) ions. When overexpressed in mammalian cells, a GFP-LpiR1 fusion protein is localized in the cytoplasm. Intracellular signaling antibody array analysis revealed small changes in the phosphorylation state of several components of the Akt signaling pathway in HEK293T cells overexpressing LpiR1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Thienopyrimidine-type compounds protect Arabidopsis plants against the hemibiotrophic fungal pathogen Colletotrichum higginsianum and bacterial pathogen Pseudomonas syringae pv. maculicola.

PubMed

Narusaka, Mari; Narusaka, Yoshihiro

2017-03-04

Plant activators activate systemic acquired resistance-like defense responses or induced systemic resistance, and thus protect plants from pathogens. We screened a chemical library composed of structurally diverse small molecules. We isolated six plant immune-inducing thienopyrimidine-type compounds and their analogous compounds. It was observed that the core structure of thienopyrimidine plays a role in induced resistance in plants. Furthermore, we highlight the protective effect of thienopyrimidine-type compounds against both hemibiotrophic fungal pathogen, Colletotrichum higginsianum, and bacterial pathogen, Pseudomonas syringae pv. maculicola, in Arabidopsis thaliana. We suggest that thienopyrimidine-type compounds could be potential lead compounds as novel plant activators, and can be useful and effective agrochemicals against various plant diseases.

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector

PubMed Central

Caplan, Jeffrey L.; Mamillapalli, Padmavathi; Burch-Smith, Tessa M.; Czymmek, Kirk; Dinesh-Kumar, S.P.

2008-01-01

Summary Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50-kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a pre-recognition complex containing the p50 effector and NRIP1. PMID:18267075

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes†

PubMed Central

Alontaga, Aileen Y.; Fenton, Aron W.

2011-01-01

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family. PMID:21261284

Platelets: versatile effector cells in hemostasis, inflammation, and the immune continuum

PubMed Central

Vieira-de-Abreu, Adriana; Campbell, Robert A.; Weyrich, Andrew S.

2015-01-01

Platelets are chief effector cells in hemostasis. In addition, however, their specializations include activities and intercellular interactions that make them key effectors in inflammation and in the continuum of innate and adaptive immunity. This review focuses on the immune features of human platelets and platelets from experimental animals and on interactions between inflammatory, immune, and hemostatic activities of these anucleate but complex and versatile cells. The experimental findings and evidence for physiologic immune functions include previously unrecognized biologic characteristics of platelets and are paralleled by new evidence for unique roles of platelets in inflammatory, immune, and thrombotic diseases. PMID:21818701

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

PubMed

Nemri, Adnane; Saunders, Diane G O; Anderson, Claire; Upadhyaya, Narayana M; Win, Joe; Lawrence, Gregory J; Jones, David A; Kamoun, Sophien; Ellis, Jeffrey G; Dodds, Peter N

2014-01-01

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

PubMed

Li, Dayong; Zhang, Huijuan; Song, Qiuming; Wang, Lu; Liu, Shixia; Hong, Yongbo; Huang, Lei; Song, Fengming

2015-06-14

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

Wind Tunnel Test of an RPV with Shape-Change Control Effector and Sensor Arrays

NASA Technical Reports Server (NTRS)

Raney, David L.; Cabell, Randolph H.; Sloan, Adam R.; Barnwell, William G.; Lion, S. Todd; Hautamaki, Bret A.

2004-01-01

A variety of novel control effector concepts have recently emerged that may enable new approaches to flight control. In particular, the potential exists to shift the composition of the typical aircraft control effector suite from a small number of high authority, specialized devices (rudder, aileron, elevator, flaps), toward larger numbers of smaller, less specialized, distributed device arrays. The concept envisions effector and sensor networks composed of relatively small high-bandwidth devices able to simultaneously perform a variety of control functions using feedback from disparate data sources. To investigate this concept, a remotely piloted flight vehicle has been equipped with an array of 24 trailing edge shape-change effectors and associated pressure measurements. The vehicle, called the Multifunctional Effector and Sensor Array (MESA) testbed, was recently tested in NASA Langley's 12-ft Low Speed wind tunnel to characterize its stability properties, control authorities, and distributed pressure sensitivities for use in a dynamic simulation prior to flight testing. Another objective was to implement and evaluate a scheme for actively controlling the spanwise pressure distribution using the shape-change array. This report describes the MESA testbed, design of the pressure distribution controller, and results of the wind tunnel test.

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

PubMed

Ashida, Hiroshi; Sasakawa, Chihiro

2015-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria

PubMed Central

Ashida, Hiroshi; Sasakawa, Chihiro

2016-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections. PMID:26779450

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

PubMed

Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

2014-01-01

Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Identification of effector-like proteins in Trichoderma spp. and role of a hydrophobin in the plant-fungus interaction and mycoparasitism.

PubMed

Guzmán-Guzmán, Paulina; Alemán-Duarte, Mario Iván; Delaye, Luis; Herrera-Estrella, Alfredo; Olmedo-Monfil, Vianey

2017-02-15

Trichoderma spp. can establish beneficial interactions with plants by promoting plant growth and defense systems, as well as, antagonizing fungal phytopathogens in mycoparasitic interactions. Such interactions depend on signal exchange between both participants and can be mediated by effector proteins that alter the host cell structure and function, allowing the establishment of the relationship. The main purpose of this work was to identify, using computational methods, candidates of effector proteins from T. virens, T. atroviride and T. reesei, validate the expression of some of the genes during a beneficial interaction and mycoparasitism and to define the biological function for one of them. We defined a catalogue of putative effector proteins from T. virens, T. atroviride and T. reesei. We further validated the expression of 16 genes encoding putative effector proteins from T. virens and T. atroviride during the interaction with the plant Arabidopsis thaliana, and with two anastomosis groups of the phytopathogenic fungus Rhizoctonia solani. We found genes which transcript levels are modified in response to the presence of both plant fungi, as well as genes that respond only to either a plant or a fungal host. Further, we show that overexpression of the gene tvhydii1, a Class II hydrophobin family member, enhances the antagonistic activity of T. virens against R. solani AG2. Further, deletion of tvhydii1 results in reduced colonization of plant roots, while its overexpression increases it. Our results show that Trichoderma is able to respond in different ways to the presence of a plant or a fungal host, and it can even distinguish between different strains of fungi of a given species. The putative effector proteins identified here may play roles in preventing perception of the fungus by its hosts, favoring host colonization or protecting it from the host's defense response. Finally, the novel effector protein TVHYDII1 plays a role in plant root colonization by T, virens, and participates in its antagonistic activity against R. solani.

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

PubMed

Moogk, Duane; Zhong, Shi; Yu, Zhiya; Liadi, Ivan; Rittase, William; Fang, Victoria; Dougherty, Janna; Perez-Garcia, Arianne; Osman, Iman; Zhu, Cheng; Varadarajan, Navin; Restifo, Nicholas P; Frey, Alan B; Krogsgaard, Michelle

2016-07-15

CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. Copyright © 2016 by The American Association of Immunologists, Inc.

Exploring a regulatory role for mast cells: 'MCregs'?

PubMed

Frossi, Barbara; Gri, Giorgia; Tripodo, Claudio; Pucillo, Carlo

2010-03-01

Regulatory cells can mould the fate of the immune response by direct suppression of specific subsets of effector cells, or by redirecting effectors against invading pathogens and infected or neoplastic cells. These functions have been classically, although not exclusively, ascribed to different subsets of T cells. Recently, mast cells have been shown to regulate physiological and pathological immune responses, and thus to act at the interface between innate and adaptive immunity assuming different functions and behaviors at discrete stages of the immune response. Here, we focus on these poorly defined, and sometimes apparently conflicting, functions of mast cells. Copyright 2010 Elsevier Ltd. All rights reserved.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

PubMed

Schmueck-Henneresse, Michael; Omer, Bilal; Shum, Thomas; Tashiro, Haruko; Mamonkin, Maksim; Lapteva, Natalia; Sharma, Sandhya; Rollins, Lisa; Dotti, Gianpietro; Reinke, Petra; Volk, Hans-Dieter; Rooney, Cliona M

2017-07-01

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO + CCR7 - effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

Endosomal protein traffic meets nuclear signal transduction head on.

PubMed

Horazdovsky, Bruce

2004-02-01

Rab5 plays a key role in controlling protein traffic through the early stages of the endocytic pathway. Previous studies on the modulators and effectors of Rab5 protein function have tied the regulation of several signal transduction pathways to the movement of protein through endocytic compartments. In the February 6, 2004, issue of Cell, Miaczynska et al. describe a surprising new link between Rab5 function and the nucleus by uncovering two new Rab5 effectors as potential regulators of the nucleosome remodeling and histone deacetylase protein complex NuRD/MeCP1.

Multiple Rap1 effectors control Epac1-mediated tightening of endothelial junctions.

PubMed

Pannekoek, Willem-Jan; Vliem, Marjolein J; Bos, Johannes L

2018-02-17

Epac1 and Rap1 mediate cAMP-induced tightening of endothelial junctions. We have previously found that one of the mechanisms is the inhibition of Rho-mediated tension in radial stress fibers by recruiting the RhoGAP ArhGAP29 in a complex containing the Rap1 effectors Rasip1 and Radil. However, other mechanisms have been proposed as well, most notably the induction of tension in circumferential actin cables by Cdc42 and its GEF FGD5. Here, we have investigated how Rap1 controls FGD5/Cdc42 and how this interconnects with Radil/Rasip1/ArhGAP29. Using endothelial barrier measurements, we show that Rho inhibition is not sufficient to explain the barrier stimulating effect of Rap1. Indeed, Cdc42-mediated tension is induced at cell-cell contacts upon Rap1 activation and this is required for endothelial barrier function. Depletion of potential Rap1 effectors identifies AF6 to mediate Rap1 enhanced tension and concomitant Rho-independent barrier function. When overexpressed in HEK293T cells, AF6 is found in a complex with FGD5 and Radil. From these results we conclude that Rap1 utilizes multiple pathways to control tightening of endothelial junctions, possibly through a multiprotein effector complex, in which AF6 functions to induce tension in circumferential actin cables.

Blockade of PD-1/PD-L1 Promotes Adoptive T-Cell Immunotherapy in a Tolerogenic Environment

PubMed Central

Kenna, Tony J.; Galea, Ryan; Large, Justin; Yagita, Hideo; Steptoe, Raymond J.

2015-01-01

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells. PMID:25741704

Blockade of PD-1/PD-L1 promotes adoptive T-cell immunotherapy in a tolerogenic environment.

PubMed

Blake, Stephen J P; Ching, Alan L H; Kenna, Tony J; Galea, Ryan; Large, Justin; Yagita, Hideo; Steptoe, Raymond J

2015-01-01

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.

CD134/CD137 Dual Costimulation-Elicited IFN-γ Maximizes Effector T Cell Function but Limits Treg Expansion

PubMed Central

Rose, Marie-Clare St.; Taylor, Roslyn A.; Bandyopadhyay, Suman; Qui, Harry Z.; Hagymasi, Adam T.; Vella, Anthony T.; Adler, Adam J.

2012-01-01

T cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies to the costimulatory receptors CD134 plus CD137 can program T cells responding to tolerogenic antigen to undergo expansion and effector T cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation, the current study utilized a model where dual costimulation programs T cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to play a pivotal role in maximizing the function of effector T cells while simultaneously limiting the expansion of CD4+CD25+Foxp3+ Tregs. In antigen-responding effector T cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4+Foxp3+ Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during dual costimulation IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T cells while simultaneously limiting Treg expansion. PMID:23295363

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions.

PubMed

Wallqvist, Anders; Wang, Hao; Zavaljevski, Nela; Memišević, Vesna; Kwon, Keehwan; Pieper, Rembert; Rajagopala, Seesandra V; Reifman, Jaques

2017-01-01

Coxiella burnetii is an obligate Gram-negative intracellular pathogen and the etiological agent of Q fever. Successful infection requires a functional Type IV secretion system, which translocates more than 100 effector proteins into the host cytosol to establish the infection, restructure the intracellular host environment, and create a parasitophorous vacuole where the replicating bacteria reside. We used yeast two-hybrid (Y2H) screening of 33 selected C. burnetii effectors against whole genome human and murine proteome libraries to generate a map of potential host-pathogen protein-protein interactions (PPIs). We detected 273 unique interactions between 20 pathogen and 247 human proteins, and 157 between 17 pathogen and 137 murine proteins. We used orthology to combine the data and create a single host-pathogen interaction network containing 415 unique interactions between 25 C. burnetii and 363 human proteins. We further performed complementary pairwise Y2H testing of 43 out of 91 C. burnetii-human interactions involving five pathogen proteins. We used the combined data to 1) perform enrichment analyses of target host cellular processes and pathways, 2) examine effectors with known infection phenotypes, and 3) infer potential mechanisms of action for four effectors with uncharacterized functions. The host-pathogen interaction profiles supported known Coxiella phenotypes, such as adapting cell morphology through cytoskeletal re-arrangements, protein processing and trafficking, organelle generation, cholesterol processing, innate immune modulation, and interactions with the ubiquitin and proteasome pathways. The generated dataset of PPIs-the largest collection of unbiased Coxiella host-pathogen interactions to date-represents a rich source of information with respect to secreted pathogen effector proteins and their interactions with human host proteins.

Primary Murine CD4+ T Cells Fail to Acquire the Ability to Produce Effector Cytokines When Active Ras Is Present during Th1/Th2 Differentiation

PubMed Central

Janardhan, Sujit V.; Marks, Reinhard; Gajewski, Thomas F.

2014-01-01

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo. PMID:25397617

Protein phosphatase AP2C1 negatively regulates basal resistance and defense responses to Pseudomonas syringae.

PubMed

Shubchynskyy, Volodymyr; Boniecka, Justyna; Schweighofer, Alois; Simulis, Justinas; Kvederaviciute, Kotryna; Stumpe, Michael; Mauch, Felix; Balazadeh, Salma; Mueller-Roeber, Bernd; Boutrot, Freddy; Zipfel, Cyril; Meskiene, Irute

2017-02-01

Mitogen-activated protein kinases (MAPKs) mediate plant immune responses to pathogenic bacteria. However, less is known about the cell autonomous negative regulatory mechanism controlling basal plant immunity. We report the biological role of Arabidopsis thaliana MAPK phosphatase AP2C1 as a negative regulator of plant basal resistance and defense responses to Pseudomonas syringae. AP2C2, a closely related MAPK phosphatase, also negatively controls plant resistance. Loss of AP2C1 leads to enhanced pathogen-induced MAPK activities, increased callose deposition in response to pathogen-associated molecular patterns or to P. syringae pv. tomato (Pto) DC3000, and enhanced resistance to bacterial infection with Pto. We also reveal the impact of AP2C1 on the global transcriptional reprogramming of transcription factors during Pto infection. Importantly, ap2c1 plants show salicylic acid-independent transcriptional reprogramming of several defense genes and enhanced ethylene production in response to Pto. This study pinpoints the specificity of MAPK regulation by the different MAPK phosphatases AP2C1 and MKP1, which control the same MAPK substrates, nevertheless leading to different downstream events. We suggest that precise and specific control of defined MAPKs by MAPK phosphatases during plant challenge with pathogenic bacteria can strongly influence plant resistance. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Immune Checkpoint Blockade for Breast Cancer.

PubMed

Swoboda, April; Nanda, Rita

An effective antitumor immune response requires interaction between cells of the adaptive and innate immune system. Three key elements are required: generation of activated tumor-directed T cells, infiltration of activated T cells into the tumor microenvironment, and killing of tumor cells by activated T cells. Tumor immune evasion can occur as a result of the disruption of each of these three key T cell activities, resulting in three distinct cancer-immune phenotypes. The immune inflamed phenotype, characterized by the presence of a robust tumor immune infiltrate, suggests impaired activated T cell killing of tumor cells related to the presence of inhibitory factors. Programmed death receptor-1 (PD-1) is an inhibitory transmembrane protein expressed on T cells, B cells, and NK cells. The interaction between PD-1 and its ligands (PD-L1/L2) functions as an immune checkpoint against unrestrained cytotoxic T effector cell activity-it promotes peripheral T effector cell exhaustion and conversion of T effector cells to immunosuppressive T regulatory (Treg) cells. Immune checkpoint inhibitors, which block the PD-1/PD-L1 axis and reactivate cytotoxic T effector cell function, are actively being investigated for the treatment of breast cancer.

An effector Peptide family required for Drosophila toll-mediated immunity.

PubMed

Clemmons, Alexa W; Lindsay, Scott A; Wasserman, Steven A

2015-04-01

In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes - the Bomanins (Boms) - that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire.

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

PubMed

Matheny, Sharon A; Chen, Chiyuan; Kortum, Robert L; Razidlo, Gina L; Lewis, Robert E; White, Michael A

2004-01-15

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

Polymicrobial sepsis impairs bystander recruitment of effector cells to infected skin despite optimal sensing and alarming function of skin resident memory CD8 T cells

PubMed Central

Shan, Qiang; Xue, Hai-Hui; Harty, John T.

2017-01-01

Sepsis is a systemic infection that enhances host vulnerability to secondary infections normally controlled by T cells. Using CLP sepsis model, we observed that sepsis induces apoptosis of circulating memory CD8 T-cells (TCIRCM) and diminishes their effector functions, leading to impaired CD8 T-cell mediated protection to systemic pathogen re-infection. In the context of localized re-infections, tissue resident memory CD8 T-cells (TRM) provide robust protection in a variety of infectious models. TRM rapidly ‘sense’ infection in non-lymphoid tissues and ‘alarm’ the host by enhancing immune cell recruitment to the site of the infection to accelerate pathogen clearance. Here, we show that compared to pathogen-specific TCIRCM, sepsis does not invoke significant numerical decline of Vaccinia virus induced skin-TRM keeping their effector functions (e.g., Ag-dependent IFN-γ production) intact. IFN-γ-mediated recruitment of immune cells to the site of localized infection was, however, reduced in CLP hosts despite TRM maintaining their ‘sensing and alarming’ functions. The capacity of memory CD8 T-cells in the septic environment to respond to inflammatory cues and arrive to the site of secondary infection/antigen exposure remained normal suggesting T-cell-extrinsic factors contributed to the observed lesion. Mechanistically, we showed that IFN-γ produced rapidly during sepsis-induced cytokine storm leads to reduced IFN-γR1 expression on vascular endothelium. As a consequence, decreased expression of adhesion molecules and/or chemokines (VCAM1 and CXCL9) on skin endothelial cells in response to TRM-derived IFN-γ was observed, leading to sub-optimal bystander-recruitment of effector cells and increased susceptibility to pathogen re-encounter. Importantly, as visualized by intravital 2-photon microscopy, exogenous administration of CXCL9/10 was sufficient to correct sepsis-induced impairments in recruitment of effector cells at the localized site of TRM antigen recognition. Thus, sepsis has the capacity to alter skin TRM anamnestic responses without directly impacting TRM number and/or function, an observation that helps to further define the immunoparalysis phase in sepsis survivors. PMID:28910403

An Improved Method for TAL Effectors DNA-Binding Sites Prediction Reveals Functional Convergence in TAL Repertoires of Xanthomonas oryzae Strains

PubMed Central

Pérez-Quintero, Alvaro L.; Rodriguez-R, Luis M.; Dereeper, Alexis; López, Camilo; Koebnik, Ralf; Szurek, Boris; Cunnac, Sebastien

2013-01-01

Transcription Activators-Like Effectors (TALEs) belong to a family of virulence proteins from the Xanthomonas genus of bacterial plant pathogens that are translocated into the plant cell. In the nucleus, TALEs act as transcription factors inducing the expression of susceptibility genes. A code for TALE-DNA binding specificity and high-resolution three-dimensional structures of TALE-DNA complexes were recently reported. Accurate prediction of TAL Effector Binding Elements (EBEs) is essential to elucidate the biological functions of the many sequenced TALEs as well as for robust design of artificial TALE DNA-binding domains in biotechnological applications. In this work a program with improved EBE prediction performances was developed using an updated specificity matrix and a position weight correction function to account for the matching pattern observed in a validation set of TALE-DNA interactions. To gain a systems perspective on the large TALE repertoires from X. oryzae strains, this program was used to predict rice gene targets for 99 sequenced family members. Integrating predictions and available expression data in a TALE-gene network revealed multiple candidate transcriptional targets for many TALEs as well as several possible instances of functional convergence among TALEs. PMID:23869221

Impaired Subset Progression and Polyfunctionality of T Cells in Mice Exposed to Methamphetamine during Chronic LCMV Infection

PubMed Central

Sriram, Uma; Hill, Beth L.; Cenna, Jonathan M.; Gofman, Larisa; Fernandes, Nicole C.; Haldar, Bijayesh; Potula, Raghava

2016-01-01

Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host’s innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses. PMID:27760221

Impaired Subset Progression and Polyfunctionality of T Cells in Mice Exposed to Methamphetamine during Chronic LCMV Infection.

PubMed

Sriram, Uma; Hill, Beth L; Cenna, Jonathan M; Gofman, Larisa; Fernandes, Nicole C; Haldar, Bijayesh; Potula, Raghava

2016-01-01

Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host's innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses.

Two-Partner Secretion: Combining Efficiency and Simplicity in the Secretion of Large Proteins for Bacteria-Host and Bacteria-Bacteria Interactions

PubMed Central

Guérin, Jeremy; Bigot, Sarah; Schneider, Robert; Buchanan, Susan K.; Jacob-Dubuisson, Françoise

2017-01-01

Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process. PMID:28536673

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

PubMed Central

2013-01-01

Background Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. Results We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. Conclusions Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. PMID:24252298

Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction*

PubMed Central

Lewis, Aurélia E.; Sommer, Lilly; Arntzen, Magnus Ø.; Strahm, Yvan; Morrice, Nicholas A.; Divecha, Nullin; D'Santos, Clive S.

2011-01-01

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3–7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions. PMID:21048195

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

PubMed

de Lourdes Mora-García, María; García-Rocha, Rosario; Morales-Ramírez, Omar; Montesinos, Juan José; Weiss-Steider, Benny; Hernández-Montes, Jorge; Ávila-Ibarra, Luis Roberto; Don-López, Christian Azucena; Velasco-Velázquez, Marco Antonio; Gutiérrez-Serrano, Vianey; Monroy-García, Alberto

2016-10-26

In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

PubMed Central

Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

2016-01-01

The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties.

PubMed

Nakhaei-Rad, Saeideh; Nakhaeizadeh, Hossein; Kordes, Claus; Cirstea, Ion C; Schmick, Malte; Dvorsky, Radovan; Bastiaens, Philippe I H; Häussinger, Dieter; Ahmadian, Mohammad Reza

2015-06-19

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

PubMed

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of Phytochemically Characterized Extracts From Syringa vulgaris and Isolated Secoiridoids on Mediators of Inflammation in a Human Neutrophil Model.

PubMed

Woźniak, Marta; Michalak, Barbara; Wyszomierska, Joanna; Dudek, Marta K; Kiss, Anna K

2018-01-01

Aim of the study: The aim of the present study was to investigate the effects of phytochemically characterized extracts connected with the traditional use (infusions and ethanolic extracts) of different parts of Syringa vulgaris (common lilac) on the pro-inflammatory functions of neutrophils. Active compounds were isolated from the most promising extract(s) using bioassay-guided fractionation, and their activity and molecular mechanisms of action were determined. Methods: The extracts were characterized using a HPLC-DAD- MS n method. The effects on ROS, MMP-9, TNF-α, IL-8, and MCP-1 production by neutrophils were measured using luminol-dependent chemiluminescence and enzyme-linked immunosorbent assay (ELISA) methods. The effects on p38MAPK, ERK1/2, JNK phosphorylation, and NF- k B p65 translocation were determined using western blots. Results: The major compounds detected in the extracts and infusions belong to structural groups, including caffeic acid derivatives, flavonoids, and iridoids. All extracts and infusions were able to significantly reduce ROS and IL-8 production. Bioassay-guided fractionation led to the isolation of the following secoiridoids: 2″-epiframeroside, oleonuezhenide, oleuropein, ligstroside, neooleuropein, hydroxyframoside, and framoside. Neooleuropein appeared to be the most active compound in the inhibition of cytokine production by attenuating the MAP kinase pathways. Conclusion: The present study demonstrated that common lilac, which is a traditionally used medicinal plant in Europe, is a valuable source of active compounds, especially neooleuropein.

A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death1[OPEN

PubMed Central

Elmore, James M.; Creer, Athena Y.; Feng, Baomin; Franco, Jessica Y.; He, Ping; Phinney, Brett

2017-01-01

Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae. Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses. PMID:27852951

A single effector protein, ArvRpt2ea from Erwinia amylovora, can cause fire blight disease symptons and induces salicylic acid dependent defense response

USDA-ARS?s Scientific Manuscript database

The AvrRpt2EA effector protein of Erwinia amylovora is important for pathogen recognition in the fire blight resistant crabapple Malus ×robusta 5; however, little is known about its role in susceptible apple genotypes. In order to study its function in planta, we expressed a plant optimized version...

Method and apparatus for loss of control inhibitor systems

NASA Technical Reports Server (NTRS)

A'Harrah, Ralph C. (Inventor)

2007-01-01

Active and adaptive systems and methods to prevent loss of control incidents by providing tactile feedback to a vehicle operator are disclosed. According to the present invention, an operator gives a control input to an inceptor. An inceptor sensor measures an inceptor input value of the control input. The inceptor input is used as an input to a Steady-State Inceptor Input/Effector Output Model that models the vehicle control system design. A desired effector output from the inceptor input is generated from the model. The desired effector output is compared to an actual effector output to get a distortion metric. A feedback force is generated as a function of the distortion metric. The feedback force is used as an input to a feedback force generator which generates a loss of control inhibitor system (LOCIS) force back to the inceptor. The LOCIS force is felt by the operator through the inceptor.

The role of effectors of biotrophic and hemibiotrophic fungi in infection.

PubMed

Koeck, Markus; Hardham, Adrienne R; Dodds, Peter N

2011-12-01

Biotrophic and hemibiotrophic fungi are successful groups of plant pathogens that require living plant tissue to survive and complete their life cycle. Members of these groups include the rust fungi and powdery mildews and species in the Ustilago, Cladosporium and Magnaporthe genera. Collectively, they represent some of the most destructive plant parasites, causing huge economic losses and threatening global food security. During plant infection, pathogens synthesize and secrete effector proteins, some of which are translocated into the plant cytosol where they can alter the host's response to the invading pathogen. In a successful infection, pathogen effectors facilitate suppression of the plant's immune system and orchestrate the reprogramming of the infected tissue so that it becomes a source of nutrients that are required by the pathogen to support its growth and development. This review summarizes our current understanding of the function of fungal effectors in infection. © 2011 Blackwell Publishing Ltd.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

PubMed

Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

2017-09-01

Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

The conserved upstream region of lscB/C determines expression of different levansucrase genes in plant pathogen Pseudomonas syringae

PubMed Central

2014-01-01

Background Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants. It produces the exopolysaccharide levan by the enzyme levansucrase. Levansucrase has three gene copies in PG4180, two of which, lscB and lscC, are expressed while the third, lscA, is cryptic. Previously, nucleotide sequence alignments of lscB/C variants in various P. syringae showed that a ~450-bp phage-associated promoter element (PAPE) including the first 48 nucleotides of the ORF is absent in lscA. Results Herein, we tested whether this upstream region is responsible for the expression of lscB/C and lscA. Initially, the transcriptional start site for lscB/C was determined. A fusion of the PAPE with the ORF of lscA (lscB UpN A) was generated and introduced to a levan-negative mutant of PG4180. Additionally, fusions comprising of the non-coding part of the upstream region of lscB with lscA (lscB Up A) or the upstream region of lscA with lscB (lscA Up B) were generated. Transformants harboring the lscB UpN A or the lscB Up A fusion, respectively, showed levan formation while the transformant carrying lscA Up B did not. qRT-PCR and Western blot analyses showed that lscB UpN A had an expression similar to lscB while lscB Up A had a lower expression. Accuracy of protein fusions was confirmed by MALDI-TOF peptide fingerprinting. Conclusions Our data suggested that the upstream sequence of lscB is essential for expression of levansucrase while the N-terminus of LscB mediates an enhanced expression. In contrast, the upstream region of lscA does not lead to expression of lscB. We propose that lscA might be an ancestral levansucrase variant upstream of which the PAPE got inserted by potentially phage-mediated transposition events leading to expression of levansucrase in P. syringae. PMID:24670199

Miniature Transposable Sequences Are Frequently Mobilized in the Bacterial Plant Pathogen Pseudomonas syringae pv. phaseolicola

PubMed Central

Bardaji, Leire; Añorga, Maite; Jackson, Robert W.; Martínez-Bilbao, Alejandro; Yanguas-Casás, Natalia; Murillo, Jesús

2011-01-01

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10−5 and 1.1×10−6, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria. PMID:22016774

Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola.

PubMed

Bardaji, Leire; Añorga, Maite; Jackson, Robert W; Martínez-Bilbao, Alejandro; Yanguas-Casás, Natalia; Murillo, Jesús

2011-01-01

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10(-5) and 1.1×10(-6), depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

The Development of Adult Innate Lymphoid Cells

PubMed Central

Yang, Qi; Bhandoola, Avinash

2016-01-01

Innate lymphoid cells (ILC) are a specialized family of effector lymphocytes that transcriptionally and functionally mirror effector subsets of T cells, but differ from T cells in that they lack clonally-distributed adaptive antigen receptors. Our understanding of this family of lymphocytes is still in its infancy. In this review, we summarize current understanding and discuss recent insights into the cellular and molecular events that occur during early ILC development in adult mice. We discuss how these events overlap and diverge with the early development of adaptive T cells, and how they may influence the molecular and functional properties of mature ILC. PMID:26871595

Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

PubMed

Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

2012-09-01

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

In-flight adaptive performance optimization (APO) control using redundant control effectors of an aircraft

NASA Technical Reports Server (NTRS)

Gilyard, Glenn B. (Inventor)

1999-01-01

Practical application of real-time (or near real-time) Adaptive Performance Optimization (APO) is provided for a transport aircraft in steady climb, cruise, turn descent or other flight conditions based on measurements and calculations of incremental drag from a forced response maneuver of one or more redundant control effectors defined as those in excess of the minimum set of control effectors required to maintain the steady flight condition in progress. The method comprises the steps of applying excitation in a raised-cosine form over an interval of from 100 to 500 sec. at the rate of 1 to 10 sets/sec of excitation, and data for analysis is gathered in sets of measurements made during the excitation to calculate lift and drag coefficients C.sub.L and C.sub.D from two equations, one for each coefficient. A third equation is an expansion of C.sub.D as a function of parasitic drag, induced drag, Mach and altitude drag effects, and control effector drag, and assumes a quadratic variation of drag with positions .delta..sub.i of redundant control effectors i=1 to n. The third equation is then solved for .delta..sub.iopt the optimal position of redundant control effector i, which is then used to set the control effector i for optimum performance during the remainder of said steady flight or until monitored flight conditions change by some predetermined amount as determined automatically or a predetermined minimum flight time has elapsed.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation.

PubMed

Kassem, Sahar; Gaud, Guillaume; Bernard, Isabelle; Benamar, Mehdi; Dejean, Anne S; Liblau, Roland; Fournié, Gilbert J; Colacios, Céline; Malissen, Bernard; Saoudi, Abdelhadi

2016-07-01

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation.

A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation

PubMed Central

Kassem, Sahar; Bernard, Isabelle; Dejean, Anne S.; Liblau, Roland; Fournié, Gilbert J.; Colacios, Céline

2016-01-01

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation. PMID:27438086

Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity.

PubMed

Mukaihara, Takafumi; Hatanaka, Tadashi; Nakano, Masahito; Oda, Kenji

2016-04-12

The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. Ralstonia solanacearum is the causal agent of bacterial wilt disease of plants. This pathogen injects virulence effector proteins into host cells to suppress disease resistance responses of plants. In this article, we report a biochemical activity of R. solanacearum effector protein RipAY. RipAY can degrade GSH, a tripeptide that plays important roles in the plant immune system, with its γ-glutamyl cyclotransferase activity. The high GSH degradation activity of RipAY is considered to be a good weapon for this bacterium to suppress plant immunity. However, GSH also plays important roles in bacterial tolerance to various stresses and growth. Interestingly, RipAY has an excellent safety mechanism to prevent unwanted firing of its enzyme activity in bacterial cells because RipAY is specifically activated by host eukaryotic thioredoxins. This study also reveals a novel host plant protein acting as a molecular switch for effector activation. Copyright © 2016 Mukaihara et al.

Robotic End Effectors for Hard-Rock Climbing

NASA Technical Reports Server (NTRS)

Kennedy, Brett; Leger, Patrick

2004-01-01

Special-purpose robot hands (end effectors) now under development are intended to enable robots to traverse cliffs much as human climbers do. Potential applications for robots having this capability include scientific exploration (both on Earth and other rocky bodies in space), military reconnaissance, and outdoor search and rescue operations. Until now, enabling robots to traverse cliffs has been considered too difficult a task because of the perceived need of prohibitively sophisticated planning algorithms as well as end effectors as dexterous as human hands. The present end effectors are being designed to enable robots to attach themselves to typical rock-face features with less planning and simpler end effectors. This advance is based on the emulation of the equipment used by human climbers rather than the emulation of the human hand. Climbing-aid equipment, specifically cams, aid hooks, and cam hooks, are used by sport climbers when a quick ascent of a cliff is desired (see Figure 1). Currently two different end-effector designs have been created. The first, denoted the simple hook emulator, consists of three "fingers" arranged around a central "palm." Each finger emulates the function of a particular type of climbing hook (aid hook, wide cam hook, and a narrow cam hook). These fingers are connected to the palm via a mechanical linkage actuated with a leadscrew/nut. This mechanism allows the fingers to be extended or retracted. The second design, denoted the advanced hook emulator (see Figure 2), shares these features, but it incorporates an aid hook and a cam hook into each finger. The spring-loading of the aid hook allows the passive selection of the type of hook used. The end effectors can be used in several different modes. In the aid-hook mode, the aid hook on one of the fingers locks onto a horizontal ledge while the other two fingers act to stabilize the end effector against the cliff face. In the cam-hook mode, the broad, flat tip of the cam hook is inserted into a non-horizontal crack in the cliff face. A subsequent transfer of weight onto the end effector causes the tip to rotate within the crack, creating a passive, self-locking action of the hook relative to the crack. In the advanced hook emulator, the aid hook is pushed into its retracted position by contact with the cliff face as the cam hook tip is inserted into the crack. When a cliff face contains relatively large pockets or cracks, another type of passive self-locking can be used. Emulating the function of the piece of climbing equipment called a "cam" (note: not the same as a "cam hook"; see Figure 1), the fingers can be fully retracted and the entire end effector inserted into the feature. The fingers are then extended as far as the feature allows. Any weight then transferred to the end effector will tend to extend the fingers further due to frictional force, passively increasing the grip on the feature. In addition to the climbing modes, these end effectors can be used to walk on (either on the palm or the fingertips) and to grasp objects by fully extending the fingers.

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

PubMed

Pecenková, Tamara; Janda, Martin; Ortmannová, Jitka; Hajná, Vladimíra; Stehlíková, Zuzana; Žárský, Viktor

2017-09-01

Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated. Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. maculicola (Psm) and P. syringae pv. tomato DC3000 (Pst), as well as Agrobacterium tumefaciens (Atu) and Escherichia coli (Eco). Root hair lengths were measured after 24 and 48h of direct exposure to each bacterial strain. Several Arabidopsis mutants with impaired responses to pathogens, impaired ethylene perception and defects in the exocyst vesicle tethering complex that is involved in secretion were also analysed. Arabidopsis seedling roots infected with Psm or Pst responded similarly to when infected with plant growth-promoting rhizobacteria; root hair growth was stimulated and primary root growth was inhibited. Other plant- and soil-adapted bacteria induced similar root hair responses. The most compromised root hair growth stimulation response was found for the knockout mutants exo70A1 and ein2. The single immune pathways dependent on salicylic acid, jasmonic acid and PAD4 are not directly involved in root hair growth stimulation; however, in the mutual cross-talk with ethylene, they indirectly modify the extent of the stimulation of root hair growth. The Flg22 peptide does not initiate root hair stimulation as intact bacteria do, but pretreatment with Flg22 prior to Psm inoculation abolished root hair growth stimulation in an FLS2 receptor kinase-dependent manner. These early response phenomena are not associated with changes in auxin levels, as monitored with the pDR5::GUS auxin reporter. Early stimulation of root hair growth is an effect of an unidentified component of living plant pathogenic bacteria. The root hair growth response is triggered in the range of hours after bacterial contact with roots and can be modulated by FLS2 signalling. Bacterial stimulation of root hair growth requires functional ethylene signalling and an efficient exocyst-dependent secretory machinery. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please email: journals.permissions@oup.com

Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine-learning approach.

PubMed

Teper, Doron; Burstein, David; Salomon, Dor; Gershovitz, Michael; Pupko, Tal; Sessa, Guido

2016-04-01

The Gram-negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85-10 strain by various experimental and computational techniques. To identify additional Xcv 85-10 effectors, we applied a genome-wide machine-learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)-dependent expression, 5' regulatory sequences, amino acid composition bias and GC content. Thirty-six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)-inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild-type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild-type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth. © 2015 BSPP and John Wiley & Sons Ltd.

Platelets as Cellular Effectors of Inflammation in Vascular Diseases

PubMed Central

Rondina, Matthew T.; Weyrich, Andrew S.; Zimmerman, Guy A.

2013-01-01

Platelets are chief effector cells in hemostasis. In addition, they are multifaceted inflammatory cells with functions that span the continuum from innate immune responses to adaptive immunity. Activated platelets have key “thromboinflammatory” activities in a variety of vascular disorders and vasculopathies. Recently-identified inflammatory and immune activities provide insights into the biology of these versatile blood cells that are directly relevant to human vascular diseases. PMID:23704217

Antigen-Specific Induction of Osteopontin Contributes to the Chronification of Allergic Contact Dermatitis

PubMed Central

Seier, Anne M.; Renkl, Andreas C.; Schulz, Guido; Uebele, Tanja; Sindrilaru, Anca; Iben, Sebastian; Liaw, Lucy; Kon, Shigeyuki; Uede, Toshimitsu; Weiss, Johannes M.

2010-01-01

Allergic contact dermatitis is a T cell-mediated immune response, which in its relapsing chronic form is of high socioeconomic impact. The phosphoglycoprotein osteopontin (OPN) has chemotactic and Th1 cytokine functions and in various models is essential for robust T cell-mediated immunity. Here we demonstrate that OPN is abundantly expressed by both effector T cells and keratinocytes in allergic contact dermatitis lesions. T cells from nickel-allergic donors secrete high levels of OPN following antigen-specific stimulation. OPN may substitute for missing IFN-γ secretion in T effector cells because low IFN-γ-producing T cell clones secrete high levels of OPN, and OPN down-modulates their interleukin-4 expression. Furthermore, interferon-γ from T effector cells augments OPN in allergic contact dermatitis by inducing OPN in keratinocytes, which in turn polarizes dendritic cells and attracts inflammatory cells. In the murine contact hypersensitivity (CHS) model for allergic contact dermatitis, OPN is strongly induced in antigen-specific proliferating T cells, and OPN null mice display a reduced chronic CHS inflammatory response due to a decreased influx of effector T cells. Importantly, because of its function for chronic allergic contact dermatitis, OPN may well be a therapeutic target, because anti-OPN antibody treatment in part suppresses established chronic CHS. PMID:20008129

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Interferon-inducible effector mechanisms in cell-autonomous immunity

PubMed Central

MacMicking, John D.

2014-01-01

Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells — a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world. PMID:22531325

Kinematic functions for redundancy resolution using configuration control

NASA Technical Reports Server (NTRS)

Seraji, Homayoun (Inventor)

1994-01-01

The invention fulfills new goals for redundancy resolution based on manipulator dynamics and end-effector characteristics. These goals are accomplished by employing the recently developed configuration control approach. Redundancy resolution is achieved by controlling the joint inertia matrix of the end-effector mass matrix that affect the inertial torques or by reducing the joint torques due to gravity loading and payload. The manipulator mechanical-advantage and velocity-ratio are also used as performance measures to be improved by proper utilization of redundancy. Furthermore, end-effector compliance, sensitivity, and impulsive force at impact are introduced as redundancy resolution criteria. The new goals for redundancy resolution allow a more efficient utilization of the redundant joints based on the desired task requirements.

The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways.

PubMed

Wathugala, Deepthi L; Hemsley, Piers A; Moffat, Caroline S; Cremelie, Pieter; Knight, Marc R; Knight, Heather

2012-07-01

• Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold- and drought-inducible gene expression and freezing- and osmotic-stress tolerance. Its identification as a component of the MEDIATOR transcriptional co-activator complex led us to address its involvement in other transcriptional responses. • Gene expression responses to Pseudomonas syringae, ultraviolet-C (UV-C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real-time PCR and susceptibility to UV-C irradiation and Pseudomonas infection were assessed. • sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV-C irradiation. They exhibited correspondingly weaker PR (pathogenesis-related) gene expression than wild-type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV-C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild-type but not sfr6 by overexpression of ERF5. • SFR6/MED16 controls both SA- and JA-mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV-C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

NpPDR1, a pleiotropic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense.

PubMed

Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

2005-09-01

Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family.

NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense1

PubMed Central

Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

2005-01-01

Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family. PMID:16126865

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

PubMed

Naor, Adit; Panas, Michael W; Marino, Nicole; Coffey, Michael J; Tonkin, Christopher J; Boothroyd, John C

2018-04-03

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins. Copyright © 2018 Naor et al.

The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice.

PubMed

Park, Chan-Ho; Chen, Songbiao; Shirsekar, Gautam; Zhou, Bo; Khang, Chang Hyun; Songkumarn, Pattavipha; Afzal, Ahmed J; Ning, Yuese; Wang, Ruyi; Bellizzi, Maria; Valent, Barbara; Wang, Guo-Liang

2012-11-01

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

Structure activity relationship of synaptic and junctional neurotransmission.

PubMed

Goyal, Raj K; Chaudhury, Arun

2013-06-01

Chemical neurotransmission may include transmission to local or remote sites. Locally, contact between 'bare' portions of the bulbous nerve terminal termed a varicosity and the effector cell may be in the form of either synapse or non-synaptic contact. Traditionally, all local transmissions between nerves and effector cells are considered synaptic in nature. This is particularly true for communication between neurons. However, communication between nerves and other effectors such as smooth muscles has been described as nonsynaptic or junctional in nature. Nonsynaptic neurotransmission is now also increasingly recognized in the CNS. This review focuses on the relationship between structure and function that orchestrate synaptic and junctional neurotransmissions. A synapse is a specialized focal contact between the presynaptic active zone capable of ultrafast release of soluble transmitters and the postsynaptic density that cluster ionotropic receptors. The presynaptic and the postsynaptic areas are separated by the 'closed' synaptic cavity. The physiological hallmark of the synapse is ultrafast postsynaptic potentials lasting milliseconds. In contrast, junctions are juxtapositions of nerve terminals and the effector cells without clear synaptic specializations and the junctional space is 'open' to the extracellular space. Based on the nature of the transmitters, postjunctional receptors and their separation from the release sites, the junctions can be divided into 'close' and 'wide' junctions. Functionally, the 'close' and the 'wide' junctions can be distinguished by postjunctional potentials lasting ~1s and tens of seconds, respectively. Both synaptic and junctional communications are common between neurons; however, junctional transmission is the rule at many neuro-non-neural effectors. Published by Elsevier B.V.

Structure activity relationship of synaptic and junctional neurotransmission

PubMed Central

Goyal, Raj K; Chaudhury, Arun

2013-01-01

Chemical neurotransmission may include transmission to local or remote sites. Locally, contact between ‘bare’ portions of the bulbous nerve terminal termed a varicosity and the effector cell may be in the form of either synapse or non-synaptic contact. Traditionally, all local transmissions between nerves and effector cells are considered synaptic in nature. This is particularly true for communication between neurons. However, communication between nerves and other effectors such as smooth muscles has been described as nonsynaptic or junctional in nature. Nonsynaptic neurotransmission is now also increasing recognized in the CNS. This review focuses on the relationship between structure and function that orchestrate synaptic and junctional neurotransmissions. A synapse is a specialized focal contact between the presynaptic active zone capable for ultrafast release of soluble transmitters and the postsynaptic density that cluster ionotropic receptors. The presynaptic and the postsynaptic areas are separated by the ‘closed’ synaptic cavity. The physiological hallmark of the synapse is ultrafast postsynaptic potentials lasting in milliseconds. In contrast, junctions are juxtapositions of nerve terminals and the effector cells without clear synaptic specializations and the junctional space is ‘open’ to the extracellular space. Based on the nature of the transmitters, postjunctional receptors and their separation from the release sites, the junctions can be divided into ‘close’ and ‘wide’ junctions. Functionally, the ‘close’ and the ‘wide’ junctions can be distinguished by postjunctional potentials lasting ~1 second and 10s of seconds, respectively. Both synaptic and junctional communications are common between neurons; however, junctional transmission is the rule at many neuro-non-neural effectors. PMID:23535140

Protecting and rescuing the effectors: roles of differentiation and survival in the control of memory T cell development

PubMed Central

Kurtulus, Sema; Tripathi, Pulak; Hildeman, David A.

2013-01-01

Vaccines, arguably the single most important intervention in improving human health, have exploited the phenomenon of immunological memory. The elicitation of memory T cells is often an essential part of successful long-lived protective immunity. Our understanding of T cell memory has been greatly aided by the development of TCR Tg mice and MHC tetrameric staining reagents that have allowed the precise tracking of antigen-specific T cell responses. Indeed, following acute infection or immunization, naïve T cells undergo a massive expansion culminating in the generation of a robust effector T cell population. This peak effector response is relatively short-lived and, while most effector T cells die by apoptosis, some remain and develop into memory cells. Although the molecular mechanisms underlying this cell fate decision remain incompletely defined, substantial progress has been made, particularly with regards to CD8+ T cells. For example, the effector CD8+ T cells generated during a response are heterogeneous, consisting of cells with more or less potential to develop into full-fledged memory cells. Development of CD8+ T cell memory is regulated by the transcriptional programs that control the differentiation and survival of effector T cells. While the type of antigenic stimulation and level of inflammation control effector CD8+ T cell differentiation, availability of cytokines and their ability to control expression and function of Bcl-2 family members governs their survival. These distinct differentiation and survival programs may allow for finer therapeutic intervention to control both the quality and quantity of CD8+ T cell memory. Effector to memory transition of CD4+ T cells is less well characterized than CD8+ T cells, emerging details will be discussed. This review will focus on the recent progress made in our understanding of the mechanisms underlying the development of T cell memory with an emphasis on factors controlling survival of effector T cells. PMID:23346085

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes

PubMed Central

Deb, Chandra; Howe, Charles L

2011-01-01

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler’s virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8+ lymphocytes that are CD45hiCD44loCD62L− and a population of spinal cord infiltrating target effector memory CD8+ lymphocytes that are CD45hiCD44hiCD62L−. These cells respond robustly to ex vivo stimulation by producing interferon γ but do not exhibit specificity for Theiler’s virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities. PMID:19596449

Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

PubMed Central

Procopio, Maria-Giuseppina; Laszlo, Csaba; Labban, Dania Al; Kim, Dong Eun; Bordignon, Pino; Jo, Seunghee; Goruppi, Sandro; Menietti, Elena; Ostano, Paola; Ala, Ugo; Provero, Paolo; Hoetzenecker, Wolfram; Neel, Victor; Kilarski, Witek; Swartz, Melody A.; Brisken, Cathrin; Lefort, Karine; Dotto, G. Paolo

2015-01-01

Stromal fibroblast senescence has been linked to aging-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased. Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control. PMID:26302407