scholarly journals Yeast Genomic Screens Identify Kinesins as Potential Targets of the Pseudomonas syringae Type III Effector, HopZ1a

2018 ◽  
Author(s):  
Amy Huei-Yi Lee ◽  
D. Patrick Bastedo ◽  
Timothy Lo ◽  
Maggie A. Middleton ◽  
Ji-Young Youn ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogen ◽  
Bacterial Pathogens ◽  
Functional Enrichment ◽  
Host Cells ◽  
Physical Interaction ◽  
Type Iii ◽  
Pathogen Fitness ◽  
Microtubule Networks ◽  
Yeast Genes

ABSTRACTGram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as promiscuous individual T3SEs that can have multiple host targets. To overcome these challenges, we conducted heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. We screened 75 T3SEs from the plant pathogen Pseudomonas syringae and identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media, including the acetyltransferase HopZ1a. We focused our further analysis on HopZ1a, which interacts with plant tubulin and alters microtubule networks. We first performed a Pathogenic Genetic Array (PGA) screen of HopZ1a against ~4400 yeast carrying non-essential mutations and found 95 and 10 deletion mutants which reduced or enhanced HopZ1a toxicity, respectively. To uncover putative HopZ1a host targets, we interrogated both the genetic- and physical-interaction profiles of HopZ1a by identifying yeast genes with PGA profiles most similar (i.e. congruent) to that of HopZ1a, performing a functional enrichment analysis of these HopZ1a-congruent genes, and by analyzing previously described HopZ physical interaction datasets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1.ARTICLE SUMMARYBacterial pathogens utilize secretion systems to directly deliver effector proteins into host cells, with the ultimate goal of promoting pathogen fitness. Despite the central role that effectors play in infection, the molecular function and host targets of most effectors remain uncharacterized. We used yeast genomics and protein interaction data to identify putative virulence targets of the effector HopZ1a from the plant pathogen Pseudomonas syringae. HopZ1a is an acetyltransferase that induces plant microtubule destruction. We showed that HopZ1a acetylated plant kinesin proteins known to regulate microtubule networks. Our study emphasizes the power of yeast functional genomic screens to characterize effector functions.

scholarly journals Bioinformatics Correctly Identifies Many Type III Secretion Substrates in the Plant Pathogen Pseudomonas syringae and the Biocontrol Isolate P. fluorescens SBW25

2005 ◽  
Vol 18 (8) ◽  
pp. 877-888 ◽  
Author(s):  
Boris A. Vinatzer ◽  
Joanna Jelenska ◽  
Jean T. Greenberg
Keyword(s):  
Amino Acids ◽  
Pseudomonas Syringae ◽  
Plant Pathogen ◽  
Type Iii Secretion ◽  
Host Cells ◽  
Protein Accumulation ◽  
Large Set ◽  
Virulence Proteins ◽  
Type Iii ◽  
C Terminus

The plant pathogen Pseudomonas syringae causes disease by secreting a potentially large set of virulence proteins called effectors directly into host cells, their environment, or both, using a type III secretion system (T3SS). Most P.syringae effectors have a common upstream element called the hrp box, and their N-terminal regions have amino acids biases, features that permit their bioinformatic prediction. One of the most prominent biases is a positive serine bias. We previously used the truncated AvrRpt281–255 effector containing a serine-rich stretch from amino acids 81 to 100 as a T3SS reporter. Region 81 to 100 of this reporter does not contribute to the secretion or translocation of AvrRpt2 or to putative effector protein chimeras. Rather, the serine-rich region from the N-terminus of AvrRpt2 is important for protein accumulation in bacteria. Most of the N-terminal region (amino acids 15 to 100) is not essential for secretion in culture or delivery to plants. However, portions of this sequence may increase the efficiency of AvrRpt2 secretion, delivery to plants, or both. Two effectors previously identified with the AvrRpt281–255 reporter were secreted in culture independently of AvrRpt2, validating the use of the C terminus of AvrRpt2 as a T3SS reporter. Finally, using the reduced AvrRpt2101–255 reporter, we confirmed seven predicted effectors from P. syringae pv. tomato DC3000, four from P. syringae pv. syringae B728a, and two from P. fluorescens SBW25.

scholarly journals What the Wild Things Do: Mechanisms of Plant Host Manipulation by Bacterial Type III-Secreted Effector Proteins

2021 ◽  
Vol 9 (5) ◽  
pp. 1029
Author(s):  
Karl J. Schreiber ◽  
Ilea J. Chau-Ly ◽  
Jennifer D. Lewis
Keyword(s):  
Pseudomonas Syringae ◽  
Structural Data ◽  
Enzymatic Activities ◽  
Effector Proteins ◽  
Host Recognition ◽  
Plant Host ◽  
Type Iii ◽  
Xanthomonas Species ◽  
Pathogen Fitness ◽  
Enzymatic Mechanisms

Phytopathogenic bacteria possess an arsenal of effector proteins that enable them to subvert host recognition and manipulate the host to promote pathogen fitness. The type III secretion system (T3SS) delivers type III-secreted effector proteins (T3SEs) from bacterial pathogens such as Pseudomonas syringae, Ralstonia solanacearum, and various Xanthomonas species. These T3SEs interact with and modify a range of intracellular host targets to alter their activity and thereby attenuate host immune signaling. Pathogens have evolved T3SEs with diverse biochemical activities, which can be difficult to predict in the absence of structural data. Interestingly, several T3SEs are activated following injection into the host cell. Here, we review T3SEs with documented enzymatic activities, as well as T3SEs that facilitate virulence-promoting processes either indirectly or through non-enzymatic mechanisms. We discuss the mechanisms by which T3SEs are activated in the cell, as well as how T3SEs modify host targets to promote virulence or trigger immunity. These mechanisms may suggest common enzymatic activities and convergent targets that could be manipulated to protect crop plants from infection.

scholarly journals Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity

2016 ◽  
Vol 29 (8) ◽  
pp. 651-660 ◽  
Author(s):  
Georgy Popov ◽  
Malou Fraiture ◽  
Frederic Brunner ◽  
Guido Sessa
Keyword(s):  
Pseudomonas Syringae ◽  
Map Kinases ◽  
Molecular Mechanisms ◽  
Inducible Promoter ◽  
Effector Proteins ◽  
Host Cells ◽  
In Planta ◽  
Callose Deposition ◽  
Type Iii ◽  
Xanthomonas Euvesicatoria

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.

scholarly journals The Plant Phenolic Compound p-Coumaric Acid Represses Gene Expression in the Dickeya dadantii Type III Secretion System

2008 ◽  
Vol 75 (5) ◽  
pp. 1223-1228 ◽  
Author(s):  
Yan Li ◽  
Quan Peng ◽  
Dija Selimi ◽  
Qi Wang ◽  
Amy O. Charkowski ◽  
...  
Keyword(s):  
Phenolic Compound ◽  
Type Iii Secretion ◽  
Bacterial Pathogens ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Hydroxyl Group ◽  
Coumaric Acid ◽  
Dickeya Dadantii ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is a major virulence factor in many gram-negative bacterial pathogens. This secretion system translocates effectors directly into the cytosol of eukaryotic host cells, where the effector proteins facilitate bacterial pathogenesis by interfering with host cell signal transduction and other cellular processes. Plants defend themselves against bacterial pathogens by recognizing either the type 3 effectors or their actions and initiating a cascade of defense responses that often results in programmed cell death of the plant cell being attacked. Here we show that a plant phenolic compound, p-coumaric acid (PCA), represses the expression of T3SS genes of the plant pathogen Dickeya dadantii, suggesting that plants can also defend against bacterial pathogens by manipulating the expression of the T3SS. PCA repressed the expression of T3SS regulatory genes through the HrpX/Y two-component system, a core regulator of the T3SS, rather than through the global regulator GacS/A, which indirectly regulates the T3SS. A further analysis of several PCA analogs suggests that the para positioning of the hydroxyl group in the phenyl ring and the double bond of PCA may be important for its biological activity.

scholarly journals TRAPPC13 is a Novel Target of Mesorhizobium amorphae Type III Secretion System Effector NopP

2021 ◽  
Author(s):  
Dongying Liu ◽  
Yantao Luo ◽  
Xiaofeng Zheng ◽  
Xinye Wang ◽  
Minxia Chou ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Nitrogenase Activity ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Type Iii ◽  
Early Stages

Similar to pathogenic bacteria, rhizobia can inject effector proteins into host cells directly to promote infection via the type III secretion system. Nodulation outer protein P (NopP), a specific type III secretion system effector of rhizobia, plays different roles in the establishment of multiple rhizobia-legume symbiotic systems. Mesorhizobium amorphae CCNWGS0123 (GS0123), which infects Robinia pseudoacacia specifically, secretes several type III secretion system (T3SS) effectors, including NopP. Here, we demonstrate that NopP is secreted through T3SS-Ⅰof GS0123 during the early stages of infection, and its deficiency decreases nodule nitrogenase activity of R. pseudoacacia nodules. A trafficking protein particle complex subunit 13-like protein (TRAPPC13) ishas been identified as a NopP target protein in R. pseudoacacia roots by screening a yeast two-hybrid library. The physical interaction between NopP and TRAPPC13 is verified by bimolecular fluorescence complementation and co-immunoprecipitation assays. In addition, subcellular localization analysis reveals that both NopP and its target, TRAPPC13, are co-localized on the plasma membrane. Compared with GS0123-inoculated R. pseudoacacia roots, some genes associated with cell wall remodeling and plant innate immunity down-regulated in ΔnopP-inoculated roots at 36 hpi. The results suggest that NopP in M. amorphae CCNWGS0123 acts in multiple process in R. pseudoacacia during the early stages of infection, and TRAPPC13 could participate in the process as a NopP target.

scholarly journals The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Lihi Shaulov ◽  
Jenia Gershberg ◽  
Wanyin Deng ◽  
B. Brett Finlay ◽  
Neta Sal-Man
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Bacterial Pathogens ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Bacterial Virulence ◽  
Effector Proteins ◽  
Host Cells ◽  
Gram Negative ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy.

scholarly journals The HopPtoF Locus of Pseudomonas syringae pv. tomato DC3000 Encodes a Type III Chaperone and a Cognate Effector

2004 ◽  
Vol 17 (5) ◽  
pp. 447-455 ◽  
Author(s):  
Libo Shan ◽  
Hye-sook Oh ◽  
Jianfu Chen ◽  
Ming Guo ◽  
Jianmin Zhou ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Whole Genome Sequence ◽  
Avirulence Gene ◽  
Bacterial Cells ◽  
Tomato Dc3000 ◽  
Virulence Proteins ◽  
Type Iii

Type III secretion systes are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcFPto and HopFPto, that are preceded by a single hrp box promoter. We present evidence here to show that ShcFPto and HopFPto encode a type III chaperone and a cognate effector, respectively. ShcFPto interacts with and stabilizes the HopFPto protein in the bacterial cell. Translation of HopFPto starts at a rare initiation codon ATA that limits the synthesis of the HopFPto protein to a low level in bacterial cells.

scholarly journals Whole-Genome Expression Profiling Defines the HrpL Regulon of Pseudomonas syringae pv. tomato DC3000, Allows de novo Reconstruction of the Hrp cis Element, and Identifies Novel Coregulated Genes

2006 ◽  
Vol 19 (11) ◽  
pp. 1167-1179 ◽  
Author(s):  
Adriana O. Ferreira ◽  
Christopher R. Myers ◽  
Jeffrey S. Gordon ◽  
Gregory B. Martin ◽  
Monica Vencato ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
De Novo ◽  
Effector Proteins ◽  
Iterative Refinement ◽  
Host Cells ◽  
Whole Genome ◽  
Wild Type ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
In The Wild

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and ΔhrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the ΔhrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated op-erons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phyto-hormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

scholarly journals Enhancer-Binding Proteins HrpR and HrpS Interact To Regulate hrp-Encoded Type III Protein Secretion inPseudomonas syringae Strains

2001 ◽  
Vol 183 (19) ◽  
pp. 5589-5598 ◽  
Author(s):  
Steven W. Hutcheson ◽  
Jamie Bretz ◽  
Thomas Sussan ◽  
Songmu Jin ◽  
Kyong Pak
Keyword(s):  
Pseudomonas Syringae ◽  
Binding Proteins ◽  
Protein Translocation ◽  
Effector Proteins ◽  
Expression Vectors ◽  
Physical Interaction ◽  
Enhancer Binding Proteins ◽  
Type Iii ◽  
Type Iii Protein Secretion ◽  
Cognate Protein

ABSTRACT In Pseudomonas syringae strains, thehrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity.hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5′ to hrpR, and reverse transcriptase PCR detected the presence of anhrpRS transcript. The hrpR promoter and coding sequence were conserved among P. syringaestrains. The coding sequences for hrpR andhrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia colitransformants carrying an hrpL′-lacZfusion. HrpS could function as a weak activator of thehrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the ς54-dependenthrpL promoter in P. syringae strains.

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