scholarly journals Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000

2017 ◽  
Vol 30 (8) ◽  
pp. 656-665 ◽  
Author(s):  
Milija Jovanovic ◽  
Christopher Waite ◽  
Ellen James ◽  
Nicholas Synn ◽  
Timothy Simpson ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogens ◽  
Functional Characterization ◽  
Direct Interaction ◽  
Negative Control ◽  
Effector Proteins ◽  
Tomato Dc3000 ◽  
Sequence Alignments ◽  
Group I ◽  
Σ Factor

The plant pathogen Pseudomonas syringae pv. tomato DC3000 uses a type III secretion system (T3SS) to transfer effector proteins into the host. The expression of T3SS proteins is controlled by the HrpL σ factor. Transcription of hrpL is σ54-dependent and bacterial enhancer-binding proteins HrpR and HrpS coactivate the hrpL promoter. The HrpV protein imposes negative control upon HrpR and HrpS through direct interaction with HrpS. HrpG interacts with HrpV and relieves such negative control. The sequence alignments across Hrp group I-type plant pathogens revealed conserved HrpV and HrpG amino acids. To establish structure–function relationships in HrpV and HrpG, either truncated or alanine substitution mutants were constructed. Key functional residues in HrpV and HrpG are found within their C-terminal regions. In HrpG, L101 and L105 are indispensable for the ability of HrpG to directly interact with HrpV and suppress HrpV-dependent negative regulation of HrpR and HrpS. In HrpV, L108 and G110 are major determinants for interactions with HrpS and HrpG. We propose that mutually exclusive binding of HrpS and HrpG to the same binding site of HrpV governs a transition from negative control to activation of the HrpRS complex leading to HrpL expression and pathogenicity of P. syringae.

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 269-280 ◽  
Author(s):  
Ute Kabisch ◽  
Angelika Landgraf ◽  
Jana Krause ◽  
Ulla Bonas ◽  
Jens Boch
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Gel Filtration ◽  
Effector Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
Effector Genes ◽  
Two Hybrid

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

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 The HopZ Family of Pseudomonas syringae Type III Effectors Require Myristoylation for Virulence and Avirulence Functions in Arabidopsis thaliana

2008 ◽  
Vol 190 (8) ◽  
pp. 2880-2891 ◽  
Author(s):  
Jennifer D. Lewis ◽  
Wasan Abada ◽  
Wenbo Ma ◽  
David S. Guttman ◽  
Darrell Desveaux
Keyword(s):  
Arabidopsis Thaliana ◽  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Pathogenic Bacteria ◽  
Functional Characterization ◽  
Defense Responses ◽  
Effector Proteins ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Virulence Protein

ABSTRACT Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1aPsyA2, HopZ1bPgyUnB647, HopZ1cPmaE54326, HopZ2Ppi895A and HopZ3PsyB728a. HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.

scholarly journals A Pseudomonas syringae pv. tomato DC3000 Hrp (Type III Secretion) Deletion Mutant Expressing the Hrp System of Bean Pathogen P. syringae pv. syringae 61 Retains Normal Host Specificity for Tomato

2003 ◽  
Vol 16 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Derrick E. Fouts ◽  
Jorge L. Badel ◽  
Adela R. Ramos ◽  
Ryan A. Rapp ◽  
Alan Collmer
Keyword(s):  
Host Range ◽  
Resistance Gene ◽  
Host Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Secretion System ◽  
Effector Proteins ◽  
Tomato Dc3000 ◽  
Tomato Plants ◽  
Type Iii

The plant pathogenic species Pseudomonas syringae is divided into numerous pathovars based on host specificity. For example, P. syringae pv. tomato DC3000 is pathogenic on tomato and Arabidopsis, whereas P. syringae pv. syringae 61 is pathogenic on bean. The ability of P. syringae strains to elicit the hypersensitive response (HR) in non-hosts or be pathogenic (or parasitic) in hosts is dependent on the Hrp (type III secretion) system and effector proteins this system is thought to inject into plant cells. To test the role of the Hrp system in determining host range, the hrp/hrc gene cluster (hrpK through hrpR) was deleted from DC3000 and complemented in trans with the orthologous cluster from strain 61. Mutant CUCPB5114 expressing the bean pathogen Hrp system on plasmid pCPP2071 retained the ability of wild-type DC3000 to elicit the HR in bean, to grow and cause bacterial speck in tomato, and to elicit a cultivar-specific (gene-for-gene) HR in tomato plants carrying the Pto resistance gene. However, the symptoms produced in compatible tomato plants involved markedly reduced chlorosis, and CUCPB5114(pCPP2071) did not grow or produce symptoms in Arabidopsis Col-0 although it was weakly virulent in NahG Arabidopsis. A hypersensitive-like collapse was produced by CUCPB5114(pCPP2071) in Arabidopsis Col-0 at 1 × 107 CFU/ml, but only if the bacteria also expressed AvrB, which is recognized by the RPM1 resistance gene in Col-0 and confers incompatibility. These observations support the concept that the P. syringae effector proteins, rather than secretion system components, are the primary determinants of host range at both the species and cultivar levels of host specificity.

scholarly journals Novel Exchangeable Effector Loci Associated with the Pseudomonas syringae hrp Pathogenicity Island: Evidence for Integron-Like Assembly from Transposed Gene Cassettes

2003 ◽  
Vol 16 (6) ◽  
pp. 495-507 ◽  
Author(s):  
James C. Charity ◽  
Kyong Pak ◽  
Charles F. Delwiche ◽  
Steven W. Hutcheson
Keyword(s):  
Pseudomonas Syringae ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Tomato Dc3000 ◽  
Gene Cassettes ◽  
Brassica Spp ◽  
Geographical Regions ◽  
Intergenic Sequences ◽  
Polymerase Chain ◽  
New Alleles

Pseudomonas syringae strains use a type III secretion system (TTSS) to translocate effector proteins that assist in the parasitism of host plant cells. Some genes for effector proteins are clustered in the exchangeable effector locus (EEL) associated with the hrp pathogenicity island. A polymerase chain reaction-based screen was developed to amplify the EEL from P. syringae strains. Of the 86 strains screened, the EEL was successfully amplified from 30 predominately North American P. syringae pv. syringae strains using hrpK and queA-derived primers and from an additional three strains using hrpL and queA-derived primers. Among the amplified EEL, ten distinct types of EEL were identified that could be classified into six families distinguishable by genetic composition, but other types of EEL may be present in strains isolated in other geographical regions. No linkage with the host range of the source strain was apparent. Gene cassettes carrying conserved flanking, coding, and intergenic sequences, present in different combinations, were identified in the characterized EEL. Six new alleles of known effectors were identified that differed from the homolog in sequence, size, or both of the gene. One of these apparently novel effector proteins, HopPsyB, retained a strongly conserved amino terminus similar to that of HopPsyA, but other regions of the two polypeptides were only weakly similar. hopPsyB was expressed from an apparent operon that included hrpK and a shcA homolog, shcB. Escherichia coli MC4100 expressing the hrp TTSS, ShcB, and HopPsyB elicited the hypersensitive response (HR) in tobacco, consistent with effector production. Indicative of translocation as an effector, P. syringae pv. tomato DC3000 expressing a HopPsyB':'AvrRpt2 fusion elicited the HR in RPS2+ Arabidopsis thaliana. P. syringae pv. tomato DC3000 carrying HopPsyB exhibited slightly enhanced virulence in several Brassica spp. These results are consistent with the hypotheses that the EEL is a source of disparate effectors functioning in pathogenicity of P. syringae strains and that it evolved independently of the hrp pathogenicity island central conserved region, most likely through integron-like assembly of transposed gene cassettes.

scholarly journals The Arabidopsis thaliana JASMONATE INSENSITIVE 1 Gene Is Required for Suppression of Salicylic Acid-Dependent Defenses During Infection by Pseudomonas syringae

2006 ◽  
Vol 19 (7) ◽  
pp. 789-800 ◽  
Author(s):  
Neva Laurie-Berry ◽  
Vinita Joardar ◽  
Ian H. Street ◽  
Barbara N. Kunkel
Keyword(s):  
Arabidopsis Thaliana ◽  
Salicylic Acid ◽  
Pseudomonas Syringae ◽  
Plant Pathogens ◽  
Symptom Development ◽  
Tomato Dc3000 ◽  
Jasmonate Signaling ◽  
Pathogenesis Related ◽  
Elevated Expression

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coi1) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1(PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus,P. syringae pv. tomatoDC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.

scholarly journals Roq1 confers resistance to Xanthomonas, Pseudomonas syringae and Ralstonia solanacearum in tomato

2019 ◽  
Author(s):  
Nicholas C. Thomas ◽  
Connor G. Hendrich ◽  
Upinder S. Gill ◽  
Caitilyn Allen ◽  
Samuel F. Hutton ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Ralstonia Solanacearum ◽  
Plant Pathogens ◽  
Field Trials ◽  
Current Control ◽  
Effector Proteins ◽  
Effective Control ◽  
Crop Species ◽  
Xanthomonas Perforans ◽  
Pathogen Effector

AbstractXanthomonas species, Pseudomonas syringae and Ralstonia solanacearum are bacterial plant pathogens that cause significant yield loss in many crop species. Current control methods for these pathogens are insufficient but there is significant potential for generating new disease-resistant crop varieties. Plant immune receptors encoded by nucleotide-binding, leucine-rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence / absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in many R. solanacearum strains. We found that Roq1 also mediates perception of RipB and confers immunity to Xanthomonas, P. syringae, and R. solanacearum when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.SummaryA single immune receptor expressed in tomato confers strong resistance to three different bacterial diseases.

scholarly journals Identification of a Twin-Arginine Translocation System in Pseudomonas syringae pv. tomato DC3000 and Its Contribution to Pathogenicity and Fitness

2005 ◽  
Vol 187 (24) ◽  
pp. 8450-8461 ◽  
Author(s):  
Philip A. Bronstein ◽  
Matthew Marrichi ◽  
Sam Cartinhour ◽  
David J. Schneider ◽  
Matthew P. DeLisa
Keyword(s):  
Pseudomonas Syringae ◽  
Virulence Factors ◽  
Fluorescent Protein ◽  
Effector Proteins ◽  
Broad Host Range ◽  
Tomato Dc3000 ◽  
Phytopathogenic Bacterium ◽  
Type Iii ◽  
Twin Arginine Translocation ◽  
Tat System

ABSTRACT The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium.

scholarly journals Pseudomonas syringae pv. tomato DC3000 Type III Effector HopAA1-1 Functions Redundantly with Chlorosis-Promoting Factor PSPTO4723 to Produce Bacterial Speck Lesions in Host Tomato

2009 ◽  
Vol 22 (11) ◽  
pp. 1341-1355 ◽  
Author(s):  
Kathy R. Munkvold ◽  
Alistair B. Russell ◽  
Brian H. Kvitko ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Secretion System ◽  
Effector Proteins ◽  
Gtpase Activating Protein ◽  
Tomato Dc3000 ◽  
Bacterial Speck ◽  
Type Iii ◽  
Bacterial Speck Disease

The ability of Pseudomonas syringae pv. tomato DC3000 to cause bacterial speck disease in tomato is dependent on the injection, via the type III secretion system, of approximately 28 Avr/Hop effector proteins. HopAA1-1 is encoded in the conserved effector locus (CEL) of the P. syringae Hrp pathogenicity island. Transiently expressed HopAA1-1 acts inside Saccharomyces cerevisiae and plant cells to elicit cell death. hopAA1 homologs were cloned and sequenced from the CEL of seven P. syringae strains representing diverse pathovars. Analysis of the sequences revealed that HopAA1-1 carries a potential GTPase-activating protein (GAP) domain, GALRA, which is polymorphic (FEN instead of LRA) in HopAA1-2, a paralogous DC3000 effector. Deleting hopAA1-1 from DC3000 reduces the formation of necrotic speck lesions in dip-inoculated tomato leaves if effector-gene cluster IX or just PSPTO4723 within this region has been deleted. A HopAA1-1 mutant in which the putative catalytic arginine in the GAP-like domain has been replaced with alanine retains its ability to kill yeast and promote the formation of speck lesions by the ΔhopAA1-1ΔIX mutant, but a HopAA1-1 mutant carrying the FEN polymorphism loses both of these abilities. Unexpectedly, PSPTO4723 does not appear to encode an effector and its deletion also reduces disease-associated chlorosis.

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