Two Novel Type III-Secreted Proteins of Xanthomonas campestris pv. vesicatoria Are Encoded within the hrp Pathogenicity Island

ABSTRACT The Hrp type III protein secretion system (TTSS) is essential for pathogenicity of gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. cDNA-amplified fragment length polymorphism and reverse transcription-PCR analyses identified new genes, regulated by key hrp regulator HrpG, in the regions flanking the hrp gene cluster. Sequence analysis revealed genes encoding HpaG, a predicted leucine-rich repeat-containing protein, the lysozyme-like HpaH protein, and XopA and XopD, which are similar in sequence to Hpa1 from Xanthomonas oryzae pv. oryzae and PsvA from Pseudomonas syringae, respectively. XopA and XopD (Xanthomonas outer proteins) are secreted by the Xanthomonas Hrp TTSS and thus represent putative effector proteins. Mutations in xopA, but not in xopD, resulted in reduced bacterial growth in planta and delayed plant reactions in susceptible and resistant host plants. Since the xopD promoter contains a putative hrp box, which is characteristic of hrpL-regulated genes in P. syringae and Erwinia spp., the gene was probably acquired by horizontal gene transfer. Interestingly, the regions flanking the hrp gene cluster also contain insertion sequences and genes for a putative transposase and a tRNAArg. These features suggest that the hrp gene cluster of X. campestris pv. vesicatoria is part of a pathogenicity island.

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The Pseudomonas syringae HopPtoV Protein Is Secreted in Culture and Translocated into Plant Cells via the Type III Protein Secretion System in a Manner Dependent on the ShcV Type III Chaperone

Journal of Bacteriology ◽

10.1128/jb.186.11.3621-3630.2004 ◽

2004 ◽

Vol 186 (11) ◽

pp. 3621-3630 ◽

Cited By ~ 12

Author(s):

Misty D. Wehling ◽

Ming Guo ◽

Zheng Qing Fu ◽

James R. Alfano

Keyword(s):

Pseudomonas Syringae ◽

Protein Secretion ◽

Plant Cells ◽

Secretion System ◽

Effector Proteins ◽

In Planta ◽

Type Iii ◽

Protein Secretion System ◽

Type Iii Protein Secretion ◽

Plant Pathogenesis

ABSTRACT The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis.

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The algT Gene of Pseudomonas syringae pv. glycinea andNew Insights into the Transcriptional Organization of thealgT-muc Gene Cluster

Journal of Bacteriology ◽

10.1128/jb.01160-06 ◽

2006 ◽

Vol 188 (23) ◽

pp. 8013-8021 ◽

Cited By ~ 14

Author(s):

Alexander Schenk ◽

Michael Berger ◽

Lisa M. Keith ◽

Carol L. Bender ◽

Georgi Muskhelishvili ◽

...

Keyword(s):

Gene Cluster ◽

Pseudomonas Syringae ◽

Azotobacter Vinelandii ◽

Regulatory Gene ◽

In Planta ◽

Phytopathogenic Bacterium ◽

Reverse Transcription Pcr ◽

Alginate Production ◽

Alginate Biosynthesis

ABSTRACT The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae, the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d-mannuronic and l-guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii. These genes encode the alternative sigma factor AlgT (σ22), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae, and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli. We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT, mucA, mucB, mucD, and algD, which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT, mucA, and mucB are cotranscribed as an operon in P. syringae. Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae.

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Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

10.1101/2021.11.22.469601 ◽

2021 ◽

Author(s):

David A Baltrus ◽

Qian Feng ◽

Brian H Kvitko

Keyword(s):

Pseudomonas Syringae ◽

Evolutionary Dynamics ◽

Effector Proteins ◽

In Planta ◽

Genomic Context ◽

Type Iii Effectors ◽

Type Iii ◽

Genome Context ◽

The Impact ◽

Multiple Type

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

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Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-16-0137-r ◽

2016 ◽

Vol 29 (8) ◽

pp. 651-660 ◽

Cited By ~ 17

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.

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PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family

Journal of Bacteriology ◽

10.1128/jb.00180-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4903-4917 ◽

Cited By ~ 28

Author(s):

Damien Meyer ◽

Sébastien Cunnac ◽

Mareva Guéneron ◽

Céline Declercq ◽

Frédérique Van Gijsegem ◽

...

Keyword(s):

Protein Secretion ◽

Ralstonia Solanacearum ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Secretion System ◽

Effector Proteins ◽

Experimental Conditions ◽

Type Iii ◽

Protein Secretion System ◽

Type Iii Protein Secretion

ABSTRACT Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum.

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The Majority of the Type III Effector Inventory of Pseudomonas syringae pv. tomato DC3000 Can Suppress Plant Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-9-1069 ◽

2009 ◽

Vol 22 (9) ◽

pp. 1069-1080 ◽

Cited By ~ 147

Author(s):

Ming Guo ◽

Fang Tian ◽

Yashitola Wamboldt ◽

James R. Alfano

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

In Planta ◽

Tomato Dc3000 ◽

Type Iii Effector ◽

Type Iii Effectors ◽

Type Iii ◽

Molecular Pattern ◽

Effector Triggered Immunity ◽

Type Iii Protein Secretion

The Pseudomonas syringae type III protein secretion system (T3SS) and the type III effectors it injects into plant cells are required for plant pathogenicity and the ability to elicit a hypersensitive response (HR). The HR is a programmed cell death that is associated with effector-triggered immunity (ETI). A primary function of P. syringae type III effectors appears to be the suppression of ETI and pathogen-associated molecular pattern–triggered immunity (PTI), which is induced by conserved molecules on microorganisms. We reported that seven type III effectors from P. syringae pv. tomato DC3000 were capable of suppressing an HR induced by P. fluorescens(pHIR11) and have now tested 35 DC3000 type III effectors in this assay, finding that the majority of them can suppress the HR induced by HopA1. One newly identified type III effector with particularly strong HR suppression activity was HopS2. We used the pHIR11 derivative pLN1965, which lacks hopA1, in related assays and found that a subset of the type III effectors that suppressed HopA1-induced ETI also suppressed an ETI response induced by AvrRpm1 in Arabidopsis thaliana. A. thaliana plants expressing either HopAO1 or HopF2, two type III effectors that suppressed the HopA1-induced HR, were reduced in the flagellin-induced PTI response as well as PTI induced by other PAMPs and allowed enhanced in planta growth of P. syringae. Collectively, our results suggest that the majority of DC3000 type III effectors can suppress plant immunity. Additionally, the construct pLN1965 will likely be a useful tool in determining whether other type III effectors or effectors from other types of pathogens can suppress either ETI, PTI, or both.

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XopC and XopJ, Two Novel Type III Effector Proteinsfrom Xanthomonas campestris pv.vesicatoria

Journal of Bacteriology ◽

10.1128/jb.185.24.7092-7102.2003 ◽

2003 ◽

Vol 185 (24) ◽

pp. 7092-7102 ◽

Cited By ~ 65

Author(s):

Laurent Noël ◽

Frank Thieme ◽

Jana Gäbler ◽

Daniela Büttner ◽

Ulla Bonas

Keyword(s):

Pseudomonas Syringae ◽

Plant Cell ◽

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Effector Proteins ◽

Type Iii ◽

Genome Wide ◽

A Genome ◽

Xanthomonas Campestris Pv.Vesicatoria

ABSTRACT Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion (TTS) system which translocates bacterial effector proteins into the plant cell. Previous transcriptome analysis identified a genome-wide regulon of putative virulence genes that are coexpressed with the TTS system. In this study, we characterized two of these genes, xopC and xopJ. Both genes encode Xanthomonas outer proteins (Xops) that were shown to be secreted by the TTS system. In addition, type III-dependent translocation of both proteins into the plant cell was demonstrated using the AvrBs3 effector domain as a reporter. XopJ belongs to the AvrRxv/YopJ family of effector proteins from plant and animal pathogenic bacteria. By contrast, XopC does not share significant homology to proteins in the database. Sequence analysis revealed that the xopC locus contains several features that are reminiscent of pathogenicity islands. Interestingly, the xopC region is flanked by 62-bp inverted repeats that are also associated with members of the Xanthomonas avrBs3 effector family. Besides xopC, a second gene of the locus, designated hpaJ, was shown to be coexpressed with the TTS system. hpaJ encodes a protein with similarity to transglycosylases and to the Pseudomonas syringae pv. maculicola protein HopPmaG. HpaJ secretion and translocation by the X. campestris pv. vesicatoria TTS system was not detectable, which is consistent with its predicted Sec signal and a putative function as transglycosylase in the bacterial periplasm.

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Expression Levels of avrBs3-Like Genes Affect Recognition Specificity in Tomato Bs4- But Not in Pepper Bs3-Mediated Perception

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-1215 ◽

2005 ◽

Vol 18 (11) ◽

pp. 1215-1225 ◽

Cited By ~ 27

Author(s):

Sebastian Schornack ◽

Kristin Peter ◽

Ulla Bonas ◽

Thomas Lahaye

Keyword(s):

Xanthomonas Campestris ◽

Transcript Abundance ◽

Effector Proteins ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Disease Resistance Gene ◽

In Planta ◽

Type Iii ◽

Recognition Specificity ◽

Bacterial Type

The tomato Bs4 disease resistance gene mediates recognition of avrBs4-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria to give a hypersensitive response (HR). Here, we present the characterization of the Bs4 promoter and its application for lowlevel expression of bacterial type III effector proteins in planta. Real-time polymerase chain reaction showed that Bs4 is constitutively expressed at low levels and that transcript abundance does not change significantly upon infection with avrBs4-containing xanthomonads. A 302-bp promoter fragment was found to be sufficient to promote Bs4 gene function. Previous studies have shown that high, constitutive in planta expression of avrBs3 (AvrBs3 and AvrBs4 proteins are 96.6% identical) via the Cauliflower mosaic virus 35S (35S) promoter triggers a Bs4-dependent HR whereas X. campestris pv. vesicatoria-mediated delivery of AvrBs3 into the plant cytoplasm does not. Here, we demonstrate that, when expressed under control of the weak Bs4 promoter, avrBs3 does not trigger a Bs4-dependent HR whereas avrBs4 does. In contrast, the pepper Bs3 gene, which mediates recognition of AvrBs3- but not AvrBs4- delivering xanthomonads, retains its recognition specificity even if avrBs4 was expressed in planta from the strong 35S promoter. Importantly, Bs4 promoter-driven expression of hax3, hax4 (two recently isolated avrBs3-like genes), avrBs3, and avrBs4 resulted in identical reactions as observed upon infection with X. campestris pv. vesicatoria strains that express the respective avr gene, suggesting that the protein levels expressed under control of the Bs4 promoter are similar to those that are translocated by the bacterial type III secretion system.

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

Journal of Bacteriology ◽

10.1128/jb.183.19.5589-5598.2001 ◽

2001 ◽

Vol 183 (19) ◽

pp. 5589-5598 ◽

Cited By ~ 100

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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Naturally Occurring Nonpathogenic Isolates of the Plant Pathogen Pseudomonas syringae Lack a Type III Secretion System and Effector Gene Orthologues

Journal of Bacteriology ◽

10.1128/jb.01757-07 ◽

2008 ◽

Vol 190 (8) ◽

pp. 2858-2870 ◽

Cited By ~ 53

Author(s):

Toni J. Mohr ◽

Haijie Liu ◽

Shuangchun Yan ◽

Cindy E. Morris ◽

José A. Castillo ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Plant Cells ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Plant Diseases ◽

In Planta ◽

Type Iii ◽

Effector Gene

ABSTRACT Pseudomonas syringae causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of P. syringae isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic P. syringae ancestor through loss of the T3SS. In the nonpathogenic isolate P. syringae 508 the genomic region that in pathogenic P. syringae strains contains the hrp-hrc cluster coding for the T3SS and flanking effector genes is absent. P. syringae 508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain P. syringae pv. syringae B728a, but none were detected. The absence of the hrp-hrc cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of P. syringae 508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the P. syringae pv. syringae 61 effector gene hopA1 increased in planta growth almost 10-fold. This suggests that P. syringae 508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.

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