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

2008 ◽  
Vol 190 (8) ◽  
pp. 2858-2870 ◽  
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.

scholarly journals Pseudomonas syringae Strains Naturally Lacking the Classical P. syringae hrp/hrc Locus Are Common Leaf Colonizers Equipped with an Atypical Type III Secretion System

2010 ◽  
Vol 23 (2) ◽  
pp. 198-210 ◽  
Author(s):  
Christopher R. Clarke ◽  
Rongman Cai ◽  
David J. Studholme ◽  
David S. Guttman ◽  
Boris A. Vinatzer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Ice Nucleation ◽  
Secretion System ◽  
Effector Proteins ◽  
Population Densities ◽  
Type Iii ◽  
Effector Genes

Pseudomonas syringae is best known as a plant pathogen that causes disease by translocating immune-suppressing effector proteins into plant cells through a type III secretion system (T3SS). However, P. syringae strains belonging to a newly described phylogenetic subgroup (group 2c) are missing the canonical P. syringae hrp/hrc cluster coding for a T3SS, flanking effector loci, and any close orthologue of known P. syringae effectors. Nonetheless, P. syringae group 2c strains are common leaf colonizers and grow on some tested plant species to population densities higher than those obtained by other P. syringae strains on nonhost species. Moreover, group 2c strains have genes necessary for the production of phytotoxins, have an ice nucleation gene, and, most interestingly, contain a novel hrp/hrc cluster, which is only distantly related to the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster appears to encode a functional T3SS although the genes hrpK and hrpS, present in the classical P. syringae hrp/hrc cluster, are missing. The genome sequence of a representative group 2c strain also revealed distant orthologues of the P. syringae effector genes avrE1 and hopM1 and the P. aeruginosa effector genes exoU and exoY. A putative life cycle for group 2c P. syringae is discussed.

scholarly journals Identification of Pseudomonas syringae pv. syringae 61 Type III Secretion System Hrp Proteins That Can Travel the Type III Pathway and Contribute to the Translocation of Effector Proteins into Plant Cells

2007 ◽  
Vol 189 (15) ◽  
pp. 5773-5778 ◽  
Author(s):  
Adela R. Ramos ◽  
Joanne E. Morello ◽  
Sandeep Ravindran ◽  
Wen-Ling Deng ◽  
Hsiou-Chen Huang ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iii

ABSTRACT Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.

scholarly journals Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

2004 ◽  
Vol 186 (2) ◽  
pp. 543-555 ◽  
Author(s):  
Lisa M. Schechter ◽  
Kathy A. Roberts ◽  
Yashitola Jamir ◽  
James R. Alfano ◽  
Alan Collmer
Keyword(s):  
Amino Acids ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Reporter System ◽  
Aliphatic Amino Acid ◽  
Type Iii

ABSTRACT Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphEPto are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.

scholarly journals Pseudomonas syringae Lytic Transglycosylases Coregulated with the Type III Secretion System Contribute to the Translocation of Effector Proteins into Plant Cells

2007 ◽  
Vol 189 (22) ◽  
pp. 8277-8289 ◽  
Author(s):  
Hye-Sook Oh ◽  
Brian H. Kvitko ◽  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Dominant Negative Effect ◽  
Lytic Transglycosylases ◽  
Type Iii

ABSTRACT Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH1-241. DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH1-241 variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection.

scholarly journals Components of the Pseudomonas syringae Type III Secretion System Can Suppress and May Elicit Plant Innate Immunity

2010 ◽  
Vol 23 (6) ◽  
pp. 727-739 ◽  
Author(s):  
Hye-Sook Oh ◽  
Duck Hwan Park ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Leaf Tissue ◽  
Secretion System ◽  
Colony Development ◽  
Plant Responses ◽  
In Planta ◽  
Callose Deposition ◽  
Type Iii

The type III secretion system (T3SS) of Pseudomonas syringae translocates into plant cells multiple effectors that suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). P. syringae pv. tomato DC3000 no longer delivers the T3SS translocation reporter AvrPto-Cya in Nicotiana benthamiana leaf tissue in which PTI was induced by prior inoculation with P. fluorescens(pLN18). Cosmid pLN18 expresses the T3SS system of P. syringae pv. syringae 61 but lacks the hopA1Psy61 effector gene. P. fluorescens(pLN18) expressing HrpHPtoDC3000 or HopP1PtoDC3000, two T3SS-associated putative lytic transglycosylases, suppresses PTI, based on multiple assays involving DC3000 challenge inoculum (AvrPto-Cya translocation, hypersensitive response elicitation, and colony development in planta) or on plant responses (vascular dye uptake or callose deposition). Analysis of additional mutations in pHIR11 derivatives revealed that the pLN18-encoded T3SS elicits a higher level of reactive oxygen species (ROS) than does P. fluorescens without a T3SS, that enhanced ROS production is dependent on the HrpK1 translocator, and that HopA1Psy61 suppresses ROS elicitation attributable to both the P. fluorescens PAMPs and the presence of a functional T3SS.

scholarly journals 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

2004 ◽  
Vol 186 (11) ◽  
pp. 3621-3630 ◽  
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.

scholarly journals Pseudomonas syringae Exchangeable Effector Loci: Sequence Diversity in Representative Pathovars and Virulence Function in P. syringae pv. syringae B728a

2003 ◽  
Vol 185 (8) ◽  
pp. 2592-2602 ◽  
Author(s):  
Wen-Ling Deng ◽  
Amos H. Rehm ◽  
Amy O. Charkowski ◽  
Clemencia M. Rojas ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Brown Spot ◽  
Dependent Manner ◽  
Type Iii ◽  
Effector Genes

ABSTRACT Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL—HopPsyC, HopPsyE, and HopPsyV—were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.

scholarly journals Transcriptional Regulation of Components of the Type III Secretion System and Effectors in Pseudomonas syringae pv. phaseolicola

2004 ◽  
Vol 17 (11) ◽  
pp. 1250-1258 ◽  
Author(s):  
R. Thwaites ◽  
P. D. Spanu ◽  
N. J. Panopoulos ◽  
C. Stevens ◽  
J. W. Mansfield
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cell Wall ◽  
Type Iii Secretion System ◽  
Regulatory Elements ◽  
Secretion System ◽  
Mrna Levels ◽  
In Planta ◽  
Type Iii ◽  
Effector Genes

Quantitative real-time polymerase chain reaction was used with specific TaqMan probes to examine transcription of selected hrp and effector genes in Pseudomonas syringae pv. phaseolicola strains 1448A (race 6) and 1449B (race 7). Transcripts examined were from genes encoding the regulators hrpR and hrpL, core structural components of the type III secretion system (TTSS) hrcC, hrcJ, hrcN, hrcU, and hrpA; the first open-reading frame of each hrp operon, including hrpF, hrpJ, hrpP, and hrpY; and also secreted effectors hrpZ, avrPphE, avrPphF, and virPphA. All genes were induced by incubation in a minimal medium and showed patterns of expression indicating regulation by HrpRS and HrpL. Basal mRNA levels and the timing of accumulation of transcripts after induction differed significantly, suggesting the operation of additional regulatory elements. However, no clear transcriptional hierarchy emerged to explain the ordered construction of the TTSS. Quantitative analysis confirmed that the rates and levels of transcript accumulation within the first 2 h after inoculation were considerably higher in planta than in vitro, and indicated that plant cell wall contact may enhance transcription of TTSS and effector genes in P. syringae pv. phaseolicola. The low-abundance hrcU mRNA had a half-life of 16.5 min, whereas other transcripts had half-lives between 3 and 8 min.

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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