Inhibitory Activity of Sedum middendorffianum-Derived 4-Hydroxybenzoic Acid and Vanillic Acid on the Type III Secretion System of Pseudomonas syringae pv. tomato DC3000

The injection of nearly 30 effector proteins by the type III secretion system underlies the ability of Pseudomonas syringae pv. tomato DC3000 to cause disease in tomato and other host plants. The search for effector functions is complicated by redundancy within the repertoire and by plant resistance (R)-gene sentinels, which may convert effector virulence activities into a monolithic defense response. On the premise that some effectors target universal eukaryotic processes and that yeast (Saccharomyces cerevisiae) lacks R genes, the DC3000 effector repertoire was expressed in yeast. Of 27 effectors tested, HopAD1, HopAO1, HopD1, HopN1, and HopU1 were found to inhibit growth when expressed from a galactose-inducible GAL1 promoter, and HopAA1-1 and HopAM1 were found to cause cell death. Catalytic site mutations affecting the tyrosine phosphatase activity of HopAO1 and the cysteine protease activity of HopN1 prevented these effectors from inhibiting yeast growth. Expression of HopAA1-1, HopAM1, HopAD1, and HopAO1 impaired respiration in yeast, as indicated by tests with ethanol glycerol selective media. HopAA1-1 colocalized with porin to yeast mitochondria and was shown to cause cell death in yeast and plants in a domain-dependent manner. These results support the use of yeast for the study of plant-pathogen effector repertoires.

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Identification of Harpins in Pseudomonas syringae pv. tomato DC3000, Which Are Functionally Similar to HrpK1 in Promoting Translocation of Type III Secretion System Effectors

Journal of Bacteriology ◽

10.1128/jb.01146-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8059-8072 ◽

Cited By ~ 88

Author(s):

Brian H. Kvitko ◽

Adela R. Ramos ◽

Joanne E. Morello ◽

Hye-Sook Oh ◽

Alan Collmer

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Pectate Lyase ◽

Secretion System ◽

Tomato Dc3000 ◽

Type Iii ◽

Lytic Transglycosylase ◽

T3ss Effector ◽

Protein Classes

ABSTRACT Harpins are a subset of type III secretion system (T3SS) substrates found in all phytopathogenic bacteria that utilize a T3SS. Pseudomonas syringae pv. tomato DC3000 was previously reported to produce two harpins, HrpZ1 and HrpW1. DC3000 was shown here to deploy two additional proteins, HopAK1 and HopP1, which have the harpin-like properties of lacking cysteine, eliciting the hypersensitive response (HR) when partially purified and infiltrated into tobacco leaves, and possessing a two-domain structure similar to that of the HrpW1 class of harpins. Unlike the single-domain harpin HrpZ1, the two-domain harpins have C-terminal enzyme-like domains: pectate lyase for HopAK1 and lytic transglycosylase for HopP1. Genetic techniques to recycle antibiotic markers were applied to DC3000 to generate a quadruple harpin gene polymutant. The polymutant was moderately reduced in the elicitation of the HR and translocation of the T3SS effector AvrPto1 fused to a Cya translocation reporter, but the mutant was unaffected in the secretion of AvrPto1-Cya. The DC3000 hrpK1 gene encodes a putative translocator in the HrpF/NopX family and was deleted in combination with the four harpin genes. The hrpK1 quadruple harpin gene polymutant was strongly reduced in HR elicitation, virulence, and translocation of AvrPto1-Cya into plant cells but not in the secretion of representative T3SS substrates in culture. HrpK1, HrpZ1, HrpW1, and HopAK1, but not HopP1, were independently capable of restoring some HR elicitation to the hrpK1 quadruple harpin gene polymutant, which suggests that a consortium of semiredundant translocators from three protein classes cooperate to form the P. syringae T3SS translocon.

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Carbon source and cell density-dependent regulation of type III secretion system gene expression in Pseudomonas syringae pathovar tomato DC3000

Research in Microbiology ◽

10.1016/j.resmic.2012.08.005 ◽

2012 ◽

Vol 163 (8) ◽

pp. 531-539 ◽

Cited By ~ 20

Author(s):

Jennifer L. Stauber ◽

Ekaterina Loginicheva ◽

Lisa M. Schechter

Keyword(s):

Gene Expression ◽

Carbon Source ◽

Cell Density ◽

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Tomato Dc3000 ◽

Density Dependent ◽

Type Iii

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Genomic Mining for Substrates of the Type III Secretion System of Pseudomonas syringae pv. tomato DC3000: New Insights into Mechanisms of Pathogenesis

Pseudomonas syringae and related pathogens ◽

10.1007/978-94-017-0133-4_39 ◽

2003 ◽

pp. 363-372

Author(s):

J. R. Alfano ◽

C. R. Buell ◽

S. T. Chancey ◽

A. Collmer ◽

A. Espinosa ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Tomato Dc3000 ◽

Type Iii

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Closing the Circle on the Discovery of Genes Encoding Hrp Regulon Members and Type III Secretion System Effectors in the Genomes of Three Model Pseudomonas syringae Strains

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-1151 ◽

2006 ◽

Vol 19 (11) ◽

pp. 1151-1158 ◽

Cited By ~ 104

Author(s):

Magdalen Lindeberg ◽

Samuel Cartinhour ◽

Christopher R. Myers ◽

Lisa M. Schechter ◽

David J. Schneider ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Active Member ◽

Tomato Dc3000 ◽

Type Iii ◽

Effector Genes ◽

Genes Encoding

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.

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Ca 2+ -Induced Two-Component System CvsSR Regulates the Type III Secretion System and the Extracytoplasmic Function Sigma Factor AlgU in Pseudomonas syringae pv. tomato DC3000

Journal of Bacteriology ◽

10.1128/jb.00538-17 ◽

2017 ◽

Vol 200 (5) ◽

Cited By ~ 11

Author(s):

Maxwell R. Fishman ◽

Johnson Zhang ◽

Philip A. Bronstein ◽

Paul Stodghill ◽

Melanie J. Filiatrault

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Dependent Manner ◽

Tomato Dc3000 ◽

Content Type ◽

Type Iii ◽

Two Component ◽

Two Component Systems

ABSTRACT 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 characterized a TCS in the plant-pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 composed of the histidine kinase CvsS and the response regulator CvsR. CvsSR is necessary for virulence of P. syringae pv. tomato DC3000, since Δ cvsS and Δ cvsR strains produced fewer symptoms than the wild type (WT) and demonstrated reduced growth on multiple hosts. We discovered that expression of cvsSR is induced by Ca 2+ concentrations found in leaf apoplastic fluid. Thus, Ca 2+ can be added to the list of signals that promote pathogenesis of P. syringae pv. tomato DC3000 during host colonization. Through chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and global transcriptome analysis (RNA-seq), we discerned the CvsR regulon. CvsR directly activated expression of the type III secretion system regulators, hrpR and hrpS , that regulate P. syringae pv. tomato DC3000 virulence in a type III secretion system-dependent manner. CvsR also indirectly repressed transcription of the extracytoplasmic sigma factor algU and production of alginate. Phenotypic analysis determined that CvsSR inversely regulated biofilm formation, swarming motility, and cellulose production in a Ca 2+ -dependent manner. Overall, our results show that CvsSR is a key regulatory hub critical for interaction with host plants. IMPORTANCE Pathogenic bacteria must be able to react and respond to the surrounding environment, make use of available resources, and avert or counter host immune responses. Often, these abilities rely on two-component systems (TCSs) composed of interacting proteins that modulate gene expression. We identified a TCS in the plant-pathogenic bacterium Pseudomonas syringae that responds to the presence of calcium, which is an important signal during the plant defense response. We showed that when P. syringae is grown in the presence of calcium, this TCS regulates expression of factors contributing to disease. Overall, our results provide a better understanding of how bacterial pathogens respond to plant signals and control systems necessary for eliciting disease.

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Multiple Approaches to a Complete Inventory of Pseudomonas syringae pv. tomato DC3000 Type III Secretion System Effector Proteins

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-1180 ◽

2006 ◽

Vol 19 (11) ◽

pp. 1180-1192 ◽

Cited By ~ 79

Author(s):

Lisa M. Schechter ◽

Monica Vencato ◽

Katy L. Jordan ◽

Sarah E. Schneider ◽

David J. Schneider ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Complex Mixture ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Dependent Manner ◽

Tomato Dc3000 ◽

Type Iii

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that translocates virulence effector proteins into host cells via a type III secretion system (T3SS). Many effector-encoding hypersensitive response and pathogenicity (Hrp) outer protein (hop) genes have been identified previously in DC3000 using bioinformatic methods based on Hrp promoter sequences and characteristic N-terminal amino acid patterns that are associated with T3SS substrates. To approach completion of the Hop/effector inventory in DC3000, 44 additional candidates were tested by the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter assay; 10 of the high-probability candidates were confirmed as T3SS substrates. Several previously predicted hop genes were tested for their ability to be expressed in an HrpL-dependent manner in culture or to be expressed in planta. The data indicate that DC3000 harbors 53 hop/avr genes and pseudogenes (encoding both injected effectors and T3SS substrates that probably are released to the apoplast); 33 of these genes are likely functional in DC3000, 12 are nonfunctional members of valid Hop families, and 8 are less certain regarding their production at functional levels. Growth of DC3000 in tomato and Arabidopsis Col-0 was not impaired by constitutive expression of repaired versions of two hops that were disrupted naturally by transposable elements or of hop genes that are naturally cryptic. In summary, DC3000 carries a complex mixture of active and inactive hop genes, and the hop genes in P. syringae can be identified efficiently by bioinformatic methods; however, a precise inventory of the subset of Hops that are important in pathogenesis awaits more knowledge based on mutant phenotypes and functions within plants.

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