Deletions in the Repertoire of Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap among Effectors

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.

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Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay between HopAD1 and AvrPtoB

Cell Host & Microbe ◽

10.1016/j.chom.2015.05.007 ◽

2015 ◽

Vol 17 (6) ◽

pp. 752-762 ◽

Cited By ~ 48

Author(s):

Hai-Lei Wei ◽

Suma Chakravarthy ◽

Johannes Mathieu ◽

Tyler C. Helmann ◽

Paul Stodghill ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Tomato Dc3000 ◽

Type Iii ◽

Type Iii Secretion Effector

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-2-0198 ◽

2010 ◽

Vol 23 (2) ◽

pp. 198-210 ◽

Cited By ~ 69

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.

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A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

Molecular Plant Pathology ◽

10.1111/mpp.12876 ◽

2019 ◽

Vol 21 (1) ◽

pp. 139-144 ◽

Cited By ~ 4

Author(s):

Megan R. O’Malley ◽

Ching‐Fang Chien ◽

Scott C. Peck ◽

Nai‐Chun Lin ◽

Jeffrey C. Anderson

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Tomato Dc3000 ◽

Type Iii

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Inhibition of the type III secretion system of Pseudomonas syringae pv. tomato DC3000 by resveratrol oligomers identified in Vitis vinifera L

Pest Management Science ◽

10.1002/ps.5764 ◽

2020 ◽

Vol 76 (7) ◽

pp. 2294-2303 ◽

Cited By ~ 2

Author(s):

Ji Eun Kang ◽

Byeong Jun Jeon ◽

Min Young Park ◽

Hye Ji Yang ◽

Jaeyoung Kwon ◽

...

Keyword(s):

Vitis Vinifera ◽

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Vitis Vinifera L ◽

Tomato Dc3000 ◽

Type Iii

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A Survey of the Pseudomonas syringae pv. tomato DC3000 Type III Secretion System Effector Repertoire Reveals Several Effectors That Are Deleterious When Expressed in Saccharomyces cerevisiae

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-4-0490 ◽

2008 ◽

Vol 21 (4) ◽

pp. 490-502 ◽

Cited By ~ 34

Author(s):

Kathy R. Munkvold ◽

Michael E. Martin ◽

Philip A. Bronstein ◽

Alan Collmer

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Dependent Manner ◽

Tomato Dc3000 ◽

Type Iii ◽

Pathogen Effector

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.5.447 ◽

2004 ◽

Vol 17 (5) ◽

pp. 447-455 ◽

Cited By ~ 22

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.

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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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.1.43 ◽

2003 ◽

Vol 16 (1) ◽

pp. 43-52 ◽

Cited By ~ 29

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.

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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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