Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 Family Member, Is Needed To Produce l-allo-Isoleucine, a Precursor for the Phytotoxin Coronatine

ABSTRACTPseudomonas syringaepv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plantNicotiana benthamianain a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed inN. benthamianawithcfa,cma, andcmaLmutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either thecmaoperon orcmaLaccumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered incmaLmutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaLmutant when it is supplemented with 50 μg/mll-allo-isoleucine, the starting unit for CMA production.cmaLis found in all other sequencedP. syringaestrains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.

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The awr Gene Family Encodes a Novel Class of Ralstonia solanacearum Type III Effectors Displaying Virulence and Avirulence Activities

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-11-0321 ◽

2012 ◽

Vol 25 (7) ◽

pp. 941-953 ◽

Cited By ~ 44

Author(s):

Montserrat Solé ◽

Crina Popa ◽

Oriane Mith ◽

Kee Hoon Sohn ◽

Jonathan D. G. Jones ◽

...

Keyword(s):

Gene Family ◽

Pseudomonas Syringae ◽

Ralstonia Solanacearum ◽

Tomato Dc3000 ◽

Type Iii Effectors ◽

Type Iii ◽

New Gene ◽

Transient Overexpression ◽

Effector Recognition

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophan-arginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (Δawr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts.

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Identification and Characterization of a Well-Defined Series of Coronatine Biosynthetic Mutants of Pseudomonas syringae pv. tomato DC3000

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.2.162 ◽

2004 ◽

Vol 17 (2) ◽

pp. 162-174 ◽

Cited By ~ 137

Author(s):

David M. Brooks ◽

Gustavo Hernández-Guzmán ◽

Andrew P. Kloek ◽

Francisco Alarcón-Chaidez ◽

Aswathy Sreedharan ◽

...

Keyword(s):

Pseudomonas Syringae ◽

High Performance ◽

Tomato Dc3000 ◽

Disease Symptoms ◽

Coronafacic Acid ◽

Tn5 Mutants ◽

Cor Genes ◽

Identification And Characterization

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.

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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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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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Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors

Molecular Plant Pathology ◽

10.1111/mpp.12655 ◽

2018 ◽

Vol 19 (7) ◽

pp. 1779-1794 ◽

Cited By ~ 11

Author(s):

Hai-Lei Wei ◽

Alan Collmer

Keyword(s):

Pseudomonas Syringae ◽

Tomato Dc3000 ◽

Type Iii Effectors ◽

Type Iii ◽

Plant Pathogenesis

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A Draft Genome Sequence of Pseudomonas syringae pv. tomato T1 Reveals a Type III Effector Repertoire Significantly Divergent from That of Pseudomonas syringae pv. tomato DC3000

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-1-0052 ◽

2009 ◽

Vol 22 (1) ◽

pp. 52-62 ◽

Cited By ~ 101

Author(s):

Nalvo F. Almeida ◽

Shuangchun Yan ◽

Magdalen Lindeberg ◽

David J. Studholme ◽

David J. Schneider ◽

...

Keyword(s):

Host Range ◽

Host Specificity ◽

Pseudomonas Syringae ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Tomato Dc3000 ◽

Type Iii ◽

Host Range Determination ◽

Range Determination

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system–independent factors also play a role in this interaction.

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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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Genome-Assisted Development of a Diagnostic Protocol for Distinguishing High Virulence Pseudomonas syringae pv. tomato Strains

Plant Disease ◽

10.1094/pdis-08-14-0833-re ◽

2015 ◽

Vol 99 (4) ◽

pp. 527-534 ◽

Cited By ~ 5

Author(s):

Lisa A. Jones ◽

Surya Saha ◽

Alan Collmer ◽

Christine D. Smart ◽

Magdalen Lindeberg

Keyword(s):

New York ◽

Pseudomonas Syringae ◽

Genome Comparison ◽

Type Iii Effectors ◽

Type Iii ◽

Host Interaction ◽

Group I ◽

Virulent Strains ◽

High Virulence ◽

Relationship Of

A severe outbreak of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, occurred in central New York in 2009. Isolate 09150, collected from this outbreak and subsequently named NYS-T1, was found to be highly virulent on tomato. To better understand the relationship of 09150 to other P. syringae strains and develop a diagnostic assay for aggressive strains of this pathogen, the 09150 genome was sequenced. Genome comparison revealed it to be highly similar to a previously sequenced isolate, T1. Genetic factors linked to host interaction including type III effectors, toxin biosynthetic genes, and elicitors of host innate immunity were identified. Type III effector repertoires were compared with other strains in the high virulence T1-like subgroup and lower virulence DC3000/P. syringae pv. maculicola subgroup within P. syringae phylogenetic Group I. Primers for conventional PCR were developed using sequences for avrA, hopW, conserved in the former subgroup and hopN, present in the latter. These were tested on isolates in the two subgroups, other pseudomonads, and other bacterial pathogens of tomato. Primers developed for avaA and hopW were diagnostic for more virulent strains of P. syringae pv. tomato while primers for hopN were diagnostic for P. syringae pv. tomato DC3000 and related P. syringe pv. maculicola strains. Primers designed against hopR distinguished both of these P. syringae subgroups from other P. syringae strains.

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