scholarly journals Molecular Determinants Required for the Avirulence Function of AvrPphB in Bean and Other Plants

2002 ◽  
Vol 15 (3) ◽  
pp. 292-300 ◽  
Author(s):  
Anastasia P. Tampakaki ◽  
Marina Bastaki ◽  
John W. Mansfield ◽  
Nickolas J. Panopoulos
Keyword(s):  
Pseudomonas Syringae ◽  
Potato Virus X ◽  
Hypersensitive Reaction ◽  
Avirulence Gene ◽  
In Planta ◽  
Mutant Proteins ◽  
Type Iii ◽  
Amino Terminal ◽  
Plant Recognition ◽  
Effector Recognition

The avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola determines incompatibility, manifested as a hypersensitive reaction (HR), on bean cultivars carrying the R3 resistance gene and also confers avirulence on other plants. The AvrPphB protein carries an embedded consensus myristoylation motif and is cleaved in bacteria and certain plants to yield fragments of about 6 and 28 kDa. We investigated plant recognition and type III translocation determinants in AvrPphB by constructing three N-terminally truncated and two site-directed mutants carrying substitutions in the conserved G63 residue of the myristoy-lation motif, which lies adjacent to the proteolytic cleavage site. The peptides were either delivered to plant cells by pseudomonads or were expressed transiently in planta via the Agrobacterium tumefaciens or Potato virus X. The 63 amino terminal residues were required for type III-mediated translocation from Pseudomonas strains to the plant, but were partially dispensable for effector recognition following in planta expression. Substitution of the G63 residue resulted in differential HR phenotypes in two different R3 cultivars of bean and abolished effector processing in Pseudomonas strains. Agrobacterium-mediated expression of the mutant proteins elicited HR in resistant bean hosts and in tomato but elicited no reaction in Nicotiana species.

scholarly journals Confocal Imaging of Pseudomonas syringae pv. phaseolicola Colony Development in Bean Reveals Reduced Multiplication of Strains Containing the Genomic Island PPHGI-1

2010 ◽  
Vol 23 (10) ◽  
pp. 1294-1302 ◽  
Author(s):  
S. A. C. Godfrey ◽  
J. W. Mansfield ◽  
D. S. Corry ◽  
H. C. Lovell ◽  
R. W. Jackson ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Genomic Island ◽  
Effective Means ◽  
Leaf Tissue ◽  
Hypersensitive Reaction ◽  
Confocal Imaging ◽  
Avirulence Gene ◽  
Colony Development ◽  
Bacterial Survival ◽  
In Planta

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

Identification of a DNA region required for growth of Pseudomonas syringae pv. tomato on tomato plants

1992 ◽  
Vol 38 (9) ◽  
pp. 883-890 ◽  
Author(s):  
Dennis P. Jackson ◽  
Douglas A. Gray ◽  
Vincent L. Morris ◽  
Diane A. Cuppels
Keyword(s):  
Organic Acids ◽  
Pseudomonas Syringae ◽  
Acid Metabolism ◽  
Carbon Sources ◽  
Hypersensitive Reaction ◽  
Wild Type ◽  
In Planta ◽  
Tomato Plants ◽  
Tartaric Acids ◽  
Nonpathogenic Strain

The prototrophic Pseudomonas syringae pv. tomato mutant DC3481, which is the result of a single-site Tn5 insertion, cannot grow and cause disease on tomato plants and cannot use the major organic acids of tomato, i.e., citric, malic, succinic, and tartaric acids, as sole carbon sources. Although nonpathogenic, strain DC3481 can still induce a hypersensitive reaction in nonhost plants. We have identified a 30-kb fragment of P. syringae pv. tomato wild-type DNA that can complement this mutant. EcoRI fragments from this region were subcloned and individually subjected to functional complementation analysis. The 3.8-kb fragment, which was the site of the Tn5 insertion, restored pathogenicity and the ability to use all the major organic acids of tomato as carbon sources. It shares sequence homology with several P. syringae pathovars but not other bacterial tomato pathogens. Our results indicate that sequences on the 3.8-kb EcoRI fragment are required for both the ability to grow on tomato leaves (and thus cause disease) and the utilization of carboxylic acids common to tomato. The 3.8-kb fragment may contain a sequence (or sequences) that regulates both traits. Key words: Pseudomonas syringae pv. tomato, phytopathogenicity, Tn5, tricarboxylic acid metabolism, bacterial speck, growth in planta.

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

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.

scholarly journals Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity

2016 ◽  
Vol 29 (8) ◽  
pp. 651-660 ◽  
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.

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

2012 ◽  
Vol 25 (7) ◽  
pp. 941-953 ◽  
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.

scholarly journals Immunogold Labeling of Hrp Pili of Pseudomonas syringae pv. tomato Assembled in Minimal Medium and In Planta

2001 ◽  
Vol 14 (2) ◽  
pp. 234-241 ◽  
Author(s):  
Wenqi Hu ◽  
Jing Yuan ◽  
Qiao-Ling Jin ◽  
Patrick Hart ◽  
Sheng Yang He
Keyword(s):  
Arabidopsis Thaliana ◽  
Pseudomonas Syringae ◽  
Minimal Medium ◽  
Leaf Tissue ◽  
Hypersensitive Reaction ◽  
Immunogold Labeling ◽  
Structural Protein ◽  
In Planta ◽  
Hrp Genes

Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.

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

2002 ◽  
Vol 184 (5) ◽  
pp. 1340-1348 ◽  
Author(s):  
Laurent Noël ◽  
Frank Thieme ◽  
Dirk Nennstiel ◽  
Ulla Bonas
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
In Planta ◽  
Type Iii ◽  
Reverse Transcription Pcr ◽  
New Genes ◽  
Type Iii Protein Secretion

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.

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

2009 ◽  
Vol 22 (9) ◽  
pp. 1069-1080 ◽  
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.

scholarly journals Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean

1997 ◽  
Vol 10 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Nakul Puri ◽  
Carol Jenner ◽  
Mark Bennett ◽  
Ruth Stewart ◽  
John Mansfield ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Induction Time ◽  
Phenotypic Expression ◽  
Constitutive Expression ◽  
Hypersensitive Reaction ◽  
Full Length ◽  
Avirulence Gene ◽  
Over Expression ◽  
E Coli ◽  
Induction Kinetics

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY - strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

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