119 research outputs found
Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity
No full textBackground: Microbe-associated molecular patterns, such as those present in bacterial flagellin, are powerful inducers of the innate immune response in plants. Successful pathogens deliver virulence proteins, termed effectors, into the plant cell where they can interfere with the immune response and promote disease. Engineering the plant immune system to enhance disease resistance requires a thorough understanding of its components. Results: We describe a high-throughput screen, using RNA sequencing and virus-induced gene silencing, to identify tomato genes whose expression is enhanced by the flagellin microbe-associated molecular pattern flgII-28, but reduced by activities of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrPto and AvrPtoB. Gene ontology terms for this category of Flagellin-induced repressed by effectors (FIRE) genes showed enrichment for genes encoding certain subfamilies of protein kinases and transcription factors. At least 25 of the FIRE genes have been implicated previously in plant immunity. Of the 92 protein kinase-encoding FIRE genes, 33 were subjected to virus-induced gene silencing and their involvement in pattern-triggered immunity was tested with a leaf-based assay. Silencing of one FIRE gene, which encodes the cell wall-associated kinase SlWAK1, compromised the plant immune response resulting in increased growth of Pst and enhanced disease symptoms. Conclusions: Our transcriptomic approach identifies FIRE genes that represent a pathogen-defined core set of immune-related genes. The analysis of this set of candidate genes led to the discovery of a cell wall-associated kinase that participates in plant defense. The FIRE genes will be useful for further elucidation of the plant immune system.Fil: Rosli, Hernan Guillermo. Boyce Thompson Institute for Plant Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Zhen, Yi. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Pombo, Marina Alejandra. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Zhong, Silin. Boyce Thompson Institute for Plant Research; Estados Unidos. University of Hong Kong; Hong KongFil: Bombarely, Aureliano. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Fei, Zhangjun. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Collmer, Alan. Cornell University; Estados Unidos. King Abdulaziz University; Estados UnidosFil: Martin, Gregory B.. Boyce Thompson Institute for Plant Research; Estados Unidos. Cornell University; Estados Unidos. King Abdulaziz University; Estados Unido
The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system
No full textErwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (\u3e50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1.61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrp/hrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector
DEFENSE AND COUNTER-DEFENSE IN THE RICE-XANTHOMONAS PATHOSYSTEM
No full text215 pagesPlants have multi-layered immune systems that survey for and respond to pathogens. Successful pathogens subvert these defenses. This defense and counter-defense dynamic leads to a molecular arms race characterized by rapid evolution of plant immunity and pathogen virulence proteins. Pathogens from the bacterial genus Xanthomonas deploy virulence proteins called transcription activator-like (TAL) effectors. TAL effectors comprise DNA binding and gene activation modules and are able to activate specific host genes. Characterized strains of rice-infecting Xanthomonas oryzae encode as many as 26 TAL effectors, with distinct specificities. Hijacking host transcription is a powerful virulence mechanism, however bacterial proteins delivered into the plant cell are at risk of being detected by host defense proteins. Carolina Gold Select rice has an immunity gene, Xo1, that recognizes TAL effectors and prevents disease. Interestingly, this variety is susceptible to some strains of Xanthomonas oryzae, suggesting a second, unidentified counter-defense pathogen protein. Here, I identify and study a class of Xanthomonas counter-defense proteins encoded by genes originally overlooked as TAL effector pseudogenes due to deletions in the N- and C-terminal coding regions. In fact, these truncated TAL effectors (truncTALEs) are non-DNA-binding virulence factors that suppress Xo1-mediated plant immunity. Further, using long-read sequencing a high-quality genome assembly of Carolina Gold Select was generated. Fourteen nucleotide binding and leucine-rich repeat (NLR) defense protein genes were identified at the Xo1 locus. Seven of these Fourteen are expressed in the presence of the pathogen, and one was a strong Xo1 candidate based on structural similarity to the functionally similar Xa1 gene. A broad comparison of NLRs revealed the presence of Xa1-like genes in diverse Oryzae species. Next, by expressing the Xo1 candidate as a transgene in a susceptible variety and observing resistance, we confirmed it to be Xo1. We showed that, like TAL effectors and truncTALEs, the Xo1 protein localizes to the nucleus. Xo1 co-immunoprecipitates with a tagged truncTALE, suggesting that suppression of Xo1-mediated defense is due to interaction of the two proteins
Gene Ontology (GO) for Microbe–Host Interactions and Its Use in Ongoing Annotation of Three Pseudomonas syringae Genomes via the Pseudomonas–Plant Interaction (PPI) Web Site
No full textCharacterization Of A Novel Effector From Blumeria Graminis F. Sp. Hordei That Suppress Host Cell Death
No full textThe interaction of barley, Hordeum vulgare L., with the powdery mildew fungus, Blumeria graminis f. sp. hordei, is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes ca. 540 predicted effectors, which are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a singlecopy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and Host-Induced-Gene-Silencing of BEC1019 restricts haustorial development in compatible interactions. Here we show that Barley stripe mosaic virusInduced Gene Silencing of BEC1019 significantly reduces fungal biomass in barley leaves, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar non-specific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 97 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and freeliving non-pathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle, and at least two highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR suppressing activity of BEC1019. When mutating another conserved motif, HRxxH, and delivered by Xanthomonas campestris pv. vesicatoria translocation mutant strain, BEC1019 failed to suppress cell death caused by Xoc, indicating that HRxxH may play a role in effector translocation
Investigation Of The Interconnected Roles Of Cmal And Hopaa1-1 In The Virulence Of Pseudomonas Syringae Pv.Tomato Dc3000 In Nicotiana Benthamiana
No full textPseudomonas syringae pv. tomato DC3000 (DC3000) is a model plant pathogenic bacterium that infects tomato and Arabidopsis thaliana. It requires the phytotoxin coronatine and the delivery of type III effector proteins (T3Es) into the host cell cytoplasm for defense suppression and virulence. CmaL is a small protein found to be necessary for coronatine production. Coronatine is a potent molecular mimic of jasmonoyl-isoleucine, a plant hormone conjugate involved in regulating plant defenses. Coronatine is constructed of two amide bond-linked moieties, coronafacic acid and coronamic acid. CmaL was shown to be required for the production of L-allo-isoleucine, a precursor for coronamic acid biosynthesis. DC3000 mutants lacking both cmaL and the T3E gene hopAA1-1 are reduced in speck formation in tomato. hopAA1-1 is member of the conserved effector locus, a group of effector genes located adjacent to the genes encoding the type three secretion apparatus that are widespread among P. syringae strains. HopAA1-1 is toxic to both plants and yeast upon expression within them. To gain insight into the basis for its toxicity in eukaryotic cells, the subcellular localization of HopAA1-1 was investigated. HopAA1-1 was found to colocalize with plant peroxisomes. Truncated derivatives of HopAA1-1 that are not cytotoxic and cannot promote symptom formation do not localize with peroxisomes. Additionally, other truncated derivatives of HopAA1-1 colocalize with the endoplasmic reticulum in addition to peroxisomes, suggesting that HopAA1 -1 interacts with the endomembrane system. A DC3000 mutant with 28 T3E genes deleted (DC3000D28E) is a recently developed tool for investigating effector functions. DC3000D28E derivatives with small sets of effector genes progressively restored show increasing virulence wh en inoculated by infiltration with a blunt syringe into the model plant Nicotiana benthamiana. Because of its location in a cluster of effector genes, cmaL was inadvertently deleted in the construction of DC3000D28E. The importance of coronatine and its partial redundancy with HopAA1-1 in promoting an early stage of pathogenesis was revealed by restoring cmaL and hopAA1-1 to selected DC3000D28E derivatives and assaying the strains by dip inoculation of N. benthamiana leaves, which requires bacteria to follow a natural infection route through stomata
Pamp-Triggered Immunity Components And Lysm-Receptor-Like Kinases
No full textIn order to identify components of Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) pathways in Nicotiana benthamiana, a largescale forward-genetics screen using virus-induced gene silencing and a celldeath-based assay for assessing PTI was performed. The assay relied on four combinations of PTI-inducing non-pathogens and cell-death-causing challenger pathogens, and was first validated in plants silenced for FLS2 or BAK1. Over 3,200 genes were screened and 14 genes were identified that, when silenced, compromised PTI. A subset of the genes was found to act downstream of FLS2mediated PTI induction and silencing of three genes compromised production of reactive oxygen species (ROS) in leaves exposed to flg22. The 14 genes encode proteins with potential functions in defense and hormone signaling, protein stability and degradation, energy and secondary metabolism and cell wall biosynthesis and provide a new resource to explore the molecular basis for the involvement of these processes in PTI. Peptidoglycan (PGN) has been shown to trigger immune responses in Arabidopsis thaliana. However, in tomato, PGN did not trigger archetypal immune responses such as mitogen-activated protein kinase activation, ROS production or protection from subsequent bacterial infections. This lack of responses suggests that PGN is not involved in activating immunity and stopping bacterial colonization in tomato. In A. thaliana, immunity against bacteria requires LysM-receptor-like kinases (LysM-RLKs). Two tomato LysMRLKs, SlBti9 and SlLyk13, were shown to be required for resistance against both pathogenic and non-pathogenic Pseudomonas syringae pv. tomato. Flagellin-mediated responses were compromised in plants silenced for SlBti9 and SlLyk13, which could explain the increased bacterial susceptibility observed in these plants. RNAi of SlBti9 and SlLyk13 also compromised chitin perception, as was reported previously for A. thaliana LysM-RLK CERK1. Autophosphorylation seems to be required for the activity of these two tomato LysMRLKs in immunity as cell death mediated by over-expression of these proteins in N. benthamiana was abolished in kinase-inactive mutants. SlBti9 and/or SlLyk13 probably function as either pattern recognition receptors for a yet uncharacterized bacterial PAMP or are part of the receptor complex to transduce the signal once the recognition event has occurred
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