VERMONT COBRE: PROJECT 5: REGULATION AND EFFECTORS OF PLANT INNATE IMMUNITY

佛蒙特州 COBRE：项目 5：植物先天免疫的调节和影响因素

• 批准号: 7720919
• 负责人: TERRENCE P DELANEY
• 金额: $ 17.66万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720919
• 关键词: Alleles; Applications Grants; Arabidopsis; Biochemical; Bioinformatics; Biological Assay; Biological Markers; Candidate Disease Gene; Centers of Research Excellence; Chimeric Proteins; Chromosomes; Cloning; Communities; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupled; DNA Sequence; Data; Data Set; Disease Resistance; Enhancers; F-Box Proteins; Funding; Gene Expression; Genes; Genetic Complementation Test; Genetic Screening; Genome; Genotype; Goals; Grant; Hand; Immune system; Insertion Mutation; Institution; Left; Libraries; Localized; Luciferases; Maps; Mediating; Mentors; Microarray Analysis; Mouse-ear Cress; Mutation; Natural Immunity; Object Attachment; Paper; Parasites; Parents; Pathway interactions; Peronospora; Phenotype; Plants; Play; Population; Precipitation; Predisposition; Protein Overexpression; Proteins; Protoplasts; Pseudomonas syringae; Publications; Regulation; Reporter; Reporter Genes; Research; Research Personnel; Resistance; Resolution; Resources; Rice; Role; Salicylic Acid; Salicylic Acids; Sampling; Sequence Analysis; Source; Suppressor Mutations; Testing; Transgenic Plants; United States National Institutes of Health; Vermont; Work; base; comparative; expectation; in vivo; member; mutant; novel; pathogen; programs; promoter; transcription factor; ubiquitin-protein ligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Plants employ multiple defense pathways to defend against pathogen attack. These include systemic acquired resistance (SAR) that is dependent upon accumulation of endogenous salicylic acid (SA) and the downstream NIM1 (also called NPR1) gene in Arabidopsis thaliana, which shows homology to the mammalian innate immunity regulator Ik-B. However, outside of SAR, other essential defence pathways contribute to plant defense, and collectively constitute the plant innate immune system. Our project goals are to elucidate SAR-independent mechanisms for plant defense. The Delaney program joined the COBRE in May 2007, and has worked toward two main objectives. The first involves the characterization and cloning of arabidopsis genes that regulate SAR-independent disease resistance and are believed encode regulators or effectors of novel plant defense pathways. Arabidopsis nim1 mutants (SAR-incompetent) were screened for enhancer mutations that exacerbate their heightened susceptibility to pathogens normally unable to grow due to SAR and other defense pathways. The test pathogen used is an obligate biotrophic oomycete parasite Peronospora parasitica. The nip1 (nim1-independent resistance to Peronospora) mutant was discovered that is hypersusceptible to H. parasitica, implicating an additional, SAR independent defense pathway active against this pathogen. We have mapped NIP1 to an approximately 17 cM region on chromosome III of Arabidopsis, using a mapping population of approximately 416 mutant plants identified from a screen of 12,000 F2 plants from a mapping cross. Of the 416, 120 plants have been analyzed using PCR-based molecular markers that distinguish the mapping-cross parents. This allowed linkage to be established between NIP1 and flanking markers 8 and 9 cM away. We are now attempting to narrow this interval so the NIP1 gene is located with sufficient precision that we can use a DNA sequencing approach to evaluate candidate loci. For this to be feasible, we must generate high-resolution maps of this locus and will then identify the gene by comparative sequence analysis of candidate genes in mutant versus wild-type plants within the region containing the gene. This will require that the gene be localized to an approx. 20 kbp region, which in Arabidopsis is usually about 0.1 cM. To accomplish this, we will identify additional molecular markers within the 17 cM interval, and map the approximately crossover points that we currently have in our mapping population. We also need additional F2 plants that are characterized for marker and NIP1 genotype. These will come from the remaining nip1 F2 plants in hand, as well as additional analysis of F2 populations from our mapping crosses. Candidate gene will be tested in planta by using complementation tests of mutant plants that have introduced into them a wild-type copy of a gene believed to be responsible for the mutant phenotype. We have previous expertise in this approach to gene isolation, having used it to clone Arabidopsis DET1, NIM1 and SON1 genes. An additional objective is to analyze the function of Arabidopsis genes that were found to be induced or suppressed in plants expressing SAR-independent resistance. We hypothesize that some of these genes play important roles in the resistant phenotype. We performed microarray analysis (whole-genome Affymetrix) of several genotype plants that express SAR-independent resistance, including a son1 single mutant, son1 nim1, and a line we produced that overexpresses a transcription factor (AtTGA5) and we found to consequently express a form of SAR-independent resistance. We also included wild-type plants in the analysis. Each genotype was either exposed to H. parasitica or left na¿ve, and a wild type plant sample was treated to induce SAR, providing 9 experimental samples (in triplicate). With assistance from Dr. Jeff Bond, in the Bioinformatics Core, we have thoroughly investigated this data set and identified sets of genes that correlate with SAR-independent resistance. We are now testing whether those genes have functions by procuring insertional mutations in most of the genes, from members of the Arabidopsis research community who have created insertion libraries. In the last 6 months, these insertion alleles have been crossed into some of the genotypes that express SAR-independent resistance (i.e. son1 nim1), and we are now beginning phenotypic tests of the multi-mutant lines that should express resistance, but because of the insertion mutation may not. Our assays involve pathogens of Arabidopsis including H. parasitica and Pseudomonas syringae. We have approximately 20 genes that were are testing in this manner, with the goal of having data on whether they have direct roles in son1-mediated resistance. In other work, we are actively pursuing study of an F-box protein in Arabidopsis, SON1, that we recovered in a forward genetic screen to identify suppressor mutations of the nim1 mutation. We have performed biochemical co-precipitation studies to try to identify substrates of a putative SCF E3 Ubiquitin ligase complex that includes SON1. Notably, plants contain a very large family of F-box proteins (approx 650 in Arabidopsis and rice). We have identified a candidate substrate using co-precipitation of a GST-SON1 fusion protein, and mass spec analysis of the isolated protein. We are currently investigating whether the candidate substrate protein is an authentic in vivo interactor with SON1. In related work we have generated SON1 promoter-reporter transgenic plants that will help us understand expression of this gene, SON1-GFP translational fusions that will help us examine subcellular localization of SON1, and are developing a protoplast transient assay that will allow is to study SON1 activity by its ability to genetically complement a son1-1 mutant protoplast, as indicated by induced expression of a SON1-dependent promoter coupled to a luciferase reporter gene. We have use this assay in past work to examine NIM1 activity in vivo. Together this work is aimed at characterizing additional pathways that contribute to plant innate immunity. Mentoring Summary: Cory Teuscher We discussed his publication record and submitting the renewal (or new application) for his NSF grant. I expressed to him that a reasonable minimal expectation would be one paper in press, one in review, and at least one NSF/NIH grant application per year until funding is obtained. I strongly encouraged him to submit a renewal for this coming cycle.