Molecular Basis of Arabidopsis Innate Immunity

拟南芥先天免疫的分子基础

• 批准号: 7995595
• 负责人: BRIAN John STASKAWICZ
• 金额: $ 10.23万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-08 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7995595
• 关键词: Achievement; Affinity Chromatography; Algorithms; Amino Acids; Animals; Arabidopsis; Arabidopsis RIN4 protein; Binding; Biochemical; Biochemical Genetics; Bioinformatics; Biological; Biological Assay; Biological Models; Cell Death; Cell Extracts; Cell membrane; Cells; Cleaved cell; Co-Immunoprecipitations; Coupled; Cysteine Protease; Data; Deletion Mutagenesis; Development; Disease; Disease Resistance; Dissection; Drosophila chb protein; Enzymes; Estradiol; Event; Evolution; Fungal Genome; Gel Chromatography; Generations; Genes; Genetic; Genome; Goals; Growth; Host resistance; Immune system; Infection; Ion Exchange; Knock-out; Laboratories; Lead; Libraries; Mass Spectrum Analysis; Modeling; Molecular; Molecular Genetics; Mouse-ear Cress; Mutation; N-terminal; Natural Immunity; Pathology; Pathway interactions; Peptide Hydrolases; Plants; Positioning Attribute; Procedures; Process; Proteins; Proteolysis; Proteolytic Processing; Pseudomonas syringae; Recombinants; Regulation; Repression; Research; Resistance; Saccharomyces cerevisiae; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Specific qualifier value; System; Tertiary Protein Structure; Testing; Tomatoes; Transgenic Organisms; Triad Acrylic Resin; Type III Secretion System Pathway; Vascular Plant; Virulence; Yeasts; assay development; base; genome sequencing; in vitro Assay; insight; leucine-rich repeat protein; member; milligram; molecular recognition; pathogen; protein complex; research study; response

DESCRIPTION (provided by applicant): Recent studies have shown that both plant and animal bacterial innate immunity share many features in common in response to pathogen infection. The Arabidopsis/Pseudomonas syringae pathosystem is a highly tractable model system to study the molecular basis of bacterial plant innate immunity. The complete genome sequence of both interacting partners has greatly facilitated many molecular approaches including; bioinformatic, genetic, biochemical, cell biological and structural biological studies. A hallmark feature of the bacterial innate immune system in Arabidopsis thaliana is the surveillance of bacterial effector proteins delivered to the plant cell via a type III secretion system by cognate resistance proteins. In our laboratory, we have focused on the RPS2 disease resistance-signaling pathway that specifically recognizes the bacterial effector protein, AvrRpt2. The RPS2 gene encodes for CC/NB/LRR protein and represents a member of one of the subclasses of the "superfamily" of NB/LRR disease resistance proteins found in all plants. The goals of this project are elucidate the molecular events that specify the recognition of bacterial effector proteins and the biochemical and cellular signaling events that determine bacterial innate immunity. In aim 1, we will identify and characterize the amino acid recognition cleavage site of the AvrRpt2 protease. We propose to employ a molecular genetic strategy that will allow us to define and characterize the AvrRpt2 protease cleavage site in AvrRpt2 and RIN4. The identification and characterization of this site will provide insight in the biochemical mechanism of proteolysis and may aid in the identification of addition protein targets in the Arabidopsis genome for this important virulence effector protein. In aim 2, we will purify the AvrRpt2 and RIN4 Proteins for Biochemical Activity Studies. The development of these assays will be critical for the development of the biochemical isolation of the proposed eukaryotic factor that is essential for protease activity described in aim 3. In aim 3, we propose to identify and characterize the putative eukaryotic factor required for AvrRpt2 protease activity by concomitant biochemical and genetic experimental strategies. In aim 4, we will define the molecular basis of RIN4 negative regulation by defining which protein domains of RIN4 are involved in the repression of RPS2 activation and which domains of RPS2 are interacting with RIN4 by utilizing deletion mutagenesis strategies in conjunction with co-immunoprecipitation experiments. Finally in aim 5, we propose to genetically identify mutations that compromise the induction of the Rps2-specified disease resistance Pathway. The genetic dissection of the Rps2 pathway coupled with the identification of RPS2-interacting proteins will put us in a strong position to uncover the molecular events controlling the Rps2 resistance-signaling pathway.

描述（由申请人提供）：最近的研究表明，动植物细菌先天免疫都在病原体感染中具有许多共同的特征。拟南芥/假单胞菌丁香病原系统是一个高度易于处理的模型系统，用于研究细菌植物先天免疫的分子基础。两种相互作用伙伴的完整基因组序列都极大地促进了许多分子方法，包括：生物信息学，遗传，生化，细胞生物学和结构生物学研究。拟南芥中细菌先天免疫系统的标志性特征是对通过同源抗性蛋白通过III型分泌系统传递到植物细胞的细菌效应蛋白的监测。在我们的实验室中，我们专注于专门识别细菌效应子蛋白AVRRPT2的RPS2抗病性信号途径。 RPS2基因编码CC/NB/LRR蛋白，代表了所有植物中发现的NB/LRR抗病性蛋白的“超家族”的子类的成员。该项目的目标是阐明了分子事件，这些事件指定了细菌效应子蛋白的识别以及确定细菌先天免疫力的生化和细胞信号事件。在AIM 1中，我们将识别并表征AVRRPT2蛋白酶的氨基酸识别裂解位点。我们建议采用一种分子遗传策略，使我们能够定义和表征AVRRPT2和RIN4中AVRRPT2蛋白酶裂解位点。该站点的鉴定和表征将为蛋白水解的生化机理提供见解，并可能有助于鉴定拟南芥基因组中这种重要的毒力效应蛋白的添加蛋白靶标。在AIM 2中，我们将纯化AVRRPT2和RIN4蛋白进行生化活性研究。这些测定的开发对于开发AIM 3中描述的蛋白酶活性至关重要的生化分离至关重要。在AIM 3中，我们建议通过和解生物化学和遗传实验策略来识别和表征AVRRPT2蛋白酶活性所需的假定真核生物。在AIM 4中，我们将通过确定RIN4的哪些蛋白质结构域参与RPS2激活的抑制以及RPS2的哪些域与RIN4相互作用，从而定义RIN4负调控的分子基础，并通过利用缺失型突变型策略与共免疫抗沉淀实验相互作用。最后，在AIM 5中，我们建议从遗传上确定损害RPS2指定疾病抗性途径的突变。 RPS2途径的遗传解剖与RPS2相互作用蛋白的鉴定相结合将使我们处于强大的位置，以发现控制RPS2电阻 - 信号信号途径的分子事件。