Molecular Basis of Arabidopsis Innate Immunity

拟南芥先天免疫的分子基础

• 批准号: 7115904
• 负责人: BRIAN John STASKAWICZ
• 金额: $ 29.69万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-29 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7115904
• 关键词: Arabidopsis; Pseudomonas; Saccharomyces cerevisiae; bacterial proteins; biological signal transduction; chemical cleavage; enzyme activity; fungal genetics; fungal proteins; gel filtration chromatography; gene expression; genetic screening; host organism interaction; immunity; immunoprecipitation; immunoregulation; ion exchange chromatography; plant proteins; protein protein interaction; protein purification; proteolysis; recombinant proteins; site directed mutagenesis; temperature sensitive mutant; transport proteins; yeast two hybrid system

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，植物和动物细菌先天免疫在应对病原体感染方面具有许多共同特征。拟南芥/丁香假单胞菌病理系统是研究细菌植物先天免疫分子基础的高度易于处理的模型系统。两个相互作用伙伴的完整基因组序列极大地促进了许多分子方法，包括：生物信息、遗传、生物化学、细胞生物学和结构生物学研究。拟南芥细菌先天免疫系统的一个标志性特征是通过同源抗性蛋白监视通过 III 型分泌系统递送至植物细胞的细菌效应蛋白。在我们的实验室中，我们重点研究专门识别细菌效应蛋白 AvrRpt2 的 RPS2 抗病信号通路。 RPS2基因编码CC/NB/LRR蛋白并且代表所有植物中发现的NB/LRR抗病蛋白“超家族”的亚类之一的成员。该项目的目标是阐明指定细菌效应蛋白识别的分子事件以及决定细菌先天免疫的生化和细胞信号传导事件。在目标 1 中，我们将鉴定并表征 AvrRpt2 蛋白酶的氨基酸识别切割位点。我们建议采用分子遗传学策略，使我们能够定义和表征 AvrRpt2 和 RIN4 中的 AvrRpt2 蛋白酶切割位点。该位点的识别和表征将提供对蛋白水解生化机制的深入了解，并可能有助于识别拟南芥基因组中这种重要毒力效应蛋白的附加蛋白靶标。在目标 2 中，我们将纯化 AvrRpt2 和 RIN4 蛋白用于生化活性研究。这些测定的发展对于目标 3 中描述的蛋白酶活性至关重要的所提出的真核因子的生化分离的发展至关重要。在目标 3 中，我们建议鉴定和表征 AvrRpt2 蛋白酶活性所需的推定真核因子通过伴随的生化和遗传实验策略。在目标 4 中，我们将通过结合免疫共沉淀实验利用缺失诱变策略来定义 RIN4 的哪些蛋白结构域参与抑制 RPS2 激活以及 RPS2 的哪些结构域与 RIN4 相互作用，从而定义 RIN4 负调控的分子基础。最后，在目标 5 中，我们建议从基因上鉴定出损害 Rps2 特异性抗病途径诱导的突变。 Rps2 通路的基因剖析与 RPS2 相互作用蛋白的鉴定相结合，将使我们能够更好地揭示控制 Rps2 耐药信号通路的分子事件。