Molecular Basis of Arabidopsis Innate Immunity

描述（由申请人提供）：最近的研究表明，植物和动物的细菌先天免疫在对病原体感染的反应中有许多共同的特征。拟南芥/丁香假单胞菌病理系统是研究植物细菌先天免疫分子基础的一个高度可控的模型系统。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；生物信息学、遗传学、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的III型分泌系统监测细菌效应蛋白传递到植物细胞。在我们的实验室中，我们专注于RPS2疾病抗性信号通路，该信号通路特异性识别细菌效应蛋白AvrRpt2。RPS2基因编码CC/NB/LRR蛋白，是所有植物中发现的NB/LRR抗病蛋白“超家族”之一亚类的成员。该项目的目标是阐明指定识别细菌效应蛋白的分子事件以及决定细菌先天免疫的生化和细胞信号事件。在目标1中，我们将鉴定和表征AvrRpt2蛋白酶的氨基酸识别切割位点。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中的AvrRpt2蛋白酶切割位点。该位点的鉴定和表征将有助于了解蛋白质水解的生化机制，并可能有助于在拟南芥基因组中鉴定这一重要毒力效应蛋白的附加蛋白靶点。在目的2中，我们将纯化AvrRpt2和RIN4蛋白用于生化活性研究。这些检测方法的发展将对提出的真核因子的生化分离的发展至关重要，真核因子对目标3中描述的蛋白酶活性至关重要。在目标3中，我们建议通过联合生化和遗传实验策略来鉴定和表征AvrRpt2蛋白酶活性所需的真核因子。在aim 4中，我们将利用缺失诱变策略结合共免疫沉淀实验，通过定义RIN4的哪些蛋白结构域参与RPS2激活的抑制，以及RPS2的哪些结构域与RIN4相互作用，来定义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损害rps2指定的抗病途径诱导的突变。Rps2通路的遗传解剖加上Rps2相互作用蛋白的鉴定将使我们处于一个强有力的位置，揭示控制Rps2抗性信号通路的分子事件。

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.