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响应病原体感染方面具有许多共同特征。拟南芥/假单胞菌致病系统是研究细菌和植物天然免疫分子基础的一个高度易处理的模型系统。两个相互作用的伴侣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。拟南芥中细菌先天免疫系统的标志性特征是通过同源抗性蛋白监视经由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，我们专注于特异性识别细菌效应蛋白AvrRpt 2的RPS 2抗病信号通路。RPS 2基因编码CC/NB/LRR蛋白，并且代表在所有植物中发现的NB/LRR抗病蛋白的“超家族”的亚类之一的成员。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。在目的1中，我们将鉴定和表征AvrRpt 2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学策略，这将使我们能够定义和表征AvrRpt 2和RIN 4中的AvrRpt 2蛋白酶切割位点。该网站的鉴定和表征将提供深入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识别这种重要的毒力效应蛋白的其他蛋白质靶点。在目标2中，我们将纯化AvrRpt 2和RIN 4蛋白用于生化活性研究。这些检测方法的开发对于目标3中所述的蛋白酶活性所必需的拟议真核因子的生物化学分离的开发至关重要。在目标3中，我们建议通过伴随的生物化学和遗传实验策略来鉴定和表征AvrRpt 2蛋白酶活性所需的推定真核因子。在目标4中，我们将通过定义RIN 4的哪些蛋白结构域参与RPS 2激活的抑制以及RIN 4与RPS 2的哪些结构域相互作用来定义RIN 4负调控的分子基础，所述RIN 4负调控通过利用缺失诱变策略结合免疫共沉淀实验来进行。最后，在目标5中，我们提出从遗传学上鉴定损害Rps 2特异性抗病途径诱导的突变。Rps 2通路的遗传解剖加上Rps 2相互作用蛋白的鉴定将使我们处于有利地位，以揭示控制Rps 2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.