Characterization of RPS5 activation and downstream interacting proteins

描述（由申请人提供）：植物已经开发了一个复杂的免疫系统，以检测和应对潜在病原体的挑战。在许多方面，这些病原体检测方法类似于脊椎动物利用的先天免疫和适应性免疫机制。参与植物免疫系统的最具特征的植物蛋白之一是RPS 5。RPS 5属于NOD-LRR类抗性蛋白，其介导植物和动物中的病原体识别。NOD-LRR蛋白如何检测病原体分子还知之甚少。RPS 5通过间接机制识别表达病原体效应子AvrPphB的假单胞菌。感染后，AvrPphB被注射到宿主细胞中，并利用其半胱氨酸蛋白酶活性切割拟南芥蛋白PBS 1。PBS 1的状态由RPS 5监控。在PBS 1裂解后，RPS 5被激活，触发下游信号转导。这导致局部细胞死亡和病原体传播的终止。参与RPS 5激活和下游信号转导的生化和细胞机制尚不清楚。该提案旨在表征导致RPS 5激活的分子和细胞事件，以及鉴定和表征RPS 5的下游相互作用伙伴。将使用几种方法来实现这些目标。将以两种方式确定响应于病原体攻击的RPS 5活化的动力学。将使用免疫共沉淀在存在或不存在PBS 1和/或AvrPphB的情况下检查RPS 5结构域的分子内相互作用。此外，将使用荧光显微镜技术确定响应于病原体识别的RPS 5、PBS 1和AvrPphB细胞定位的动力学。最后，将使用酵母双杂交分析和蛋白质复合物纯化来鉴定下游相互作用伙伴。将通过监测病原体生长和疾病症状的发展来检查这些伴侣的突变等位基因以确定它们在RPS 5介导的抗病性中的作用。这些伴侣蛋白的功能范围将通过确定它们在其他充分表征的细菌和真菌疾病抗性途径中的需求来检查。动物和植物NOD-LRR蛋白均参与疾病抗性，并且NOD-LRR蛋白中的突变与许多人类疾病如克罗恩病和Blau综合征相关。植物NOD-LRR蛋白，如RPS 5的表征，将导致更好地了解植物和动物的抗病性。此外，这些实验可能会发现以前在动物系统中未知的抗病途径，这可能会导致人类疾病的新疗法。

DESCRIPTION (provided by applicant): Plants have developed a complex immune system to detect and respond to challenge by potential pathogens. In many ways these pathogen detection methods resemble the innate immunity and adaptive immunity mechanisms utilized by vertebrate animals. One of the best characterized plant proteins involved in the plant immune system is RPS5. RPS5 belongs to the NOD-LRR class of resistance proteins, which mediate pathogen recognition in both plants and animals. How NOD-LRR proteins detect pathogen molecules is poorly understood. RPS5 recognizes Pseudomonas syringae expressing the pathogen effector, AvrPphB, through an indirect mechanism. Upon infection, AvrPphB is injected into host cells and utilizes its cysteine protease activity to cleave the Arabidopsis protein PBS1. The state of PBS1 is monitored by RPS5. Upon PBS1 cleavage, RPS5 becomes activated, triggering downstream signal transduction. This results in localized cell death and termination of pathogen spread. The biochemical and cellular mechanisms involved in RPS5 activation and downstream signal transduction are unknown. This proposal aims to characterize the molecular and cellular events that lead to RPS5 activation as well as identify and characterize downstream interacting partners of RPS5. Several methods will be used to achieve these goals. The dynamics of RPS5 activation in response to pathogen challenge will be determined in two ways. The intramolecular interactions of RPS5 domains will be examined in the presence or absence of PBS1 and/or AvrPphB using co-immunoprecipitation. Additionally, the dynamics of RPS5, PBS1, and AvrPphB cellular localization in response to pathogen recognition will be determined using fluorescence microscopy techniques. Finally, downstream interacting partners will be identified using yeast two-hybrid analysis and protein complex purification. Mutant alleles of these partners will be examined to determine their role in RPS5-mediated disease resistance by monitoring pathogen growth and development of disease symptoms. The range of function of these partner proteins will be examined by determining their requirement in other well characterized bacterial and fungal disease resistance pathways. Both animal and plant NOD-LRR proteins are involved in disease resistance and mutations in NOD-LRR proteins are associated with a number of human diseases such as Crohn's disease and Blau syndrome. Characterization of plant NOD-LRR proteins, such as RPS5, will lead to a better understanding of disease resistance in both plants and animals. In addition, these experiments may identify disease resistance pathways previously unknown in animal systems, which could lead to novel treatments for human disease.