Characterization of RPS5 activation and downstream interacting proteins

RPS5 激活和下游相互作用蛋白的表征

• 批准号: 7406451
• 负责人: Brody J DeYoung
• 金额: $ 5.13万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7406451
• 关键词: Address; Affect; Alleles; Animals; Arabidopsis PBS1 protein; Area; Binding; Biochemical; Biochemical Genetics; Biological Sciences; Cell Death; Cell Nucleus; Cell membrane; Cells; Class; Cleaved cell; Co-Immunoprecipitations; Complex; Crohn' s disease; Cysteine Protease; Disease; Disease Resistance; Event; Fluorescence Microscopy; Goals; Growth and Development function; Hydrolysis; Immune system; Immunity; Infection; Lead; Leucine-Rich Repeat; Light; Localized; Mediating; Methods; Molecular; Monitor; Mouse-ear Cress; Movement; Mutation; Natural Immunity; Nucleotides; Numbers; Pathogen detection; Pathway interactions; Plant Proteins; Plants; Process; Protein Analysis; Proteins; Pseudomonas syringae; Range; Regulation; Research Project Grants; Research Training; Resistance; Role; Signal Transduction; Symptoms; Syndrome; System; Techniques; Vertebrates; Yeasts; defense response; experience; human disease; leucine-rich repeat protein; mutant; novel; pathogen; research study; response; tool; yeast two hybrid system

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.

描述（由申请人提供）：植物已经发展出复杂的免疫系统来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使用的先天免疫和适应性免疫机制。 RPS5 是植物免疫系统中特征最明确的植物蛋白之一。 RPS5 属于 NOD-LRR 类抗性蛋白，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尚不清楚。 RPS5 通过间接机制识别表达病原体效应子 AvrPphB 的丁香假单胞菌。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来裂解拟南芥蛋白 PBS1。 PBS1 的状态由 RPS5 监控。 PBS1 裂解后，RPS5 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终止。 RPS5 激活和下游信号转导涉及的生化和细胞机制尚不清楚。该提案旨在表征导致 RPS5 激活的分子和细胞事件，以及识别和表征 RPS5 的下游相互作用伙伴。将使用多种方法来实现这些目标。 RPS5 响应病原体攻击的激活动态将通过两种方式确定。将使用免疫共沉淀在存在或不存在 PBS1 和/或 AvrPphB 的情况下检查 RPS5 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 RPS5、PBS1 和 AvrPphB 细胞定位响应病原体识别的动态。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。将检查这些伙伴的突变等位基因，通过监测病原体生长和疾病症状的发展来确定它们在 RPS5 介导的抗病性中的作用。这些伙伴蛋白的功能范围将通过确定它们在其他已充分表征的细菌和真菌抗病途径中的需求来检查。动物和植物的 NOD-LRR 蛋白都参与抗病性，NOD-LRR 蛋白的突变与许多人类疾病（例如克罗恩病和布劳综合征）有关。植物 NOD-LRR 蛋白（例如 RPS5）的表征将有助于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动物系统中以前未知的抗病途径，这可能会导致人类疾病的新疗法。