Elucidating RIN4-Mediated Immune Signaling Cascades in Arabidopsis

描述（由申请人提供）：天然免疫系统的多个关键成分在真核生物中是保守的。在植物中，先天免疫系统是抑制病原体进入和繁殖的屏障。尽管先天免疫系统很重要，但科学家们对植物免疫复合物如何组装和调节以响应病原体感知的理解仍然有限。植物免疫系统的一个关键调控因子是拟南芥基因RIN4。RIN4在所有陆地植物中都是保守的，并参与调节细菌病原体丁香假单胞菌pv的免疫感知。拟南芥中的番茄。该应用程序的初步数据证明了在没有和存在病原体刺激的情况下纯化RIN4蛋白复合物。通过质谱分析鉴定了15个新蛋白，随后通过酵母双杂交发现了多个蛋白与RIN4相互作用。拟南芥中这三种RIN4相关蛋白的敲除或过表达系对丁香疫病毒的防御反应发生了改变。这表明它们是植物免疫反应的重要组成部分。一种与RIN4相关的蛋白是H+ atp酶AHA1。实验表明，RIN4可以直接调控AHA1酶的活性。在先天免疫应答过程中，RIN4可以与AHA1协同调节叶片气孔打开，从而阻断细菌病原体进入叶片内部。本研究的中心假设是，RIN4复合物成分将是控制先天免疫信号的关键成分。几个拟议的实验试图利用紫丁香-拟南芥病理系统了解RIN4蛋白复合物组装和RIN4介导的细胞信号级联反应。拟南芥具有丰富的遗传资源、快速的生成时间、植物的先天免疫系统与其他真核生物的相似之处，是研究真核生物先天免疫信号的良好模型系统。本研究申请的具体目的是：1)阐明RIN4调控质膜H+ atp酶活性的机制；2)研究RIN4蛋白网络的时空组成；3)拟南芥RIN4相关蛋白的功能表征。

DESCRIPTION (provided by applicant): Multiple key components of the innate immune system are conserved across eukaryotes. In plants, the innate immune system serves as a barrier to inhibit both pathogen entry and multiplication. Despite the importance of the innate immune system, scientists still have a limited understanding of how plant immune complexes are assembled and regulated in response to pathogen perception. A key regulator of the plant immune system is the Arabidopsis gene RIN4. RIN4 is conserved among all land plants and acts to regulate immune perception of the bacterial pathogen Pseudomonas syringae pv. tomato in Arabidopsis. Preliminary data within this application demonstrate the purification of RIN4 protein complexes in the absence and presence of pathogen stimulus. Fifteen novel proteins were identified by mass spectrometry and multiple proteins were subsequently shown to interact with RIN4 by yeast two-hybrid. Arabidopsis knockout or overexpression lines for three of these RIN4 associated proteins display altered defense responses to P. syringae pv. tomato, suggesting that they are important components of the plant immune response. One RIN4 associated protein is the H+ATPase AHA1. Experiments indicate that RIN4 can directly regulate AHA1 enzymatic activity. RIN4 can work in concert with AHA1 to regulate leaf stomatal opening during the innate immune responses, thus blocking the entry of bacterial pathogens into the leaf interior. The central hypothesis of the proposed research is that RIN4 complex constituents will be key components controlling innate immune signaling. Several proposed experiments seek to understand RIN4 protein complex assembly and RIN4-mediated cellular signaling cascades using the P. syringae-Arabidopsis pathosystem. This pathosystem is an excellent model system to study eukaryotic innate immune signaling because of the extensive genetic resources available, the fast generation time of Arabidopsis, and the similarities between innate immune systems in plants and other eukaryotes. The specific aims of this research application are the following: 1) Elucidate the mechanism RIN4 uses to regulate plasma membrane H+ATPase activity; 2) Investigate the spatial and temporal components of the RIN4 protein network; 3) Functionally characterize Arabidopsis RIN4 associated proteins. PUBLIC HEALTH RELEVANCE: RIN4 is a central player in the regulation and activation of membrane-localized plant innate immune protein complexes. Multiple RIN4 associated proteins are widely conserved among eukaryotes and several have been implicated in immune signaling in mammals. Because there are significant similarities between innate immune systems in plants and other eukaryotes, we anticipate our findings will be broadly relevant to NIH's mission.