Elucidating RIN4-Mediated Immune Signaling Cascades in Arabidopsis

描述(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。尽管天然免疫系统很重要，但科学家们对植物免疫复合体是如何组装和调节以响应病原体感知的了解仍然有限。植物免疫系统的一个关键调节因子是拟南芥基因RIN4。RIN4在所有陆地植物中都是保守的，并调节细菌病原体紫丁香假单胞菌的免疫感受。拟南芥中的番茄。这一应用中的初步数据表明，在没有病原体刺激和存在病原体刺激的情况下，RIN4蛋白复合体可以纯化。用质谱仪鉴定了15个新的蛋白质，随后通过酵母双杂交证实了多个蛋白质与RIN4相互作用。这些RIN4相关蛋白中的三个蛋白的拟南芥基因敲除或过表达系对紫丁香疫霉的防御反应发生了改变。番茄，表明它们是植物免疫反应的重要组成部分。一种与RIN4相关的蛋白质是H+ATPase AHA1。实验表明，RIN4可以直接调节AHA1的酶活性。RIN4可以与AHA1协同工作，在天然免疫反应中调节叶片气孔的打开，从而阻止细菌病原体进入叶片内部。这项研究的中心假设是，RIN4复杂成分将是控制先天免疫信号的关键成分。一些拟议的实验试图了解RIN4蛋白复合体的组装和RIN4介导的细胞信号级联反应，使用紫丁香疫霉-拟南芥病理系统。该系统是研究真核生物天然免疫信号的良好模型系统，因为它具有丰富的遗传资源、拟南芥的快速产生时间以及植物天然免疫系统与其他真核生物的相似性。本研究的具体目的如下：1)阐明RIN4调节质膜H+ATPase活性的机制；2)研究RIN4蛋白网络的空间和时间成分；3)功能鉴定拟南芥RIN4相关蛋白。

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.