Elucidating RIN4-Mediated Immune Signaling Cascades in Arabidopsis

描述（由申请人提供）：先天免疫系统的多个关键组分在真核生物中是保守的。在植物中，先天免疫系统作为屏障来抑制病原体进入和繁殖。尽管先天免疫系统的重要性，科学家们仍然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有限。植物免疫系统的关键调节因子是拟南芥基因RIN 4。RIN 4在所有陆地植物中是保守的，并且用于调节细菌病原体假单胞菌致病变种的免疫感知。拟南芥中的番茄本申请中的初步数据证明了在不存在和存在病原体刺激的情况下RIN 4蛋白复合物的纯化。通过质谱分析鉴定了15个新蛋白，并通过酵母双杂交证实了多个蛋白与RIN 4相互作用。这些RIN 4相关蛋白的三个拟南芥敲除或过表达株系显示对P.这表明它们是植物免疫反应的重要组成部分。一种RIN 4相关蛋白是H+ ATP酶AHA 1。实验表明，RIN 4可以直接调节AHA 1酶活性。RIN 4可以与AHA 1协同工作，在先天免疫反应期间调节叶片气孔开放，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RIN 4复合物成分将是控制先天免疫信号传导的关键成分。几个提出的实验试图理解RIN 4蛋白复合物组装和RIN 4介导的细胞信号级联使用P. dichlorinagae-拟南芥病理系统。该病理系统是研究真核生物先天免疫信号传导的极好模型系统，因为可获得的广泛遗传资源、拟南芥的快速世代时间以及植物和其他真核生物先天免疫系统之间的相似性。本研究的具体目的如下：1）阐明RIN 4调节质膜H+ ATP酶活性的机制; 2）研究RIN 4蛋白网络的时空组成; 3）对拟南芥RIN 4相关蛋白进行功能表征。 RIN 4是调节和激活膜定位的植物先天免疫蛋白复合物的核心参与者。多种RIN 4相关蛋白在真核生物中广泛保守，并且其中几种涉及哺乳动物的免疫信号传导。由于植物和其他真核生物的先天免疫系统之间存在显著的相似性，我们预计我们的发现将与NIH的使命广泛相关。

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.