Elucidating RIN4_Mediated Immune Signaling Cascades in Arabidopsis

描述(由申请人提供)：先天免疫系统，或生殖系编码，是一种古老的进化保守的防御系统，调节人类、动物和植物中的病原体识别。一些先天免疫受体识别保守的微生物特征。科学研究表明，由于趋同进化，在不同的宿主中可以识别出相似的微生物特征。例如，在动物、人类和植物中，细菌鞭毛蛋白是由富含亮氨酸的重复受体识别的，导致MAPK级联反应的下游激活和抗病活性的激活。细胞内免疫受体具有核苷酸结合、富含亮氨酸的重复结构域(NLR)，可识别进入植物细胞的病原体效应蛋白。在人类和动物中，NLR受体可以识别微生物的特征，在某些情况下，还可以识别效应器。尽管在不同的生物体中都有类似的免疫受体参与，但控制受体激活和相互连接的下游信号节点的分子机制仍然难以捉摸。这可能是由于功能冗余和关键信令扇区潜在的致命性。在这一应用中，我们试图了解植物中NLR受体激活的早期事件，使用芥菜近缘植物拟南芥和细菌病原菌紫丁香假单胞菌作为模型。应用将集中在RIN4上，这是一种保守的植物蛋白，可以调节微生物特征的感知方面，可以与NLR受体RPM1和RPS2相互作用，并成为多个病原体效应器的靶标。我们能够纯化出具有生物活性的RPM1及其相关蛋白，这些蛋白将用于生化研究NLR受体的体外激活。RPM1复合体将在静止和激活状态下从拟南芥中分离出来，相关蛋白质将通过质谱学进行鉴定。我们已经优化了RPM1复合体的纯化条件，并拥有几个有趣的信号蛋白，用于下游的生化、细胞生物学和遗传验证实验。控制效应诱导的RIN4磷酸化和裂解以促进敏感基因的病原菌毒力的分子机制将被研究。更深入地了解植物免疫受体如何识别病原体将从根本上促进我们对不同生物体的受体识别的理解，这是开发新的、环境友好型疾病控制策略所必需的。该项目的具体目标是：1.确定免疫受体RPM1在体外激活时复杂组装、酶活性和已知蛋白质结合的变化。2.鉴定和鉴定处于静息和激活状态的NLR蛋白复合体。3.研究效应器诱导的RIN4修饰对与宿主蛋白相互作用的重要性。

DESCRIPTION (provided by applicant): The innate, or germline encoded, immune system is an ancient and evolutionary conserved defense system mediating pathogen recognition in humans, animals, and plants. Some innate immune receptors recognize conserved microbial features. Scientific studies have revealed that similar microbial features are recognized across diverse hosts as a result of convergent evolution. For example, in animals, humans and plants bacterial flagellin is recognized by leucine-rich repeat receptors leading to the downstream activation of MAPK cascades and activation of disease resistance. Intracellular immune receptors possessing nucleotide binding, leucine- rich repeat domains (NLRs) act to recognize pathogen effector proteins delivered into plant cells. In humans and animals, NLR receptors can recognize microbial features and, in some cases, effectors. Despite the involvement of analogous immune receptors across diverse organisms, the molecular mechanisms controlling receptor activation and interconnected downstream signaling nodes remain elusive. This is likely due to functional redundancy and lethality underlying key signaling sectors. In this application, we seek to understand the early events underlying NLR receptor activation in plants using the mustard relative, Arabidopsis thaliana, and the bacterial pathogen Pseudomonas syringae as a model. The application will focus on RIN4, a conserved plant protein that can regulate aspects of perception of microbial features, can interact with the NLR receptors RPM1 and RPS2, and is targeted by multiple pathogen effectors. We are able to purify biologically active RPM1 and associated proteins, which will be used to biochemically, investigate NLR receptor activation in vitro. The RPM1 complex will be isolated from Arabidopsis at a resting and activated state and associated proteins will be identified by mass spectrometry. We have optimized conditions for RPM1 complex purification and have several interesting signaling proteins in hand for downstream biochemical, cell biological and genetic validation experiments. The molecular mechanisms controlling effector-induced RIN4 phosphorylation and cleavage for promoting pathogen virulence in susceptible genotypes will be investigated. A greater understanding of how plant immune receptors recognize pathogens will fundamentally advance our understanding of receptor recognition across diverse organisms and is required for the development of novel, environmentally friendly disease control strategies. The specific aims of the project are: 1. Identify changes in complex assembly, enzymatic activity, and known protein associations upon activation of the immune receptor RPM1 in vitro. 2. Identify and characterize NLR protein complexes in a resting and activated state. 3. Investigate the importance of effector-induced RIN4 modifications for interaction with host proteins.