Elucidating RIN4_Mediated Immune Signaling Cascades in Arabidopsis

描述（由申请人提供）：先天或种系编码的免疫系统是一种古老且进化的保守防御系统，介导人类、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，不同宿主可以识别出相似的微生物特征。例如，在动物、人类和植物中，细菌鞭毛蛋白被富含亮氨酸的重复受体识别，导致下游 MAPK 级联的激活和抗病性的激活。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），可识别递送到植物细胞中的病原体效应蛋白。在人类和动物中，NLR 受体可以识别微生物特征，在某些情况下还可以识别效应器。尽管涉及不同生物体的类似免疫受体，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。这可能是由于关键信号部门的功能冗余和致命性造成的。在本申请中，我们以芥菜近缘植物拟南芥和细菌病原体丁香假单胞菌为模型，试图了解植物中 NLR 受体激活的早期事件。该申请将重点关注 RIN4，这是一种保守的植物蛋白，可以调节微生物特征的感知，可以与 NLR 受体 RPM1 和 RPS2 相互作用，并且是多种病原体效应子的目标。我们能够纯化具有生物活性的 RPM1 和相关蛋白，这些蛋白将用于生化研究体外 NLR 受体的激活。将从静息和激活状态的拟南芥中分离出 RPM1 复合物，并通过质谱法鉴定相关蛋白。我们优化了 RPM1 复合物纯化的条件，并拥有几种有趣的信号蛋白，可用于下游生化、细胞生物学和遗传验证实验。将研究控制效应子诱导的 RIN4 磷酸化和裂解以促进易感基因型病原体毒力的分子机制。更好地了解植物免疫受体如何识别病原体将从根本上增进我们对不同生物体受体识别的理解，并且是开发新型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. 识别体外激活免疫受体 RPM1 后复杂组装、酶活性和已知蛋白质关联的变化。 2. 识别和表征静息和激活状态下的 NLR 蛋白复合物。 3. 研究效应子诱导的 RIN4 修饰对于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.