植物免疫受体蛋白RPM1信号起始机制的研究

The Arabidopsis immune receptor RPM1 is activated at the plasma membrane through recognizing the phosphorylation of RIN4, conferring plants hypersensitive response and disease resistance. Recent results demonstrate that the activated RPM1 initiates signaling at the plasma membrane, however the molecular mechanism of RPM1 signaling is not clear yet. We will research the effects of the intra- and inter-cellular interactions of RPM1 on its function to understand how RPM1 initiates the signal. We will research the associated proteins and their functions to elucidate the mechanism of RPM1 signaling. For those known RPM1 associated proteins, we will determine the phosphorylation site of RIN4 through mass spectrometry and site mutations, and study the biochemical characteristics of RPM1-interacting protein RIN13 and its effects on RPM1 function to understand the specificity of RPM1 activation and the multiplicity of RPM1 signaling. Through mass spectrometry assay to analyze RPM1 protein complex, and reverse genetic method to determine the participation of members of the Rop G protein family to RPM1 signal transduction, we try to find more RPM1 signal transduction components , thus predicting the mechanism of RPM1 signaling from their functions. This project will enable us to establish molecular connections between RPM1 activation and the early events of RPM1 signal transduction. This will enrich our knowledge on plant disease resistance，and provide guidance to breed disease resistant crops through integrating multiple disease resistance genes.

植物免疫受体蛋白RPM1在细胞膜上感知RIN4的磷酸化而被激活，引起植物超敏反应和抗病性。最近研究证明激活后的RPM1是在细胞膜上起始信号转导的，但其分子机制仍不清楚。本项目一方面通过研究RPM1结构与功能的关系来阐明RPM1自身如何起始信号；另一方面通过研究RPM1结合蛋白及其功能，来阐明RPM1起始信号的转导机制。利用质谱分析及点突变的方法确定RPM1结合蛋白RIN4磷酸化位点，以及对RIN13生化性质及功能的研究有利于明确RPM1激活的特异性和信号转导途径的多样性。通过质谱分析RPM1蛋白复合物，以及利用反向遗传学方法检测Rop类G蛋白家族成员对RPM1功能的影响，可以发现更多参与RPM1信号转导的蛋白，进而从其功能阐明RPM1信号起始机制。本项研究将建立起RPM1激活与信号转导早期事件间的分子联系，对于认识植物抗病机理及通过有效整合抗病基因培育抗性农作物具有重要理论和实践意义。

植物免疫受体蛋白RPM1 在细胞膜上感知RIN4 的磷酸化而被激活，引起植物超敏反应和抗病性。本项目在于阐明RPM1激活的分子机制。我们证明RPM1在效应因子激活前和激活后均能寡聚化，并建立了一个模型用来解释RPM1的蛋白寡聚化可以使单个效应因子激活多个RPM1分子，从而有效地增强了抗病信号输出，提高了RPM1对病原菌效应因子的敏感性。RPM1互作蛋白RIN13可以抑制RPM1激活引起的HR反应，但能增强了RPM1介导的抗性，说明HR反应并不是抗病反应所必需的。我们证明RIN13定位于植物细胞核中并揭示了RIN13能够增强植物抗病性的主要原因在于抑制植物叶表面气孔的开度，阻止了病原菌的侵染。植物激素水杨酸（SA） 及高水平H2O2的积累对RIN13引起的气孔关闭有介导作用。RPM1的守卫蛋白RIN4可以被病原菌释放的多种效应因子修饰或水解，说明其在植物抗病反应中发挥重要作用。有研究发现Exo70E2蛋白会与植物抗病相关蛋白RIN4发生相互作用，我们以此为切入点，发现RIN4的第131-180氨基酸片段会与Exo70E2的第201-639个氨基酸片段发生相互作用，这种相互作用会使Exo70E2蛋白的含量降低。从胞内外分布来看，RIN4会使质外体中的Exo70E2分布变多。总的来说，RIN4可能控制着胞内物质非常规性地吐出的过程，这些被吐出的物质很可能是抗病相关物质，这一系列的研究能为阐述RIN4在植物天然免疫中的重要作用提供有价值的信息。通过对RPM1复合物的纯化及分析，我们发现磷脂酶D和C与RPM1存在蛋白相互作用关系。并且在体内及体外验证了RPM1与PLD的相互作用。实验结果显示PLD过量表达可以降低RPM1的蛋白含量，同时抑制RPM1介导的抗病反应。.