转录因子MoMsn2介导的稻瘟病菌HOG与CWI信号途径的交叉对话机制研究

Eukaryotic cells have developed complex signaling networks to sense extracellular signals and integrate them into intrinsic signal transduction pathways, thus response and adaptation different environmental stress situations. High-osmolarity glycerol (HOG: Ssk2-Pbs2-Hog1) and cell wall integrity (CWI: Mck1-Mkk1-Mps1) signaling pathways, which are important for the growth, stress response, cell wall integrity and infection in the rice blast fungus Magnaporthe oryzae, are the main pathways for response high osmotic stress and cell wall stress in this pathogen. Our previous studies revealed that transcription factor MoMsn2, which played crucial roles in the development and pathogenicity of M. oryzae, physically interacts with the key member of HOG pathway MoHog1. In addition, we found that HOG pathway might have crosstalk with CWI pathway in M. oryzae. The phosphorylation level of MoHog1 was regulated by the members of CWI. However, why MoHog1 interacts with MoMsn2? What is the mechanism of HOG and CWI pathways interplay? To answer these two questions, firstly, protein phosphorylation assay will be used to analysis whether MoMsn2 phosphorylated by MoHog1, and the phosphorylation site in MoMsn2 also will be identified and characterized. Secondly, under stress and no stress treatment conditions, western blot assays will be adopted to analyze the phosphorylation level of MoHog1 and MoMps1 in the mutants of the key members of HOG and CWI pathway. Thirdly, protein affinity purification assays will be further used to identify the binding proteins of MoHog1 and MoMps1. Furthermore, the role and mechanism of the candidate binding proteins during the interplay of HOG and CWI pathways will be characterized. Our results will finally clarify the biological significance of the interaction between MoHog1 and MoMsn2; reveal the regulatory mechanism of interplay between HOG and CWI pathways. The results will also provide potential fungicide targets based on the HOG and CWI pathways for novel fungicide designment.

高渗透胁迫(HOG: MoSsk2-MoPbs2-MoHog1)途径与细胞壁完整性(CWI: MoMck1-MoMkk1-MoMps1)途径是稻瘟病菌应答外界渗透和细胞壁胁迫的主要途径。项目组前期发现，转录因子MoMsn2与MoHog1互作，调控病菌的生长发育及致病力，但其互作的生物学意义还不清楚。同时，发现CWI和HOG途径存在交叉对话关系，但其对话机制仍不清楚。本项目拟分析MoMsn2是否被MoHog1磷酸化，并鉴定和阐明MoMsn2中的磷酸化位点及其功能；分析MoHog1与MoMps1在两途径关键组分突变体中的磷酸化水平，阐明两途径的关系；鉴定MoHog1和MoMps1的结合蛋白，阐明结合蛋白的功能并解析其在HOG与CWI途径对话中的调控机制。研究结果可望揭示MoMsn2与MoHog1互作的生物学意义及HOG与CWI途径对话的分子机制；为开发以该途径重要蛋白为靶标的新型药剂提供依据。

高渗透胁迫(HOG: MoSsk2-MoPbs2-MoHog1)途径与细胞壁完整性(CWI: MoMck1-MoMkk1-MoMps1)途径是稻瘟病菌应答外界渗透和细胞壁胁迫的主要途径。项目负责人前期发现转录因子MoMsn2与MoHog1互作，调控病菌的生长发育及致病力，但其互作的生物学意义还不清楚。同时，发现CWI和HOG途径存在交叉对话关系，但其对话机制仍不清楚。在本项目的资助下，项目组初步明确了MoMsn2是MoHog1的一个磷酸化底物，并鉴定和解析了MoMsn2中的磷酸化位点及其功能；分别鉴定到一系列与MoHog1和MoMps1相互结合的蛋白，阐明了其中二个关键蛋白的生物学功能，并解析了这两个关键蛋白在病菌生长发育及致病过程中介导HOG与CWI途径对话的分子机制。研究结果不仅揭示了MoMsn2与MoHog1互作的生物学意义及HOG与CWI途径对话的分子机制，而且对以该信号途径关键蛋白为靶标的新型杀菌剂的开发及病害新防治策略的制定具有重要的理论和实践指导意义。主要研究结果如下：.一、阐明了MoHog1与MoMsn2互作的生物学意义，发现MoMsn2是MoHog1的一个磷酸化底物，鉴定到7个潜在磷酸化位点并初步明确了其生物学功能。.二、鉴定到一系列分别与MoMps1和MoHog1相互结合的蛋白。功能预测发现这些蛋白参与病原菌的信号传导、能量代谢、细胞壁完整性等多种生理生化及代谢过程。.三、解析了MoMps1结合蛋白MoKnr4及其不同结构域在稻瘟病菌生长发育、胁迫应答、细胞壁完整性及致病过程中的作用机制。同时，初步解析了MoKnr4参与菌株变异的分子机制。.四、揭示了MoHog1途径相关蛋白MoTip41通过调控TOR和CWI途径交叉对话，从而控制稻瘟病菌致病力的分子机制。发现MoTip41参与TOR和CWI途径，通过缓解TOR关键蛋白MoTap42对蛋白磷酸酶MoPpe1的抑制作用激活CWI通路，从而控制病菌的致病力。