稻瘟病菌G蛋白调控因子MoRgs7的结构域及其互作蛋白的功能分析

Signal transduction cascades are the primary means by which external cues are communicated to the nuclei of eukaryotic organisms including fungi. Heterotrimeric guanine-nucleotide binding protein (G-protein) signaling is one of the most important mechanisms by which eukaryotic cells sense extracellular signals and integrate them into intrinsic signal transduction pathways. Regulators of G-protein signaling (RGS) proteins contain a conserved domain of 120 amino acids that are required for activity and function as key negative regulators of G-protein signaling pathways. Our previous study revealed that eight RGS proteins play important roles in growth, development and pathogenicity of Magnaporthe oryzae. MoRgs7, one of RGS proteins which containing a signal peptide, a GPCR(7 transmembranes)domain as well as a RGS domain, is required for full virulence in this phytopathogen. Therefore, function analysis the domains of MoRgs7 and identify its binding proteins will help us further understand the molecular mechanism of G protein signaling pathway in rice blat disease. In this study, we will construct the vectors expression different domains of MoRgs7 and transferred to the ?Morgs7 mutant, respectively. The biological phenotypes of the resulting transformants will be analysed in comparison with wild type Guy11 and the ?Morgs7 mutant. These data will clarify the function of the domains of MoRgs7 in growth, development and pathogenicity of M. oryzae. To further explore the binding proteins of MoRgs7, yeast two hybrid system will be adopted to screen the cDNA library of Magnaporthe using MoRgs7 RGS domain as a bait protein. The candidate binding proteins will be analysed by bioinformatics and their interaction with MoRgs7 RGS domain will further confirmed by co-IP. The confirmed candidates will be selected to knock out and functional characterized. These results will not only reveal the signaling pathways involving in growth, development and virulence of M. oryzae which regulated by MoRgs7, but also provide insight into the underlying molecular mechanisms that will directly benefit efforts to design fungicide and aid in the development of new disease management strategies.

异三聚体G蛋白信号途径是真核细胞感受外源信号，并将外源信号整合到胞内信号系统中最重要的信号传导途径之一，G蛋白调控因子RGS是该信号途径的负调控因子。项目组前期研究发现稻瘟病菌含有8个RGS:调控该病菌的生长发育及对水稻的致病性，其中参与致病过程的MoRgs7预测含有信号肽、7次跨膜的GPCR结构域和RGS 结构域。本项目拟阐明MoRgs7的三个结构域在该病菌生长发育和致病中的作用；采用酵母双杂交和co-IP 技术鉴定和验证MoRgs7的互作蛋白，并解析其生物学功能，从而阐明MoRgs7调控的信号途径在该病菌生长发育和致病中的作用机制。研究结果不仅有助于深入揭示G蛋白信号途径介导的分子机制，而且可为开发以MoRgs7互作蛋白为靶标的高效、低毒的杀菌剂提供参考。

异三聚体G 蛋白信号途径是真核细胞感受外源信号，并将外源信号整合到胞内信号系统中最重要的信号传导途径之一，G 蛋白调控因子RGS 是该信号途径的负调控因子。项目组前期研究发现稻瘟病菌含有8 个RGS:调控该病菌的生长发育及对水稻的致病性，其中参与致病过程的MoRgs7 预测含有信号肽、7 次跨膜的GPCR 结构域和RGS 结构域，但这些结构域的生物学功能及MoRgs7的调控机制并不清楚。因此。本项目的主要研究内容是解析MoRgs7 的三个结构域在该病菌生长发育和致病中的作用；采用酵母双杂交和co-IP 技术鉴定和验证MoRgs7 的互作蛋白，并解析其生物学功能，从而阐明MoRgs7 调控的信号途径在稻瘟病菌生长发育和致病中的作用机制。项目组通过3年的研究，取得了以下主要研究进展： 1）发现RGS结构域是MoRgs7蛋白的核心结构域，对MoRgs7的生物学功能具有决定作用，参与病菌的致病过程；而其它几个结构域的缺失并不影响MoRgs7的功能。2）鉴定到35个与MoRgs7相互结合的蛋白，并对其中的R7IP-2（Rgs7-Interacting Protein）的生物学功能进行了深入分析，发现R7IP-2通过与MoRgs7相互作用，共同调控病菌的营养生长、无性繁殖、附着胞侵入及致病性等生物学过程。3）鉴定到45个受MoRgs7调控的蛋白，并对其中的MoLys20和MoIlv1的生物学功能进行了深入分析。研究结果发现MoLys20和MoIlv1通过分别参与稻瘟病菌赖氨酸和异亮氨酸的生物合成，从而参与调控病菌的营养生长、无性繁殖、侵染菌丝生长和致病过程。上述研究结果不仅有助于深入揭示G 蛋白信号途径介导的分子机制，而且可为开发以MoRgs7 互作蛋白获调控蛋白为靶标的高效、低毒的杀菌剂提供参考。共发表SCI研究论文4篇。