稻瘟病菌SNARE蛋白MoVam7与MoSec22的结合蛋白及其调控的分泌蛋白生物学功能研究

Exocytosis and endocytosis mediated by Solube N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins are essential for cellular process of eukaryotic cells including fungal organisms. Some secreted proteins including avirulence effectors, extracellular hydrolytic enzymes as well as secretory proteins play critical roles in virulence in phytopathogens. Although many evidences revealed that exocytosis and endocytosis play an important role in growth, development and pathogenesis, the molecular mechanisms of exocytosis and endocytosis is poorly understood in Magnaporthe oryzae. Our previous studies found two SNARE proteins, MoSec22 and MoVam7 play important roles in the development, growth, exocytosis, endocytosis and pathogenicity of M. oryzae. In this project, the first objective is to identify the binding proteins of these two SNARE proteins by co-IP and elucidate the biological function of the binding proteins in development, exocytosis, endocytosis, virulence, vacuole and vesicle assembly. The second objective is to use dimensional electrophoresis and mass spectrometric analysis to identify the secretory proteins regulated by MoVam7 and MoSec22, respectively. And we will further characterize the functions of the secretory proteins in development, growth and pathogenicity. The third objective is to elucidate the effect of MoVam7 and MoSec22 on the vacuole and vesicle proteomics. Taken together, the results above will help to reveal the role of the signaling networks mediated by MoVam7 and MoSec22 in the molecular aspects of exocytosis, endocytosis and pathogenicity of M. oryzae. Moreover, it will provide potential targets to design new fungicide to prevent and control the rice blast fungus and other fungi.

分泌和胞吞是真核细胞内的两条重要的运输途径，受SNARE蛋白介导的囊泡运输调控。不少分泌蛋白在稻瘟病菌的生长发育和致病过程中具有重要作用，但是其分泌机制仍不清楚。项目组在前期工作中发现，SNARE蛋白MoSec22和MoVam7通过调控囊泡运输控制胞吞与分泌，从而参与该病菌的生长发育与致病过程。本项目拟采用co-IP技术鉴定MoVam7和MoSec22的结合蛋白，并阐明这些蛋白在该病菌生长发育、胞吞、分泌、致病中的作用机制；采用蛋白质双向电泳鉴定受MoVam7、MoSec22调控的泌出蛋白种类，并阐明这些蛋白的生物学功能；比较分析MoVam7和MoSec22对病菌囊泡和液泡蛋白组分的调控，并鉴定受其调控的囊泡蛋白的生物学功能。研究结果有助于揭示MoVam7和MoSec22介导的基因调控网络在该病菌胞吞、分泌和致病中的作用分子机制，同时为开发防治稻瘟病的药剂提供新的靶标。

分泌和胞吞是真核细胞内的两条重要的运输途径，受SNARE 蛋白介导的囊泡运输调控。不少分泌蛋白在稻瘟病菌的生长发育和致病过程中具有重要作用，但是其分泌机制仍不清楚。项目组在前期工作中发现，SNARE 蛋白MoSec22 和MoVam7 通过调控囊泡运输控制胞吞与分泌，从而参与该病菌的生长发育与致病过程。本项目采用co-IP 技术在稻瘟病菌中鉴定SNARE蛋白MoVam7和MoSec22 的结合蛋白，对这些结合蛋白进行生物信息学分析，获得候选基因，进一步采用基因敲除技术(致死突变的基因采用沉默技术)，阐明这些蛋白组分在稻瘟病菌生长发育、胞吞、分泌、致病、以及囊泡组装中的作用机制；同时以基因敲除突变体ΔMovam7 和ΔMosec22 及野生型亲本菌株为材料，采用蛋白质双向电泳技术和质谱分析法鉴定MoVam7和MoSec22 调控的外泌蛋白种类，并进一步采用基因敲除技术阐明这些蛋白的生物学功能，同时揭示MoVam7和MoSec22 及其具有明显生物表型的结合蛋白对效应蛋白分泌的影响；进一步采用基因敲除技术阐明MoVam7 和MoSec22 对稻瘟病菌囊泡和液泡组装的影响，及差异蛋白组分的生物学功能。研究结果有助于揭示SNARE 蛋白MoVam7 和MoSec22 介导的基因网络在稻瘟病菌胞吞、分泌和致病中的作用分子调控机制，同时为开发防治稻瘟病的新型药剂提供新的靶标基因。