橡胶树胶孢炭疽菌效应蛋白CgBAS的致病机制研究

The rubber anthrax caused by Colletotrichum gloeosporioides has brought serious economic loss to the rubber industry of Hainan. Recent researches showed that the effector proteins play important roles in the invasion and colonization of pathogen in host plant. Functions of effector have become a research focus to understand the mechanism of pathogenicity in the international field of plant pathology. Previously, we have predicted the effector protein family CgBAS (which contains two members, CgBAS2 and CgBAS4) of C. gloeosporioides by genome sequencing and bioinformatics. The growth rate and the pathogenicity of the CgBAS2 knockout mutant were seriously impaired, and the mutant lost the ability to generate conidia. In this project we are going to analyze the proteins regulated by CgBAS and their functions in regulating growth and conidiation of C. gloeosporioides. Secondly, we would isolate the targets of the CgBAS in the host cell and research the functions of the isolated targets by using the rubber protoplast transient expression system. This project will not only provide the new evidence for the molecular mechanism of CgBAS in regulating the pathogenicity of C. gloeosporioides, but also is helpful to build up new control strategy on rubber anthrax. The innovativeness of this project is that the pathogenic mechanism of CgBAS will be studied in both aspects of the pathogen and the host cell, based on using protoplast transient expression system on pathology of rubber tree.

胶孢炭疽菌引起的橡胶树炭疽病是造成海南橡胶产业减产的主要因素之一。效应蛋白在病原菌对寄主植物的侵染和病害发生中起着重要作用。效应蛋白的研究是当前国际植物病理学领域的研究热点。本课题组对一个强致病胶孢炭疽菌进行基因组测序，利用比较基因组学方法发现了含有两个成员的CgBAS效应蛋白家族。其中CgBAS2的缺失突变株表现为致病力显著下降且丧失产生分生孢子的能力。本项目计划通过筛选CgBAS在胶孢炭疽菌中的互作蛋白来研究CgBAS调控其生长和孢子产生等生理功能的机制；通过橡胶树原生质体瞬时表达系统研究CgBAS在植物细胞中的互作因子及其功能。以上研究结果将全面解析效应蛋白CgBAS在胶孢炭疽菌致病中的功能，对于建立新型病害防控策略具有理论意义。本项目从病原菌和寄主植物两个层次解析CgBAS的致病机制，同时将原生质体瞬时表达系统用于橡胶树病理学研究，在研究思路和研究方法上具有创新性。

效应蛋白在病原真菌对寄主植物的致病过程中起着重要作用。本课题分析了橡胶树胶孢炭疽菌活体寄生相关效应蛋白（BAS）调控该病原菌致病力的分子机制，通过构建两个蛋白编码基因的敲除突变株、以及对突变株表型分析发现，CgBAS2在调控胶孢炭疽菌生长、产孢以及致病力中都起着重要作用。首先，通过同源重组原理对CgBAS2及CgBAS4基因的编码序列全长进行了敲除，并分别获得了3个敲除突变株。在离体条件下培养发现，CgBAS2的缺失会导致胶孢炭疽菌生长速率降低、产孢量下降；而CgBAS4的缺失没有明显影响。之后，通过构建CgBAS2-GFP融合表达菌种以及在橡胶树原生质体中瞬时表达，发现CgBAS2在胶孢炭疽菌中为胞外分泌蛋白，而在寄主细胞中，该蛋白主要定位于细胞质中。进一步通过胶孢炭疽菌胞外分泌蛋白组分析，发现CgBAS2能够调节一系列与致病力相关的胞外蛋白的丰度。综合上述结果，本研究阐明了CgBAS2调控胶孢炭疽菌致病力的分子机制。