橡胶树胶孢炭疽病菌效应蛋白基因的分离及致病机理分析

Anthracnose of rubber tree was caused by Colletotrichum gloeosporioides and lead to serious economic losses in rubber planting area in China. It has been reported that effectors play crucial role in the invasion (recognition), colonization and disease development during host-pathogen interactions. However, the pathogenic mechanisms of anthracnose disease of rubber tree are not clear. We have found that the virulence of different C. gloeosporioides is various significantly. We have sequenced the genome of C. gloeosporioides with high virulence and bioinformatics analyses has been done. Three of putative effector genes were isolated and their function was studied primarily. In this project we propose to finish the genome sequencing of C. gloeosporioides with weak virulence. Differential effectors in two fungus genomes will be identified by comparative genomics. Functions of these effectors will be investigated. Additionally the targets in host cell of these key effectors will be isolated by yeast-two-hybrid and/or co-immunoprecipitation to understand how effector trigger or suppress defense responses of rubber tree. It will be very helpful to build up new control strategy for this serious fungal disease of rubber tree. The innovativeness of this project is the rapid isolation of key effectors of rubber anthracnose fungus by comparative genomics and the use of protoplast transient expression system to study the regulation of effectors on innate immunity of rubber tree.

胶孢炭疽属丝状真菌，能够引起橡胶树炭疽病，该病在橡胶园区发生日趋严重，年均橡胶产量损失10-15%。病原菌的效应蛋白对其在寄主植物的侵染（识别）、定殖和病害发生都至关重要，然而炭疽病菌对橡胶树的致病机理还不清楚，对其效应蛋白的研究也极其有限。 我们的前期研究发现不同胶孢菌株对橡胶树的致病力差异显著，并对一个强致病力胶孢炭疽菌株基因组进行了测序，利用生物信息学方法预测了候选效应蛋白基因，对3个候选基因功能进行了初步分析。本项目计划对一个弱致病胶孢菌株的基因组测序，通过比较基因组学分离强致病性炭疽效应蛋白基因并进行功能分析，进而寻找其在寄主细胞中的作用靶标，阐明其调控相关信号通路的生化机理。这对了解炭疽菌对橡胶树的致病机理、建立新型病害防控策略具有理论意义。本项目利用比较基因组学方法快速分离胶孢炭疽效应蛋白基因，通过原生质体瞬时表达系统研究效应蛋白对橡胶树先天免疫反应的影响，具有明显的创新性。

胶孢炭疽菌和尖孢炭疽病菌都能够引起橡胶树的炭疽病，对橡胶生产造成严重的经济损失。在海南省胶孢炭疽菌的致病性远远强于尖孢炭疽菌对橡胶树的致病性。效应蛋白是病原菌产生的小分子胞外分泌蛋白，在植物与病原微生物的相互作用中发挥重要作用。但目前对于橡胶树胶孢炭疽病菌效应蛋白的致病机理还不清楚。本研究对胶孢炭疽菌和尖胞炭疽菌的基因组进行了测序，利用生物信息学方法预测到415个胶孢炭疽菌和301个尖孢炭疽菌候选效应蛋白基因。从中筛选出4个胶孢炭疽菌候选效应蛋白基因（分别命名为CgE2、CgE5、CgE8和CgE12）和2个胶孢炭疽菌特有的效应蛋白基因CgSPE23和CgSPE26进行克隆和功能分析。我们根据同源重组的原理构建了这些基因的敲除突变体，对突变体的遗传学表型和在橡胶树上的致病性进行了分析。结果表明，这6个候选效应蛋白基因的功能敲除突变体对橡胶树叶片的致病性较野生型菌株明显减弱，说明这些候选效应蛋白基因在胶孢炭疽菌对橡胶树的致病过程中发挥作用，是重要的致病因子。此外，实验结果证明这些基因也参与了胶孢炭疽菌的生长、发育和代谢过程,包括孢子产量、黑色素的生成、对Fe2+的吸收调控以及菌丝体的生长等。以上研究结果有助于了解胶孢炭疽菌对橡胶树的致病机理、对于建立新型病害防控策略具有重要理论意义。