山茶刺盘孢长线形病毒引起寄主致病力衰退的分子机理研究

Colletotrichum camelliae causes leaf blight diseae of Camellia sinensis, spreading in the tea-cultivation area worldwide and severely affacting the tea production of most varieties in the summer and autumn. With the increasement of organic tea cultivation, the disease has truned severer year by year. Therefore, it is particularly important to develop new biocontrol measures. Virus, as an important biocontrol agent, has been successfully applied to prevention and controlment of chestnut blight and rape sclerotinia rot disease. In previous studies, we have found a new filamentous dsRNA virus in a attenuated strain of C. camelliae, named Colletotrichum camelliae filamentous virus 1 (CcFV-1), and clarified that CcFV-1 can signficantly induce the virulence of the host, but the relevant mechanism remains unclear. In this project, methylation and expression profiles of the host genes mediated by CcFV-1 inoculations will be screened by whole-genome methylation sequencing and transcriptome sequencing, further confirmed by other methods, and the candidate genes will be subjected to identifying their functions. It will put into effect to understand the virulence-related genomic changes induced by CcFV-1 from two aspects including transcription and post-transcriptional pathways, uncover the key genes and metabolic pathways involved in the host virulence attenuated by CcFV-1, and then clarify the molecular mechanism of the host virulence attenuation, which is expected to build theoretical base for insight into the breakthrough point of controlment of tea fungal diseases using viruses or morden molecular biological techniques.

山茶刺盘孢菌引起的茶云纹叶枯病在世界茶区广泛分布，严重危害大部分主栽品种的夏秋茶产量。随着有机茶园日益增多，该病危害逐年加重，寻找新的生防措施显得尤为重要。病毒作为一种重要的生防资源，已经应用于板栗疫病和油菜菌核病的防治。本研究在山茶刺盘孢弱毒菌株中首次发现一种新的长线形dsRNA病毒，命名为山茶刺盘孢线形病毒（CcFV-1），并明确该病毒可引起寄主致病力显著下降，但相关机理尚不明确。本项目拟采用甲基化和转录组测序比较分析CcFV-1侵染前后寄主基因甲基化和转录水平的变化，然后，采用其它方法对测序结果验证，进而对所推测的靶基因进行功能鉴定，从转录和转录后两个层次分析CcFV-1侵染对寄主致病力相关基因的调控，明确CcFV-1导致寄主致病力下降的关键基因和代谢途径，解析CcFV-1引起寄主致病力衰退的表观遗传机理，为采用病毒或现代分子生物学技术控制茶树真菌性病害探寻突破点奠定理论基础。

为解析山茶刺盘孢线形病毒（Colletotrichum camelliae filamentous virus 1, CcFV1）引起寄主致病力衰退的分子机理，加深人们对弱毒现象的深入了解，从而加以改进完善，推动在生物防治中的实际应用。本研究项目首先对山茶刺盘孢菌（Colletotrichum camelliae）进行了全基因组测序，组装，并进行了注释。以CcFV-1和山茶刺盘孢菌带毒、脱毒和传毒菌株为研究对象，将脱毒菌株和带毒菌株经茶叶诱导后分别进行了全基因甲基化测序和转录组测序，并对测序结果进行了验证。全基因甲基化测序结果显示，受病毒侵染后，寄主全基因组甲基化水平降低，整体甲基化无毒菌株约为0.543%，转染带毒菌株约为0.457%，无论是在基因功能区域还是基因上下游，脱毒菌株的甲基化水平较传毒菌株高。一共鉴定出108个差异甲基化区域（DMR），主要分布在基因间区，多数为高甲基化。通过对差异甲基化区域进行锚定，一共锚定到18个基因，包括叉头蛋白sep1转录因子（Forkhead protein sep1）、碱性亮氨酸拉链（Basic lecucine zipper, bZIP）转录因子AP-1、漆酶、镁离子转运蛋白、过氧化物酶体还原酶等。转录组测序一共鉴定到1015个差异基因（DEG），并对DEG进行了GO富集和KEGG富集，GO主要富集在丝氨酸型肽酶，辅酶结合，离子结合，氧化还原酶，四吡咯结合，亚铁血红素结合等功能。KEGG主要富集在戊糖和葡萄糖醛酸酯转换，酪氨酸代谢，淀粉和糖代谢，糖酵解和糖异生，脂肪酸代谢等通路。对候选靶基因叉头蛋白转录因子sep1编码基因（Ccsep1）、组蛋白甲基转移酶（CcSET2）和钙调素依赖蛋白激酶1（CcCMAK1）进行了敲除与回补，发现这三个基因均与菌株的营养生殖和致病力相关，可能参与不同的生物胁迫途径。该研究表明CcFV-1侵染可引起真菌寄主转录因子区域甲基化水平改变（主要包括叉头蛋白sep1转录因子和碱性亮氨酸拉链转录因子AP-1等），并通过钙调素依赖蛋白激酶1和组蛋白甲基转移酶影响蛋白质磷酸化和甲基化水平，进而影响致病相关基因表达，影响寄主真菌的致病性，为进一步明确CcFV-1 引起山茶刺盘孢菌致病力衰退的分子机理奠定了基础。