介导梨树轮纹病菌（Botryosphaeria dothidea）致病力衰退相关病毒（BdCV1）与寄主分子互作的研究

Pear ring rot, caused by Botryosphaeria species, has led to substantial economic losses by creating a large number of fruit rots and stem canker, and causing svere recession of tree growth in the main pear producing areas in China.It is urgent to find new, safe and effective measures to control pear ring rot disease increasing in recent years. Mycovirus-mediated hypovirulence in plant pathogenic fungi becomes an important biological way to control of fungal diseases. This is the first report of chrysovirus BdCV1 and partitivirus BdPV1 infectiion hypovirulent B.dothidea LW-1 strain in our study. The protoplast transfection and horizontal transmission experiment results showed BdCV1 was responsible to the hypovirulence of plant phytopathogenic fungi.The project aims to verify BdCV1 key genes responsible to host biological functions recession by genetic transformation. The important candidate genes from the host related to BdCV1 infection will be selected by RNA-seq， whose expression profiles in the presence of BdCV1 and in the absence of BdCV1 will be analyzed by real-time PCR and northern-blot hybridization. Furhter, the key candidate genes will be transiently expressed by implementing the strategies of transformation overexpression vector and RNAi vector to confirm their biological functions related to host pathogenicity. In addition, the host protein factors interaction with BdCV1 will be selected and identified by high-throughput yeast two-hybrid system, whose roles respond to virus infection will be analyzed. The signal transduction pathways and key genes related to virus and pathogenicity of plant fungi will be obtained. The performance of the project will reveal the molecular mechanism of host hypovirulence associated mycovirus BdCV1 theoretically, and provide new ideas and strategies for safe controlling pear ring rot disease in practice.

梨轮纹病是由葡萄座腔菌引起，造成梨树枝干溃疡、果实腐烂，导致树势早衰，给梨产业带来严重的经济损失。梨轮纹病危害逐年加重，寻找新的、安全有效的措施防治梨轮纹病显得尤为迫切，弱毒相关病毒导致病原真菌致病力衰退，可作为真菌病害生防的另一条重要途径。申请者首次在梨轮纹病菌弱毒株中发现真菌病毒BdCV1和BdPV1，明确了BdCV1病毒是引起致病力衰退的主要因子。本项目通过正反向遗传转化的方法，明确引起寄主致病力衰退的BdCV1的关键基因及生物学功能，采用RNA-seq和酵母双杂方法，分析BdCV1对寄主致病相关基因表达影响、鉴定与BdCV1互作的寄主蛋白，明确互作蛋白在应答病毒侵染中的作用；旨在获得与病毒互作和梨轮纹病菌致病相关的关键基因和信号转导途径，为梨轮纹病的防治挖掘潜在的基因资源。项目完成在理论上可解析BdCV1引起寄主致病力衰退机理，在实践上为果树轮纹病害的安全防治提供了新的思路和策略。

梨轮纹病由葡萄座腔菌（Botryosphaeria dothidea）引起，在梨产区造成烂果，枝干粗皮、溃疡，严重影响产量和品质。轮纹病防治一直是研究的难点,迄今对轮纹病菌的致病因子及其致病机理尚不明确，严重制约了轮纹病害防治。弱毒相关病毒导致寄主致病力衰退，可作为梨轮纹病生防的一条重要途径。. 本研究建立了快速、简便、灵敏的高通量室内鉴定梨轮纹病菌等致病性测定方法；明确了梨轮纹病原菌携带真菌病毒存在丰富多样性，普遍感染产黄青霉病毒BdCV1。从前期复合感染BdCV1和双分体病毒（BdPV1）LW-1中分离获得BdCV1的弱毒菌株LW-C，首次证实了BdCV1是引起梨轮纹病菌弱致病力因子。重点开展了以LW-1，单独感染BdCV1和BdPV1菌株LW-C和LW-P，及无毒菌株Mock为研究材料，分析了病毒与梨轮纹病菌互作的生物学特性，明确了BdCV1比BdPV1对寄主生物学特性影响明显，BdCV1引起LW-C和LW-1菌丝畸形，细胞变小并溃解，不易产孢以及致病力减弱等。采用RNA-seq和RT-qPCR法，显示了病毒诱导梨轮纹病菌差异基因表达模式，明确了BdCV1对基因表达影响最大，解析了参与病毒互作的信号途径，获得了应答病毒侵染候选基因。采用二代和三代测序获得了46.34M高质量基因组序列，包含14,091个基因，重复序列占9.3%，存在甲基化；与葡萄座腔菌科成员聚为一个分支。功能注释明确了参与代谢途径，PHI途径，信号转导等，阐明梨轮纹病菌致病分子机理。证实了轮纹病菌中存在基因沉默组份（Ago1,2,3,4，Dcl1,2和RdRp1,2,3），受病毒诱导表达，结合BdCV1阻止BdPV1传入LW-C的LW-C / LW-P水平间转染结果，揭示了寄主中存在功能性小RNA并触发寄主基因沉默反应。比较基因组sRNA分析，明确了BdCV1对轮纹病菌中存在的Bd-milRNA表达量影响最明显，WEGO揭示病毒诱导Bd-milRNA调控的mRNA基因参与了代谢，细胞，生物学途径等。. 综合以上梨轮纹病菌全基因组，转录组以及milRNA/mRNA分析，揭示了抗病毒基因沉默防卫反应，Bd-milRNA及其介导调控转录水平mRNA均参与寄主与病毒分子互作，调控了轮纹病菌生物学功能。项目完成为防治果树轮纹病害提供新的思路，并为轮纹病防治挖掘了潜在基因资源。