真菌病毒改变灰葡萄孢与重寄生菌盾壳霉非寄主互作机理的研究

Coniothyrium minitans is a mycoparasite of fungi that belong to the genus Sclerotinia, while it cannot parasitize Botrytis cinerea,a destructive fungal plant pathogen. Phylogenetically, Botrytis cinerea (Botryotinia fuckeliana) is very closely related to fungi in the genus Sclerotinia.There exists a non-host recognition interaction system between B. cinerea and C. minitans, however,the mechanism is still unknown. In previous research, we isolated a wildtype strain 10HN454 from a diseased rapeseed plant. We found that strain 10HN454 could be parasitized efficiently by C. minitans,and C. minitans could grow on the colony of strain 10HN454 and produced plentiful conidia there; we further found that strain 10HN454 was co-infected by two distinct mycoviruses, one is a member of Totivius, and the other is closely related to Hypovirus phylogenetically.Thus, we suspect that mycoviruses in strain 10HN454 modify the non-host recognition interaction system. Therefore, 10HN454 is a unique material to study the interaction of non-host fungus and mycoparasite. In this proposal, based on the unique interaction system of 10HN454 and C. minitans, the relativity of carrying mycovirus and parasitism will be determined, and the molecular mechanism that mycoviruses modify the recognition between B. cinerea and C. minitans will be studied with yeast two-hybrid, Co-IP techniques and so on. The expected results will enrich our knowledge on the interaction between mycovirus and fungus, between fungus-fungus and between virus and fungus-fungus.

盾壳霉是核盘菌属真菌的重寄生真菌，不能寄生与核盘菌亲缘关系相近的灰葡萄孢，灰葡萄孢与盾壳霉之间存在非寄主互作机制。目前有关重寄生真菌与非寄主真菌互作机制尚不明了。前期我们分离获得了灰葡萄孢野生菌株10HN454，发现它可被盾壳霉高效寄生，并在其菌落上产生大量分生孢子，同时也发现菌株10HN454感染了两种病毒。我们怀疑病毒改变了灰葡萄孢与盾壳霉的非寄主互作系统。菌株10HN454的发现为非寄主真菌与重寄生真菌分子互作研究提供了独特的材料。本项目拟以病毒-灰葡萄孢-盾壳霉互作体系为材料，进一步确定病毒与"灰葡萄孢/盾壳霉"非寄生关系改变的相关性，研究病毒对灰葡萄孢的影响，采用超量表达病毒基因、酵母双杂交、Co-IP等技术探讨病毒改变灰葡萄孢/盾壳霉非寄生互作系统的分子机理。项目的完成将为建立真菌与重寄生真菌非寄主互作理论奠定基础、丰富病毒学知识、也将为拓宽重寄生真菌的寄主范围提供新思路。

盾壳霉是核盘菌属真菌的重寄生真菌，不能寄生与核盘菌亲缘关系相近的灰葡萄孢，灰葡萄孢与盾壳霉之间存在非寄主互作机制。葡萄孢野生菌株10HN454可被盾壳霉高效寄生，同时也感染了两种病毒。本研究探索盾壳霉-葡萄孢寄生的机理。首先明确了菌株10HN454 的分类地位，根据ITS、G3PDH、HSP60和RPB2基因的核苷酸序列，将其鉴定为拟蚕豆葡萄孢（Botrytis fabiopsis），但另一个拟蚕豆葡萄孢菌株BG-55却几乎不能被盾壳霉寄生；克隆了菌株10HN454中两个病毒BfTV1/10HN454和BfHV1 /10HN454，BfHV1 /10HN454编码的蛋白中存在一个保守结构域表皮生长因子受体底物蛋白EGFRS，该因子的超量表达使核盘菌、灰葡萄孢和盾壳霉表型出现明显的变化。在核盘菌菌株1980和灰葡萄孢菌株B05.10中，通过免疫沉淀及蛋白胶条测序技术鉴定到核盘菌和灰葡萄孢中共有181个蛋白，其中有47个蛋白为可能非特异性互作的核糖体蛋白，其他134个候选蛋白。对蛋白功能的解析将为真菌病毒BfHV1/10HN454与寄主互作的机制提供线索。盾壳霉寄生菌株10HN454过程中可以自寄主中获得两个病毒，病毒对盾壳霉没有显著的影响，同时染毒的盾壳霉不能将病毒重新传回核盘菌和拟蚕豆葡萄孢菌株。分析了盾壳霉与核盘菌、灰葡萄孢和10HN454互作的转录组，筛选了在核盘菌和10HN454中抑制表达而在灰葡萄孢中增强表达的基因进行功能分析。研究了葡萄孢基因BC1G_00904、BC1G_12653和盾壳霉基因CmVps39 和CmNox1，发现BC1G_12653、CmVps39 和CmNox1的缺失影响盾壳霉的寄生。项目的完成为建立真菌与重寄生真菌非寄主互作理论奠定了基础，丰富了病毒学知识、也为拓宽重寄生真菌的寄主范围提供了新思路。