冬虫夏草菌芽生孢子在寄主体腔内一致性萌发的机理研究

Ophiocordyceps sinensis and ghost moth is a unique parasitic system. The research in the development of O. sinensis in the host body is very limited because of the difficulties in rearing and infection, and long life period. We begin to pay close attention to the development of this fungy after we resolved the problems in the rearing and infection of ghost moth. Our previous research suggested that the develop progress of O. sinensis within host body is quite different from most other Hypocreales fungus. After infection, O. sinensis persists and proliferates in host hemocole as blastospores. The feeding, activity and appearance of ghost moth shows no change. This symbiosis last for more half year until the concurrent germination of hyphal bodies when host develop to last instar larva. Therefore, we put forward a hypothesis that the blastospore germination initiated from certain specific stimulation from host, possibly related to the metamorphosis start. This project will systematically study the developing progress of O. sinensis in the hemocole of ghost moth, basing on the technologies of transcriptome, gene expressing and function, and physiology to construct the gene regulation networks of blastospore differentiation and hyphal body germination, indicate the key pathway and element, especially the host related factor involved in initiation and regulation mechanisms, clarify the growth progress and hyphal bodies development initiation mechanisms of O. sinensis in the hemocole of host. This research will expand our knowledge about the development and pathogenesis mechanisms of insect pathogenic fungus, deepen our understanding of coevolution mechanism between fungus and hosts, facilitate the phylogeny and evolution study of fungus, provide theoretical guidance for the artificial culture of Dong chong xia cao.

冬虫夏草菌及其寄主蝙蝠蛾是一对非常独特的寄生系统。我们发现冬虫夏草菌成功侵染蝙蝠蛾幼虫后，发育进程与大部分肉座菌目真菌不同，是以芽生孢子的形式在寄主体腔内存在、增殖，二者和谐共存长达半年以上，直到寄主进入末龄期后，芽生孢子一致性萌发形成菌丝体，最终致死寄主。为此，我们提出科学假设，冬虫夏草菌芽生孢子分化、萌发的启动受某种寄主因素的调控，很可能与寄主的变态发育启动相关。本项目拟系统研究冬虫夏草菌入侵蝙蝠蛾幼虫体腔后至僵虫形成的发育进程，依托转录组技术，结合基因表达和功能研究及生理生化研究，构建冬虫夏草菌芽生孢子分化、萌发的基因调控网络，以及同期寄主的差异基因表达谱，揭示调控冬虫夏草菌芽生孢子分化、萌发的关键路径和调控元件，特别是寄主相关因素在其中的启动和调控机制，丰富和发展虫生真菌的发育、致病的基础理论，加深对虫生真菌寄主适应性及虫菌协同进化机制的理解，并为冬虫夏草的人工培育提供理论指导。

冬虫夏草菌侵染蝠蛾后，以芽生孢子（芽孢）在幼虫体腔内和谐共存达半年以上，直到宿主进入末龄期后，芽孢一致性萌发形成菌丝体才致死宿主。为揭示此生长型侵染策略的协同进化机制，我们对虫草菌的型态发生与分子机制开展了系统研究。通过持续形态学观察，阐明了虫草菌的型态发生进程，首次在虫生真菌中提出前菌丝这一型态类型，其独特的形态特征与增殖延申方式，及其在芽孢向菌丝型变过程中的中介作用，丰富了虫生真菌形态发生与发育理论。通过对虫草菌不同发育时期的菌、虫转录组和代谢组的整合分析，揭示了其基因表达和代谢特征，构建了虫草菌型态发生与转换的基因调控网络。芽孢泛性激活了蝠蛾免疫系统，抗菌肽、防御素等上调表达，但特异性沉默机制如免疫识别蛋白IML2的无应答，使染菌蝠蛾的细胞和体液免疫反应受到持续抑制，为虫草菌与蝠蛾的长期共存提供了保障。对芽孢microRNA的测序与分析表明存在蝠蛾来源的microRNA，可通过降低虫草菌促菌丝生长因子如OsMep2的表达，使虫草菌长期维持芽孢形态。芽孢主要利用蝠蛾血淋巴中的营养物质完成自身的发育、增殖，致使蝠蛾血淋巴氨基酸类、蛋白质和甘油磷脂类代谢物显著低于健康蝠蛾，而脂肪体的磷脂及三酰甘油含量显著高于健康蝠蛾，并体重增加，利于虫草菌的后期发育。增殖期芽孢和菌丝期的碳水化合物和氨基酸代谢旺盛，对应于快速生长增殖。成熟期芽孢脂代谢增强，细胞内脂滴大量积累。我们构建了虫草菌脂肪酸合成通路，其中乌头酸水合酶下调为关键步骤。前菌丝期上调表达的基因主要富集于折叠、排序、降解、转录、翻译，提示发生了众多分子事件，包括细胞壁、抗生素合成基因上调表达。在前菌丝和菌丝期，脂肪酸降解增强，提示脂类物质在菌丝萌发过程中的重要作用。MAPK通路调控虫草菌的型态发生与转换过程，营养匮乏可能是促成菌丝发育的一个启动条件。本项目成果丰富发展了虫生真菌发育生物学，加深了对生长型侵染策略的虫菌协同进化机制的理解，并为冬虫夏草的人工培育提供了理论指导。