蛹虫草白毛病病原菌致病机理及蛹虫草对其侵染的响应机制研究

The white-furred defection is a common fungal disease in the cultivation of Cordyceps militaris. Pathogenic mechanism of fungi and host response are the basis of disease prevention and control and breeding of disease-resistant varieties. At present, the understanding of the interaction between the parasitic fungi and the host is very limited. In the previous study, Calcarisporium cordycipiticola was identified as the pathogen and an obligate parasite of C. militaris. In this study, C. cordycipiticola was labeled by green fluorescent protein (GFP) and observed by the confocal microscopy to reveal the infection process. The interaction between C. cordycipiticola and C. militaris was studied using Dual RNA-seq, and the identification, parasitism and disease-related genes were screened. Pathogenic mechanism of C. cordycipiticola was analyzed in combination with biological characteristic, infection process and proteomics analysis. The defense enzyme activities of C. militaris after being infected were analyzed. Gene expression of C. militaris after being infected was analyzed by excavating Dual RNA-seq data. This study will reveal the interaction between pathogens and hosts and mechanism of disease resistance of edible fungi, being favorable to the prevention and control of diseases and the breeding of disease resistant varieties.

白毛病是蛹虫草栽培中普遍发生的真菌性病害，病原菌致病机理和宿主响应机制是病害防控和培育抗病品种的基础，目前对于食用菌寄生菌和宿主的相互作用认识十分有限。申请者在前期研究中确认Calcarisporium cordycipiticola为白毛病病原菌并寄生蛹虫草。本项目以绿色荧光蛋白标记C. cordycipiticola，共聚焦显微镜跟踪观察，揭示其侵染过程；Dual RNA-seq技术研究C. cordycipiticola与蛹虫草的相互作用，筛选与识别、寄生和致病相关的基因，对关键基因进行功能验证；结合侵染过程和蛋白组、代谢产物比较分析，解析其致病机理；分析蛹虫草被病原菌侵染后防御酶系活性变化，Dual RNA-seq数据分析病原菌侵染后蛹虫草基因表达的响应，揭示其抗病机制。本研究对于揭示病原菌和宿主相互作用及食用菌抗病机制，开展抗病品种选育及病害防控具有重要的科学意义和实践价值。

虫草生齿梗孢为蛹虫草“白毛病”病原菌，重寄生蛹虫草，是蛹虫草栽培中的“头号杀手”。本实验室前期对其生物学、侵染特性和发病特点进行了研究，然而，关于虫草生齿梗孢致病机理及蛹虫草的响应机制一直未知。本项目构建了虫草生齿梗孢高效转化体系，通过荧光标记、电镜观察了其侵染过程；SMRT解析其全基因组，通过Dual RNA-seq分析，筛选与虫草生齿梗孢致病、蛹虫草抗病相关基因，并对部分基因功能进行研究，同时利CRISPR/Cas9介导获得蛹虫草抗病菌株。本项目取得重要结果如下：.虫草生齿梗孢通过蛹虫草子实体菌丝间隙侵入子实体内部、极少数穿透子实体菌丝，无特殊结构形成；蛹虫草菌丝细胞壁溶解，细胞变形，细胞器丢失，最终细胞破碎，病原菌丝在子实体中肆意穿梭，导致子实体内外完全被病原菌侵占，倾向于死体营养。全基因组测序、组装至近染色体水平，初步判定有7条染色体，基因组大小34.52-Mb，编码基因10443个。对虫草生齿梗孢侵染蛹虫草子实体不同阶段进行Dual RNA-seq分析，发现虫草生齿梗孢铁载体产生核心基因Ccnps6，Ccacs1和水平转移基因Cccyt，蛹虫草疏水蛋白基因Cmhyd1在互作转录中高表达。对Ccnps6，Ccacs1和Cccyt进行敲除、回补和过表达，确认Ccnps6，Ccacs1和Cccyt调控虫草生齿梗孢致病力。Ccnps6除调控虫草生齿梗孢生长发育外，其参与合成的铁载体可能作为信号分子调控其他致病因子从而协同影响其致病力。虫草生齿梗孢敲除Cccyt后，菌落形态、菌丝生长、分生孢子和对非生物胁迫的抗性等未发生明显改变，定位在在虫草生齿梗孢细胞壁上的CcCYT蛋白可进入蛹虫草细胞与其隔膜结合，并与蛹虫草CmHsp90蛋白结合发挥致病作用。毕赤酵母外源表达CmHYD1可提高蛹虫草对白毛病的抗性。通过CRISPR/Cas9基因编辑技术在蛹虫草基因组的安全超表达位点CmSH1超表达潜在的抗病基因Cmhyd1，创制了对白毛病抗性增强的无痕编辑蛹虫草菌株，且不影响子实体的商品性。本研究对于揭示寄生性病原菌致病机理和食用菌抗病机制，开展抗病品种选育以及病害的防控具有重要的科学意义和实践价值。