单股负链RNA病毒SsNSRV-1引起植物病原真菌核盘菌衰退的分子机制研究

Mycoviruses are widespread in nature. Previously, we isolated a mononegavirus SsNSRV-1 from a hypovirulent strain of fungal plant pathogen Sclerotinia sclerotiorum, and we found SsNSRV-1 could cause debilitation on its host. When infected by this virus, S. sclerotiorum grew slowly on PDA medium and lost the ability to produce sclerotia, a structure for dormancy and sexual reproduction; more importantly, the virus-infected strain also lost its virulence. This was the first time we demonstrated negative RNA virus could infect fungi; our finding expanded our knowledge on host range of negative RNA virus. In this proposal, our objectives are:①understanding the mechanism that SsNSRV-1 causes the debilitation of S. sclerotiorum; and ②identifying the viral determinant for the debilitation of S. sclerotiorum. First, we will understand the changes of key signal pathways and key metabolite pathways invirus-infected S. sclerotiorum with RNA_Seq technique and KEGG PATHWAY analysis; Second, we also will identify key genes which expressions are regulated by virus infection from the changed signal or metabolite pathways, and we will further study these genes function on hyphal growth, sclerotial formation and virulence with RNAi silence technique. Hopefully, we will understand why S. sclerotiorum could debilitate after infection of virus. We also may understand the mechanism for S. sclerotiorum to coordinate the hyphal growth, sclerotial formation and virulence on molecular level. Second, we will identify S. sclerotiorum-debilitation determinant of SsNSRV-1.The viral genes will be cloned and integrated on chromosome of S. sclerotiorum with Agrobacterium-mediated transformation system. The gene which could cause a similar debilitation of S. sclerotiorum will be looked as viral sclerotiorum-debilitation determinant. We also will study the mechanism of the determinant through observing the subcellular location of the viral determinant using immune colloidal gold technique and RFP gene fusion technique, and identify the host proteins which could interact with the determinant with peptide mass fingerprinting, Co-IP, yeast two-hybridization, BiFC and other techniques. Our studying is likely to help us understanding the coordination of S. sclerotiorum during hyphal growth, sclerotial formation and virulence; it also may lead to the discovery of a resistance gene for genetic modification of crops to enhance the resistance against S. sclerotiorum. Furthermore, our study will enhance our understanding on interaction between negative RNA viruses and their hosts.

真菌病毒在自然界广泛存在，前期申请者从核盘菌中发现了一种单股负链RNA病毒SsNSRV-1，发现该病毒可以引起核盘菌衰退，如生长缓慢、不产生菌核和丧失致病力等。我们的发现证实负链RNA病毒可以感染真菌，拓宽了对负链RNA病毒寄主范围的认识。本申请项目拟采用RNA-Seq及相关的生物信息学技术、转基因技术、蛋白互作研究技术等从整体水平上分析感染SsNSRV-1后，核盘菌关键信号通路和代谢通路的变化，分析调控核盘菌生长、菌核形成和致病的关键基因；同时，逐个分析病毒的6个基因对核盘菌衰退的作用，从中鉴定出导致核盘菌衰退的病毒基因，即核盘菌衰退决定因子，并明确该决定因子的作用机制。项目的完成将在整体水平上阐述核盘菌生长、致病和菌核形成以及它们之间的交互关系，有可能为作物抗菌核病育种提供潜在的基因材料；也将促进对负链RNA病毒与寄主相互作用的认识。

前期从核盘菌中发现了一种单股负链RNA病毒SsNSRV-1，发现该病毒可以引起核盘菌衰退，如生长缓慢、不产生菌核和丧失致病力等。本项目目标从SsNSRV-1的基因组中鉴定出对核盘菌衰退具有显著影响的基因。取得了一下主要的结果：（1）采用RNA-Seq及相关的生物信息学技术鉴定出核盘菌菌株AH98中共携带6个病毒SsNSRV-1， SsHV1， SsDelV2， SsMV6，SsMV7和一个新的负链病毒Sclerotinia sclerotiorum negative-stranded RNA virus 5（SsNSRV5），表明AH98的衰退与其中的病毒群体有关。（2）明确了感染SsNSRV-1后核盘菌关键信号通路和代谢通路的变化，分析了调控核盘菌生长、菌核形成和致病的关键基因。（3）通过转基因技术逐个分析病毒SsNSRV-1的5个基因ORF 1、ORF 2、ORF 3、ORF 4和ORF 6在核盘菌中的功能，从中鉴定出导致核盘菌衰退的病毒基因ORF 1，即核盘菌衰退决定因子。（4）通过转录组测序技术，解析了ORF 1调控核盘菌衰退的作用机制。功能预测的结果表明蛋白ORF 1含有一个核定位信号和两个DNA结合位点。通过比较表达ORF 1的突变菌株和野生型菌株1980的转录组数据，鉴定了686个响应ORF 1的DEGs。富集分析结果表明，被ORF 1表达调控的DEGs参与了转录，跨膜转运，蛋白质生物合成，修饰和代谢过程等多个重要生物学过程。（5）同时还鉴定出大量与分泌蛋白或致病性有关的DGEs。结果提示ORF 1蛋白可能调控核盘菌基因的转录，以促进病毒的增殖并降低宿主的毒力。ORF 1为作物抗菌核病育种提供了潜在的基因材料。（6）发现真菌（核盘菌）具有识别病毒的RNA并将其剪切的功能，预示真菌中可能存在鲜为人知的抗病毒机制。研究结果对认识病毒及其与寄主互作将具有良好的促进作用。