烟草扭脉病毒协助烟草丛顶病毒经烟蚜传播的作用机理

Tobacco bushy top disease occurred in Yunnan is a severe disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), the satellite RNA of TBTV (TBTV-satRNA) and TVDV associated RNA (TVDVaRNA) . The disease is mainly transmitted by Myzus persicae in nature, and TVDV plays an important role in the aphid transmission. In consideration of the specific characteristics of the complex agents and the importance about aphid transmission in the disease occurrence and epidemic, this proposal aims to reveal the mechanism of TVDV assisting TBTV in the process of aphid transmission on the base of the previous related progresses on tobacco bushy top disease. This proposal also wants to discribe the relationship and interaction of different causal agent in Myzus persicae transmission through construction of the infectious clones and deletion mutants possibly related to aphid transmission of the complex agents. To show the infection and circulation route of TVDV in Myzus persicae and identify the key aphid transmission-related proteins encoded by TVDV using immunofluorescent labeling and RNA interference (RNAi) techniques. To discuss the roles about TVDV coat protein (CP) and readthrough (RT) protein in Myzus persicae transmission and demonstrate the encapsidation of TVDV CP for TBTV genomic RNA, and therefore reveal the mechanism about TVDV acting as an assistant virus of TBTV for Myzus persicae transmission. The results obtained will provide the foundation for the further study about the interaction between related viruses and aphid, and finally reveal the aphid transmission mechanism of tobacco bushy top disease agent complex.

发生于云南的烟草丛顶病是一类特殊的病害，由烟草丛顶病毒（TBTV）、烟草扭脉病毒（TVDV）、烟草丛顶病毒卫星RNA（TBTV-satRNA）及烟草扭脉病毒相关RNA（TVDVaRNA）等病原物复合侵染引起。该病害在自然情况下主要通过烟蚜传播，TVDV在烟蚜传播中起着非常重要的作用。鉴于烟草丛顶病病原物的特殊性及烟蚜传播对病害发生流行的重要性，本项目拟在前期工作基础上，通过构建烟草丛顶病主要病原物侵染性克隆及其可能与烟蚜传毒相关基因的缺失突变体，分析各主要病原物在烟蚜传播中的相互关系及作用；并利用免疫荧光标记技术与RNAi技术，解读TVDV在烟蚜体内的侵染循回过程，鉴定TVDV编码的与烟蚜传毒相关的关键因子，探讨TVDV的CP及其通读蛋白在烟蚜传播TBTV中的作用，分析TVDV CP对TBTV基因组RNA的异源包裹作用，揭示TVDV协助TBTV进行烟蚜传播的作用机理。

通过项目研究，构建了烟草丛顶病4种病原物的植物侵染性克隆，以及TBTV、TVDV昆虫杆状病毒表达系统的侵染性克隆。建立了烟草丛顶病4种病原物的RT-qPCR检测技术体系，发现在4种病原物共同侵染的烟株中，TBTVsatRNA的相对含量在4种病原物中最高，TVDV的相对含量最低。首次发现TVDV可以通过甘蓝蚜、豌豆蚜、豆蚜、棉蚜等蚜虫进行传播，其中烟蚜对TVDV的获取和传播效率最高。阐明了TVDV在介体蚜虫体内的侵染循回路径：由口针进入中肠，突破中肠上皮细胞后进入血腔，最后由血腔进入副唾液腺。TVDV进入蚜虫消化道最短时间为30min，在蚜虫体内的循回期为24h。TVDV的CP和RTP在蚜虫传毒中起到了关键的作用，CP蛋白是介导TVDV突破蚜虫消化道屏障的关键蛋白，RTP蛋白不仅能帮助TVDV突破蚜虫消化道上皮细胞屏障也能突破蚜虫唾液腺屏障。TVDV CP、RTP蛋白可以协助TBTV经蚜虫传播，CP蛋白可以将TBTV带入蚜虫体内并突破蚜虫消化道屏障，RTP蛋白可以将TBTV带入蚜虫体内，也能够协助TBTV经蚜虫传播，而且还会加重TBTV在烟草上的症状。另外，TVDV的CP蛋白不仅能和TBTV的基因组RNA互作，还能与TBTV的P3蛋白发生互作，这些互作可能是TVDV通过异源包裹作用帮助TBTV进行蚜虫传播的主要方式。研究结果首次阐明了TVDV在烟蚜体内的侵染循回过程，明确了TVDV编码的与烟蚜传毒相关的关键因子，并揭示了TVDV协助TBTV进行烟蚜传播的作用机理。所获得的研究结果，进一步丰富了幽影病毒在黄症病毒的帮助下经介体昆虫传播的新机制，也将为进一步深入开展相关病毒与介体昆虫的互作关系奠定坚实的理论基础。