小麦黄条纹病毒干扰介体异沙叶蝉传播小麦矮缩病毒的机制研究

Under natural condition, vector insects can often transmit more than one plant virus. But, the mechanism of interaction between viruses in the vector insects and impact on epidemics and outbreaks of viral diseases has not been investigated. In previous work, we found that acquiring wheat yellow striate virus (WYSV) could cause a reduction for the ability to wheat dwarf virus (WDV) transmission in the same vector leafhopper Psammotettix alienus. Also, we determined the dissemination route of WDV in the leafhopper vector and identified crucial vector proteins involved in WDV transmission. On the basis of the above results, further research will be developed aimed at interaction between WYSV and WDV with their vector P. alienus. Firstly, subcellular localization and dissemination route of virus will be examined by transmission electron microscopy and immunofluorescence microscopy when the leafhopper acquired WYSV alone or both WYSV and WDV, which led to illuminate the interaction mechanism at histological level. Second, the putative proteins interacted with WYSV in leafhopper will be screened from the cDNA library of P. alienus constructed by a yeast two-hybrid system, and the competition binding receptors will be identified as compared to proteins involved in WDV transmission. Furthermore, function of the identified receptors in transmission of WYSV or WDV will be investigated by colocalization, RNAi knockdown and qPCR detection. In the addition, it will be explored that the mechanism of RNA-antiviral defense in the vector insect respond to co-infection with WYSV and WDV. This study will contribute to better understanding the mechanism about interaction relationship and co-evolutionary on multiple viruses and their vector insects. Meanwhile, it would provide theoretical basis for designing new strategies to control the viral diseases based on disturbing leafhopper to transport virus in the future.

在自然条件下，一种介体昆虫往往可传播多种植物病毒，但所传病毒间的互作机制及对病毒病害暴发流行的影响尚不清楚。申请者前期研究发现异沙叶蝉获得小麦黄条纹病毒（WYSV）后，传播小麦矮缩病毒（WDV）的能力显著下降。本项目拟以异沙叶蝉及其传播的这两种病毒为对象，在已明确WDV在虫体内的循回途径与传毒关键蛋白的基础上，首先采用透射电镜和免疫荧光共定位技术研究WYSV以及WYSV与WDV共存时病毒在虫体内的亚细胞定位和循回机制，从组织学水平阐明互作机制；其次是利用已构建的异沙叶蝉酵母双杂交cDNA文库筛选与WYSV互作的介体蛋白，比较与WDV互作蛋白的差异，并通过共定位、RNA干扰和qPCR等技术验证其传毒功能；探索异沙叶蝉体内响应WYSV和WDV复合侵染的RNAi机制。研究结果可为解析多种病毒与介体昆虫的互作关系和协同进化机制奠定基础，同时为建立基于阻断介体传毒的新型抗病毒防治策略提供理论依据。

异沙叶蝉可传播的小麦矮缩病毒和新发现的小麦黄条纹病毒，这两种亲缘关系较远的小麦病毒在异沙叶蝉体内的互作机制及可能对病害流行学的影响尚不明确。本项目研究首先明确了介体异沙叶蝉传播小麦黄条纹病毒的基本特性，同时发现小麦黄条纹病毒在异沙叶蝉体内能够增殖并可经卵传播。制备了小麦黄条纹病毒多克隆抗体，通过免疫荧光以及电镜观察揭示了小麦黄条纹病毒与小麦矮缩病毒在介体体内分布位点以及循回路径的异同。基于建立的小麦黄条纹病毒和小麦矮缩病毒双重RT-PCR和核酸斑点杂交等检测技术体系，通过生物学传毒效率实验和荧光定量以及免疫定位分析，进一步确定了小麦黄条纹病毒的存在降低了小麦矮缩病毒在介体体内的浓度，从而干扰异沙叶蝉传播小麦矮缩病毒。利用转录组测序技术明确异沙叶蝉受小麦黄条纹病毒侵染后差异基因的转录本变化，并从蛋白组学水平鉴定分析发现小麦黄条纹病毒和小麦矮缩病毒在突破异沙叶蝉中肠上皮细胞进行胞吐和囊泡转运过程中可能存在受体蛋白竞争。初步探索了异沙叶蝉先天免疫系统响应小麦黄条纹病毒和小麦矮缩病毒两种病毒共侵染的机制，发现两种病毒共存仍能诱导异沙叶蝉RNAi免疫反应。研究结果为解析多种病毒与介体昆虫的互作关系和协同进化机制奠定基础，同时为建立基于阻断介体传毒的新型抗病毒防治策略提供理论依据。