利用兽棚病毒研究水稻病毒介体昆虫飞虱和叶蝉中病毒DNA和vsiRNA产生机制

Antiviral RNA interference (RNAi) clears virus infection in both plants and animals by a highly conserved genetic pathway.The antiviral RNAi pathway begins with Dicer-mediated processing of virus-specific double-strand RNA (dsRNA) into viral-derived small interfering RNAs (vsiRNAs). Subsequently, these vsiRNAs guide specific viral RNA clearance by an Argonaute protein in RNA induced silencing complex.Effective antiviral RNAi in plants and nematodes depends on the amplification of the vsiRNAs by a related family of host RNA dependent RNA polymerases (RdRps) following the biogenesis of the primary vsiRNAs processed from viral dsRNA replicative intermediates. However, it has been unclear why the total vsiRNAs are as abundant in fruit flies as in nematodes, which do not encode an RdRp homolog. Recent studies demonstrate the production of vsiRNAs in the infected fruit flies templated by the viral extrachromosomal circular DNA (eccDNA), providing a novel pathway for the biogenesis of the vsiRNAs.Rice viruses are transmitted by insect vectors planthopper or leafhopper in a persistent way, genome of rice viruses are complex which make it hard for infectious clone construction and difficult for studying of interaction mechanism between virus and insect vectors，we found that FHV is successfully replicate in Nilaparvata lugens, Sogatella furcifera, Recilia dorsalis and Nephotettix cincticeps. Here, FHV is used as a viral tool to develop an infection system to study the interaction between virus and planthopper or leafhopper. It is aimed to clarify the type of viral DNA in planthopper or leafhopper and its role in vsiRNA biogenesis and provide a theoretical basis for further understanding how siRNA antiviral pathway regulates the mechanism of persistent and efficient transmission of rice virus by insect vectors.

RNA沉默是动植物抗病毒的一种保守机制，该途径在植物和线虫中由RdRp产生次级vsiRNA提供更多RNA降解模板。由病毒DNA形成的vsiRNA，可能是昆虫产生viRNA的另外一个来源，用以替代缺失RdRp的作用。感染昆虫兽棚病毒（FHV)的果蝇和S2细胞可以产生源于病毒DNA的vsiRNA，这可能是寄主产生vsiRNA的另外一个来源。水稻病毒由介体昆虫叶蝉或飞虱以持久增殖型方式传播，水稻病毒基因组复杂，无侵染性克隆导致研究病毒与介体互作的机制非常困难，前期发现FHV在褐飞虱、白背飞虱、电光叶蝉和黑尾叶蝉体内复制和致病。因此，本项目将以FHV为病毒工具，构建研究病毒与飞虱或者叶蝉互作的侵染系统，通过研究可以明确FHV在飞虱或者叶蝉中病毒DNA的类型及其在viRNA产生中的作用，为进一步深入解析siRNA抗病毒途径如何调控介体昆虫持久高效传播水稻病毒的机制奠定基础。

水稻病毒由介体昆虫飞虱或叶蝉以持久增殖型方式传播，水稻病毒基因组复杂，无侵染性克隆导致研究病毒与介体互作的机制非常困难。RNA沉默是动植物抗病毒的一种保守机制，RNA沉默抗病毒途径首先由Dicer形成长度为21-24nt的小RNA称为vsiRNA，继而Argonaute蛋白以这些vsiRNA为模板降解病毒RNA。由病毒DNA形成的vsiRNA，可能是昆虫产生viRNA的另外一个来源，用以替代缺失的RNA依赖的RNA聚合酶产生次级vsiRNA。兽棚病毒是研究最详尽的病毒之一，本项目以FHV为病毒工具，通过显微注射的方式构建研究病毒与飞虱/叶蝉互作的侵染系统，结果表明在昆虫中肠、脂肪体、头部、卵巢等各种组织均检测到病毒的存在，并且昆虫体内可以产生21nt的特征性病毒小RNA，说明FHV可以成功的在介体昆虫飞虱或叶蝉中建立侵染。进一步研究表明病毒侵染受病毒沉默抑制子B2表达的影响，B2缺陷的FHV∆B2无法建立侵染；通过双链RNA干扰寄主的AGO2表达后，FHV∆B2则可在昆虫体内恢复病毒积累，并且产生相应的21nt的病毒小RNA，说明飞虱或叶蝉抗病毒是与RNA沉默通路相关的。此外，预先经过FHV产生DNA处理的飞虱，可以有效的降低FHV侵染后病毒RNA和蛋白的积累，说明FHV产生的DNA具有一定抗病毒的作用。