与南方水稻黑条矮缩病毒P5-2蛋白互作的白背飞虱蛋白鉴定及其在病毒复制和传播中的功能研究

The white-backed planthopper (WBPH), Sogatella furcifera (Horvath), is an oligophytophagous insect that mainly feed on rice plants. In additin to damage rice plant by sucking the plant sap, WBPH also serves as vector for transmitting plant viruses such as Southern rice black-streaked dwarf virus (SRBSDV) in a persistent-propagative manner. SRBSDV is a non-enveloped RNA virus of the genus Fijivirus with ten linear dsRNA genomic segments that is closely related to rice black-streaked dwarf virus (RBSDV). Although much progress has been made towards understanding the interactions between vectors and virus pathogens of human and animal diseases, studies investigating SRBSDV–WBPH interactions remain limited...The segment 5 of SRBSDV encodes two proteins, P5-1 and P5-2. The P5-2 is composed of 205 amino acids and is referred as a non-structural protein. So far, the function of P5-2 is unknown. In the pilot study, we performed immunoprecipitation with antibody against P5-2 fusion protein, assayed the precipitated protein complex by SDS-PAGE, identified the host proteins binding with P5-2 by mass spectrometry. A total of 68 WBPH candidates proteins binding with P5-2 were obtained finally. Five candidates were selected and the interaction with P5-2 were further assayed by co-immunoprecipitation, and the Vps29 and Hsc70-4 proteins interacting with P5-2 were confirmed. Silencing the two genes by dsRNA injection led to decreased viral replication of SRBSDV in WBPH, suggesting that both genes play roles in regulating the replication or dissemination of SRBSDV in vivo. The proposal is going to verify the interaction between P5-2 and other protein candidates, and evaluate whether these interacting protein play roles in modulating viral replication and dissemination of SRBSDV in WBPH. These authentic interacting proteins will be further investigated to answer the detailed mechanisms involved in the regulating of virus life cycle in vivo. The project will enhance the understanding of P5-2 roel in modulating the balance between insect vector fitness and the persistent propagation of virus in vivo. Meanwhile, the study will also provide new protein targets to develop strategies for protection from SRBSDV infection transmitted by WBPH.

白背飞虱及其传播的SRBSDV是严重危害水稻生产的害虫和水稻病毒，但目前国内外对白背飞虱与SRBSDV的互作机制研究才刚刚起步。SRBSDV的S5片段编码的非结构蛋白P5-2功能未知。在前期研究中，我们通过免疫共沉淀、质谱分析和生物信息学分析，获得了与P5-2互作的68个白背飞虱候选蛋白信息。选择其中5个候选蛋白进行实验验证，确认了Hsc70-4和Vps29蛋白与P5-2蛋白互作，基因沉默这两个基因导致白背飞虱体内SRBSDV浓度降低，表明它们可能参与调控SRBSDV在白背飞虱体内增殖和传播等过程。本申请拟进一步实验验证其它候选蛋白与P5-2蛋白互作，筛选参与调控SRBSDV在白背飞虱体内增殖和传播过程的互作蛋白，并研究一些典型互作蛋白具体的分子作用机制等。本课题研究将加深对SRBSDV在白背飞虱体内持久性增殖但不致病现象的分子机制的理解，也为生物防治SRBSDV传播提供新的靶标蛋白。

白背飞虱是我国农业上重要的水稻害虫，其通过刺吸水稻汁液直接危害水稻，同时以持久增殖型方式传播南方水稻黑条矮缩病毒(SRBSDV)，严重危害我国的水稻生产。本课题以SRBSDV编码的P5-2蛋白为研究对象，鉴定与其互作的白背飞虱蛋白，并研究这些互作蛋白的功能及其在病毒复制和传播中的作用。我们主要完成了以下工作：1）通过与白背飞虱的cDNA酵母文库筛选，我们获得与P5-2互作的57个候选白背飞虱蛋白; 通过一对一互作验证，最终获得了7个白背飞虱-SRBSDV病毒P5-2互作蛋白。2）通过RNAi技术分析了互作蛋白对白背飞虱体内病毒复制和传毒能力的影响。3）验证了组织蛋白酶D（CathD）有助于SRBSDV在白背飞虱体内复制。4）鉴定了来源于白背飞虱基因组中3,057个位点的3,102个lncRNA转录本和1706个白背飞虱circRNA的特征,分析了在白背飞虱不同发育阶段的表达谱。5）发现了果蝇抗病毒lncRNA VINR和RNAi沉默抑制子DCV-1A结合，保护VINR不被细胞内RNA酶作用，从而增加VINR在细胞内的含量，启动VINR-Cactin介导的一条非经典免疫通路。6）通过对水稻宏病毒组数据进行分析，鉴定发现了一种由禾谷缢管蚜传播的新水稻病毒RTIV，水稻感染该病毒后分蘖水平降低。我们对SRBSDV编码的P5-2蛋白与白背飞虱蛋白互作研究的结果，为深入研究SRBSDV与介体昆虫白背飞虱之间的免疫互作及传播机制奠定了良好的基础。到2022年年底，本课题相关的研究成果已整理发表了4篇SCI论文, 包括Cell Host Microbe、BMC genomics等行业内知名杂志，2篇论文已被中国科技期刊全文数据库（CNKI）核心期刊接受。另外一篇投稿PloS Pathogens的论文正在进行二轮评审。培养博士研究生3名，硕士研究生1名。目前有1名硕士生和1名博士生继续进行本课题的后续研究。本项目达到预期的研究目标。