水稻黑条矮缩病毒P6和P9-1蛋白抑制灰飞虱RNAi免疫机理

Whether virus can suppress effectively the RNAi mediated antiviral immunity in insect vector is crucial for virus-transmission. Rice black streaked dwarf virus (RBSDV) has been reported to cause severe rice black streaked dwarf disease in rice fields in China, and RBSDV is transmitted by small brown planthopper (Laodelphax striatellus Fallén, SBPH). At present, little is known about how RBSDV fights effectively the RNAi antiviral immunity in insect vector. Our preliminary study showed that the RNA silencing suppressor P6 and P9-1 proteins encoded by RBSDV might be contributed to suppress RNAi immunity in SBPH. For further analysis of the related mechanism, in the project, a RNAi-mediated reaction system using SBPH Embryo Lysate is planned to be firstly established, and RNA silencing suppressor activities of P6 and P9-1 proteins and their binding with dsRNA and siRNA are further analyzed. Afterward, whether P6 and P9-1 influence on the functions of core RNaseⅢ and PAZ domains of Dicer-2 (a crucial protein in RNAi pathway) and the assembly of RNA-induced silencing complex (RISC) are investigated. Finally, a potential mechanism that RBSDV suppresses the RNAi immunity in SBPH was proposed. Research results will be helpful for understanding the mechanisms that RBSDV overcomes RNAi immunity in SBPH and the reason that SBPH acquires RBSDV with efficient ability, which is expected to provide the new ideas for plant virus disease control.

病毒能否有效抑制介体昆虫的RNAi抗病毒免疫对其介体传播至关重要。重要致灾病害水稻黑条矮缩病（病原RBSDV）由灰飞虱传播，目前RBSDV高效适应灰飞虱RNAi抗病毒免疫的相关机制知之甚少。前期研究发现，该病毒编码的沉默抑制子P6和P9-1蛋白对抑制介体RNAi免疫具有重要贡献。为进一步解析相关机理，本项目拟建立基于灰飞虱胚胎提取物的RNAi反应体系，利用该体系鉴定P6和P9-1蛋白对RNAi的沉默抑制活性，并分析两蛋白与dsRNA和siRNA的结合能力；解析P6和P9-1蛋白对Dicer-2（RNAi通路关键蛋白）核心功能域RNaseⅢ和PAZ活性的影响及对RNA沉默复合体（RISC）组装的影响，以明确RBSDV抑制灰飞虱RNAi免疫机理。研究结果有助于揭示RBSDV克服灰飞虱RNAi免疫机制，阐明灰飞虱高效获毒（RBSDV）原因，并有望为病毒病害的控制提供新的思路。

由灰飞虱传播水稻黑条矮缩病毒（Rice black streaked dwarf virus，RBSDV）引起的水稻黑条矮缩病是水稻重要病害之一，对水稻生产构成极大威胁，深入研究灰飞虱对RBSDV的传播机制，对于阻断病毒的传播、控制病毒病害具有重要意义。本项目围绕RBSDV对灰飞虱免疫系统的影响，尤其是P6和P9-1蛋白抑制灰飞虱RNAi通路的机理展开研究，取得重要进展。细化了不同灰飞虱群体对RBSDV获毒能力，比较了灰飞虱体内RNAi免疫通路关键基因的表达水平，发现RBSDV可以高效抑制灰飞虱的抗病毒免疫反应。分析了灰飞虱Dicer-2蛋白结构域，表达纯化了RNaseⅢ及PAZ两个关键功能域，并验证RNaseⅢ具有降解dsRNA的功能活性以及PAZ具有结合siRNA的功能活性。建立了可介导RNAi反应的灰飞虱Embryo Lysate体系，利用该体系证明了RBSDV编码的P6和P9-1蛋白在灰飞虱体内具有基因沉默抑制子活性，进一步证实了抑制机理为P6和P9-1蛋白可通过蛋白互作分别抑制Dicer-2蛋白RNaseⅢ-b和PAZ功能域的活性，阻断了RNAi免疫通路2个关键步骤：1）切割dsRNA产生siRNA和2）结合siRNA组装RISC，从而抑制了灰飞虱对病毒的RNAi免疫。.本项目揭示了灰飞虱高效获毒（RBSDV）的原因，可为控制昆虫传播病毒、防治病毒病害提供新的理论依据和新思路。项目执行期间，负责人作为第一、通讯作者发表论文6篇，其中SCI论文5篇，培养硕士研究生1名。项目负责人入选农业农村部“杰出青年农业科学家”2018年度资助项目人选和江苏省第五期“333高层次人才培养工程”第三层次培养对象，达到了预期目标。