昆虫RNAi抗病毒免疫途径调控南方水稻黑条矮缩病毒与不同介体间存在亲和性差异的机制研究

Southern rice black-streaked dwarf disease caused by Southern rice black-streaked dwarf virus (SRBSDV) recently has become one of the most important viral diseases of rice in China. SRBSDV was efficiently transmitted by the white-backed planthopper (WBPH) in a persistent-propagative manner, and the small brown planthopper (SBPH) can acquire but can not transmit SRBSDV. Our previous study showed that the multiplication levels of SRBSDV in the cultured insect cells derived from WBPH and SRBSDV were significantly different, which may caused by insect RNA interference (RNAi) antiviral innate immunity pathway. In this proposal, we would develop the system combined the insect cell cultures and RNAi induced by synthesized dsRNAs. By using this system, we will screen and identify the key factors of insect RNAi pathway, which could mediate the persistent infection of SRBSDV in two different insect vector cultured cells. Based on the results, we want to confirm whether the key factors of insect RNAi pathway would be involved in highly efficient transmission of virus by WBPH. Furthermore, we also want to test whether the key factors of insect RNAi pathway also could prevent viral transmission by SBPH. Our results would reveal the mechanism that insect RNAi antiviral innate immunity pathway would mediate the significantly different components between SRBSDV and its two different insect vectors. All these data would provide theory basis for designing efficient control disease strategies in future.

由南方水稻黑条矮缩病毒（SRBSDV）引起的南方水稻黑条矮缩病是目前我国发生严重的水稻病毒病之一。该病毒由介体白背飞虱以持久增殖型方式高效传播，同时灰飞虱也可低效带毒但不传毒。前期研究表明，SRBSDV在白背飞虱和灰飞虱培养细胞内的增殖水平存在着显著的差异，推测与昆虫RNA干扰（RNAi）抗病毒免疫途径的调控有关。本项目拟利用白背飞虱和灰飞虱细胞培养体系和体外合成的dsRNA诱导的RNAi技术体系，筛选鉴定调控SRBSDV在不同昆虫培养细胞内持久侵染的昆虫RNAi途径关键因子；在此基础上，在白背飞虱和灰飞虱体内鉴定参与白背飞虱高效传毒或抵御灰飞虱传毒的昆虫RNAi途径关键因子。研究结果将解析昆虫RNAi抗病毒免疫途径调控SRBSDV与不同介体昆虫间存在亲和性差异的机制，探索用于阻断白背飞虱高效传毒的抗病新策略。

由南方水稻黑条矮缩病毒（SRBSDV）引起的南方水稻黑条矮缩病是目前我国发生严重的水稻病毒病之一。该病毒由介体白背飞虱以持久增殖型方式高效传播，同时灰飞虱也可低效带毒但不传毒。前期研究发现SRBSDV在白背飞虱和灰飞虱培养细胞内的增殖水平存在着显著的差异，推测与昆虫RNA干扰（RNAi）抗病毒免疫途径的调控有关。本项目利用昆虫细胞培养体系和dsRNA诱导的RNAi技术体系，首先鉴定出调控SRBSDV在不同昆虫培养细胞内持久侵染的昆虫RNAi途径关键因子；在此基础上，解析了Dicer2是参与白背飞虱高效传毒或抵御灰飞虱传毒的昆虫RNAi途径关键因子。一方面，Dicer2参与的siRNA抗病毒天然免疫途径可有效控制SRBSDV在白背飞虱内的积累量，使其低于导致昆虫死亡的阈值，维持病毒在介体内的持久侵染，解析了昆虫免疫系统是如何应对病毒侵染并与之"和平共处"的机制；另一方面，siRNA 抗病毒免疫途径控制SRBSDV在灰飞虱体内的侵染，使其在非亲和性介体灰飞虱中肠中的滴度低于让病毒突破中肠释放屏障的阈值，导致病毒的侵染局限于昆虫的中肠上皮细胞而无法释放到外层的肌肉组织，该研究首次提出中肠siRNA抗病毒途径调控病毒-介体亲和性的概念。此外，本项目还鉴定了SRBSDV复制的关键因子，阐明了斐济病毒属病毒的循回期与P7管状结构蛋白的形成速率有关。本项目关于RNAi抗病毒免疫途径调控SRBSDV在介体内侵染的机制，为阻断介体昆虫传播病毒奠定了理论基础。研究结果发表5篇研究论文，其中SCI论文3篇，授权发明专利1项。基于课题组在水稻病毒虫传机制方面的良好研究基础，项目负责人2017年获得国家自然科学基金面上项目的资助，并以第一作者在Current Opinion of Virology上以“Vector mediated transmission of persistently transmitted plant viruses”为题发表了评论文章（2018.28: 128-132）。