水稻黑条矮缩病毒P7-2蛋白的功能分析

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, causes rice black-streaked dwarf and maize rough dwarf diseases in our country, and leads to severe yield losses of crops. Previously, we found P7-2, an uncharacterized protein encoded by RBSDV, as well as a conserved protein among plant-infecting fijiviruses, drastically enhanced the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana when expressed through the PVX-based vector.Further investigation indicated there is a strong interaction between P7-2 and SKP1 protein, a key subunit of the multicomponent SCF (SKP1/Cullin1/F-box/Rbx1) E3 ubiquitin ligase. Taken together, we propose that P7-2 might be a potential F-box protein encoded by RBSDV and functions in the same manner as cellular F-box proteins, redirecting the SCF ubiquitination ligase machinery to degrade its specific host targets which might be involved in hormone responses or virus resistance process. Co-immunoprecipitation assay and yeast two-hybrid assay will be performed to determine whether P7-2 is associated with the SCF complex as an F-box protein and the exact position of the F-box motif. The functional relevance between P7-2-SKP1 interaction and the pathogenicity of P7-2 will be investigated using virus-based expressing system and RNA interfering. The potential target proteins of P7-2 will be identified by cDNA library screens, and their activities will also be analyzed to elucidate the biological function and the molecular mechanism of RBSDV P7-2.The study will contribute to a further understanding of the functional mechanism by which RBSDV interferes with or usurps the host machinery to create a suitable cell environment for virus replication or to cause viral symptoms. It will also provide new scientific evidence for us to control diseases caused by RBSDV and other fijiviruses.

水稻黑条矮缩病毒（RBSDV）引起我国重要粮食病害水稻黑条矮缩病和玉米粗缩病。本研究前期工作发现，RBSDV未知功能蛋白P7-2与泛素连接酶SCF复合体重要组分SKP1互作，并加重异源病毒致病性，推测P7-2是病毒编码的一种F-box蛋白，参与植物蛋白泛素化降解途径，干扰植物正常生理过程。本项目拟以烟草和水稻为主要试验材料，利用酵母双杂交、免疫共沉淀等方法，确定P7-2与蛋白泛素化的关系及F-box基元的位置；通过异源病毒侵染和RNAi等技术，分析P7-2-SKP1互作与致病性的关系；进一步筛选玉米、水稻cDNA文库，鉴定P7-2特异识别的靶蛋白，探析该蛋白的作用及两者互作对植物的影响，初步阐明P7-2的生物学功能与作用机理。该结果有助于揭示RBSDV在病毒-寄主互作、致病等方面的复杂过程，加深对病毒诱导植物产生症状机理的理解，为防治RBSDV及其它斐济病毒引起的重要作物病害提供科学依据。

水稻黑条矮缩病毒（RBSDV）引起我国重要粮食病害水稻黑条矮缩病和玉米粗缩病，其编码的非结构蛋白P7-2功能未知。生物信息学分析发现，该基因在能侵染植物的斐济病毒属病毒的基因组中保守存在，而在侵染昆虫的斐济病毒属病毒中却缺少与之对应的基因。前期研究发现P7-2加重异源病毒致病性，并可与泛素连接酶SCF复合体组分SKP1互作，推测该基因（及其同源蛋白）可能是植物斐济病毒在长期进化过程中形成的分子武器，通过泛素化途径干扰或影响植物正常发育或防御过程，从而加剧病毒症状。本研究以P7-2为研究对象，通过酵母双杂交、三杂交和免疫共沉淀等实验，证明RBSDV P7-2与多个植物SKP1家族成员互作，共同参与泛素连接酶复合体的形成，并明确了P7-2、SKP1参与P7-2-SKP1互作的关键蛋白区域；系统地调研了植物产生矮化表型所涉及的信号转导通路以及其中参与泛素化途径的基因，克隆相关候选基因，通过酵母小量结合试验进行筛选。发现P7-2可与一种重要的植物激素合成通路中的关键调控蛋白Factor X互作，免疫共沉淀实验表明两者可在体外产生互作；Western blot结果显示，RBSDV侵染的植株中Factor X的蛋白表达水平明显低于健康对照，推测P7-2通过泛素化途径使Factor X产生降解，从而导致寄主植物出现矮化表型；转基因和RNA干扰实验发现，过表达P7-2可严重影响转基因水稻的正常发育，干扰SKP1基因的表达则导致植株表现出矮化、皱缩的表型，表明P7-2-SKP1互作与病毒的致病性存在相关性。上述结果初步验证了前期提出的假设，为揭示RBSDV在病毒-寄主互作、致病等方面的复杂过程，加深对病毒诱导植物产生症状机理的理解提供科学依据。