TYLCV编码的C4与寄主番茄slAnnD2互作在病毒运动中的作用机理研究

Whitefly-transmitted geminivirus (WTGs) encoded nuclear shuttle protein (NSP) and movement protein (MP) responding to intra- and inter-cellular trafficking. It is well-known that C4 is one of the most important virus-encoded proteins involved in cell-to-cell movement in monopartite WTGs, but it is still not clear the mechanism of inter-cellular trafficking in monopartite WTGs mediated by C4. Tomato yellow leaf curl virus (TYLCV), a typical monopartite WTGs, caused devastating losses worldwide, including China in recent years. In our previous research, a tomato Annexin (slAnnD2), a superfamily of proteins associated with membrane lipid and cytoskeletal components involved in membrane organization, vesicle trafficking and signaling et al., was found interacting with C4 protein encoded by TYLCV. The biological significance of the interaction during the process of TYLCV movement is still unknown. To study the function of these proteins in the virus movement process, we propose to use movement-deficient virus to see if C4 can complement of the movement of the deficient virus, use fluorescence labeling method to clear the protein subcellular distribution and whether they were co-located with each other, use the methods such as Y2H, pulldown, BIFC to conform their interaction and identify the motif or sites responsible for the interaction and characterization of the effects of the motif or sites on subcellular localization and function, use VIGS (virus induced gene silencing) and transgene method to clear the correlation of the expression level of modulated slAnnD2 with efficiency of infection and spread of TYLCV. These results are expected to characterize the proteins involved in TYLCV movement, and to promote the understanding on the movement mechanism of TYLCV and other monopartite WTGs.

粉虱传双生病毒在寄主体内的运动分为核质转运和胞间移动两个过程。对于单组分粉虱传双生病毒，其编码的C4是参与胞间运动的重要组分，但作用机理尚不清楚。项目前期在番茄黄化曲叶病毒（TYLCV）上的研究发现C4与番茄膜联蛋白（slAnnD2）存在互作，而植物膜联蛋白是一种重要的膜相关蛋白，能与F-Actin结合，因此推测二者互作参与了TYLCV的胞间运动。本项目拟采用运动缺陷型病毒互补试验研究C4运动特征；利用激光共聚焦显微技术研究C4与slAnnD2定位及共定位；通过Y2H、pull-down、BiFC等方法研究二者互作，明确其互作关键功能域/位点，分析其对细胞定位及功能的影响；通过VIGS及转基因手段研究slAnnD2表达受调控后对TYLCV系统扩散的影响，最终阐明二者互作在病毒运动扩散中的作用方式。研究结果对揭示TYLCV运动模式将有重要意义，对单组分粉虱传双生病毒运动模型研究也有推动作用。

粉虱传双生病毒在寄主体内的运动分为核质转运和胞间移动两个过程。对于单组分粉虱传双生病毒，其编码的C4是参与胞间运动的重要组分，但作用机理尚不清楚。番茄黄化曲叶病毒（TYLCV）是一种世界范围发生危害的粉虱传单组份双生病毒，项目选择TYLCV作为研究对象，在利用酵母双杂交系统其编码的C4蛋白的寄主候选因子进行研究时发现其与番茄膜联蛋白（slAnnD2）存在互作，而植物膜联蛋白是一种重要的膜相关蛋白，因此推测二者互作可能在病毒运动及系统侵染上起重要作用。基于这一假设，项目克隆了番茄slAnnD2，并进一步采用BiFC、Co-IP、LucA等方法对二者之间的互作进行了研究，确认了二者之间存在互作，亚细胞定位观察结果显示C4与slAnnD2在细胞膜上存在共定位，说明二者可能通过互作一起行使功能。为了解析其互作关键位点，我们构建了14个点突变体，发现G2突变后会导致C4丧失与SlAnnD2互作的特征，说明G2对C4与SlAnnD2的互作起着关键作用。亚细胞定位观察结果显示互作缺陷型突变体C4G2A丧失了细胞膜定位特征，说明G2同时对其亚细胞定位很关键，构建含有C4G2A点突变的侵染性克隆，接种寄主番茄后发现G2A的突变会导致病毒侵染后寄主植物显症迟，接种植株新叶病毒积累量低，说明G2A突变影响病毒的系统侵染。荧光定量PCR结果显示在TYLCV侵染寄主番茄时会诱导番茄SlAnnD2表达量下调，而沉默SlAnnD2的植株接种TYLCV后植株显症较迟，症状较轻，接种株新叶病毒积累量相对较低；利用CRISPR敲除该基因后的转基因番茄接种TYLCV也表现显症迟、接种株新叶病毒积累量较低等类似的试验结果，说明C4接合slAnnD2在病毒系统侵染中起重要作用，该基因下调后会抑制病毒运动阻碍TYLCV系统侵染，导致病害症状减轻。同时研究还发现SlAnnD2与Actin之间存在互作，干扰Actin后也会影响病毒的扩散，暗示了SlAnnD2与Actin在病毒系统侵染时可能协同起作用。这些研究结果初步揭示二者互作在TYLCV运动扩散中的作用方式，对深入解析TYLCV的运动模式，推动单组分粉虱传双生病毒运动模型研究都有重要意义。