C4结合slβ-COP利用COPI转运途径协助TYLCV系统侵染的作用方式研究

番茄黄化曲叶病毒（TYLCV）是一种世界范围发生危害的粉虱传双生病毒，其编码的C4是重要的运动蛋白，但具体作用机理尚不清楚。项目前期研究发现C4与外被体蛋白β亚基（slβ-COP）存在互作，沉默该基因会延迟病毒系统侵染，而β-COP是细胞内COPI（负责高尔基到内质网及高尔基体内转运）的重要组分，因此推测C4可能通过接合slβ-COP利用COPI转运途径参与病毒的运动扩散。本项目拟采用运动缺陷型病毒互补试验研究C4运动特征；通过Y2H、BiFC、Co-IP等方法研究二者互作，明确互作关键位点；构建缺陷型病毒载体研究互作对病毒运动的影响；利用共聚焦技术研究COPI（slβ-COP）对C4及病毒转运的影响；通过转基因研究slβ-COP对C4及TYLCV系统扩散的影响，最终阐明二者在病毒运动中的作用方式。研究结果将有益于进一步阐释TYLCV在寄主细胞内的转运方式，推动粉虱传双生病毒运动模型解析。

Tomato yellow leaf curl virus (TYLCV), a typical monopartite whitefly-transmitted geminivirus (WTGs), caused devastating losses worldwide, including China in recent years. It is well-known that C4 is one of the most important virus-encoded proteins involved in the movement, but it is still not clear about the mechanism of movement in monopartite WTGs mediated by C4. In our previous research, a tomato coatomer subunit (slβ-COP) was found interacting with C4 protein encoded by TYLCV and silencing slβ-COP induced delayed symptom appearance when the plants inoculated by TYLCV. β-COP is the β subunit of COPI (the coat protein complex I), which is well known for retrograde transport from trans-Golgi network to cis-Golgi network and endoplasmic reticulum. All these results give rise to an interesting hypothesis that C4 might be involved in intra-cellular trafficking via COPI pathway by binding β-COP to fulfill its function in the movement of TYLCV. To confirm its function in virus movement and clarify the possible role involved in the movement, we propose to use movement-deficient virus to characterize the role of C4 in complementation of the movement of deficient virus, use the methods such as Y2H, BIFC, Co-IP to conform their interaction and identify the motif or sites responsible for the interaction, construct infectious clones of interaction-deficient TYLCV mutants to clarify the effects of the interaction to the movement of TYLCV; use fluorescence labeling method to clear the effects of slβ-COP or vesicular transport mediated by COPI system to the Intra-cellular traffic of C4 or TYLCV, use transgene method to clarify the effects of slβ-COP to the movement and systemic infection of TYLCV. These results are expected to characterize the function of C4 involved in the movement of TYLCV, and to promote the understanding on the movement mechanism of TYLCV and other monopartite WTGs.