Characterization of the intercellular trafficking of Tobacco mosaic cirus movement protein and identification of interacting factors

植物细胞通过胞间连丝(PD)进行通讯，胞间连丝是植物细胞壁中的通道，为相邻细胞之间提供细胞质的连续性。在植物的叶脉和茎中，PD系统是一个细胞-细胞和系统通讯网络，它控制着各种分子的运输，包括在植物发育和防御病原体方面发挥作用的调节蛋白质和RNA分子。植物RNA病毒已经适应了这种通信网络，并编码运动蛋白(MPS)，促进病毒RNA基因组(VRNA)在细胞之间的传播。因此，研究病毒的传播和病毒的相互作用伙伴！运动蛋白为研究细胞间通讯机制提供了极好的机会。我希望应用生化研究来寻找病毒MP的新的相互作用伙伴，并应用生命细胞显微镜中的新方法，如双分子荧光互补(BIFC)和可光激活的GFP，来研究动态的蛋白质：蛋白质和蛋白质：vRNA的相互作用及其在体内的运输。此外，基于先前的报道表明MP是通过泛素/26S蛋白酶体途径降解的，我还将应用BIFC来研究感染过程中泛素化MP的形成和细胞内定位。本质上，拟议的研究将阐明与RNA运输有关的病毒宿主细胞功能，因此将允许对细胞间通信和病毒发病机制获得新的见解。

Plant cells communicate via plasmodesmata (PD), channels in the plant cell wall that provide cytoplasmic continuity between adjoining cells. With connected phloem in the plant veins and stems, the system of PD represents a cell-cell and systemic communication network that controls the trafficking of a variety of molecules, including regulatory proteins and RNA molecules that play a role in plant development and defence against pathogens. Plant RNA viruses have adapted to this communication network and encode movement proteins (MPs) that facilitate the spread of the viral RNA genome (vRNA) between cells. Thus, studying viral transport and the interaction partners of vira! movement proteins provides an excellent opportunity to investigate the intercellular communication machinery. I wish to apply biochemical studies to identify novel interaction partners of viral MP and to apply novel approaches in life cell microscopy, such as Bimolecular Fluorescence Complementation (BiFC) and photo-activatable GFP, to investigate dynamic protein:protein and protein:vRNA interactions and their trafficking in vivo. Moreover, based on previous reports indicating that the MP is degraded by the ubiquitin/26S proteasome pathway, I will apply BiFC also to investigate the formation and intracellular location of ubiquitinylated MP during infection. In essence, the proposed studies will elucidate viral host cell functions that are involved in RNA transport and therefore will allow to gain new insights in cell-cell communication and viral pathogenesis.