Synaptotagmin function in virus movement and plant development

描述（由申请人提供）：植物病毒移动蛋白通过改变连接相邻植物细胞的胞间连丝（transwall pore），将病毒基因组转运穿过细胞壁。突触结合蛋白（Synaptotagmins）是动物特有的一个多基因家族，是调节突触囊泡内吞/外吞的钙（Ca ~（2+））感受器。我们的长期目标是了解synaptotagmins如何调节病毒的运动和细胞间运输，以控制抗病性，RNAi信号的传播和细胞命运。SYTA是5种拟南芥突触结合蛋白（SYT A-E）中的1种。SYTA结合由菜豆病毒CaLCuV（MPCaLCuV）和SqLCV以及烟草病毒TMV（30 K）编码的不同运动蛋白。我们已经表明，SYTA调节早期内体的形成和细胞到细胞的运输MPCaLCuV和TMV 30 K。我们建议，SYTs调节大分子运输到胞间连丝通过内吞再捕获途径，并可能进入和退出孔以及。我们将使用转基因植物研究和基于细胞的表达测定相结合，以调查如果SYTs A，C和E是植物病毒运动和细胞间运输的关键调节因子。生化和感染性研究以及基于叶的细胞运输试验将检查SYTA是否单独调节大多数病毒的细胞间运动，或者SYTC和SYTE是否也参与其中，无论是独立于SYTA还是与SYTA合作。SYTC和SYTE在囊泡运输中的作用将通过免疫定位以及使用基于细胞和叶的分泌测定来确定。SYTA本身的多光子成像，或与MPCaLCuV和30 K，或SYTC和SYTE，将显示SYTA内体如何将运动蛋白运送到胞间连丝，以及SYTC或SYTE如何在此过程中合作。植物细胞中SYT A、C和E作用的知识将通过启动子：：GUS融合研究与发育调控相联系，以确定它们的空间和时间表达，并分析其中每种表达被RNAi或T-DNA插入抑制的转基因系。我们证明了SYTA调节的早期内体形成和细胞间运输之间的功能联系，为胞间连丝功能是如何控制的提供了新的见解。除了工程抗病植物的农艺学影响，我们的研究与人类健康有关，因为它们可以为细胞间通讯和发育中的动物突触结合蛋白的研究提供信息和加速研究，以及逆转录病毒重定向内体机制以使病毒成熟并从受感染的细胞中退出的机制。这项研究调查了植物和动物细胞之间运输的共同途径的调节。因此，它可以通过改善营养，提供有关生长缺陷的知识以及确定限制艾滋病毒和其他逆转录病毒传播的新方法来影响公共卫生。

DESCRIPTION (provided by applicant): Plant virus movement proteins transport viral genomes across the cell wall by altering plasmodesmata, transwall pores that connect adjacent plant cells. Synaptotagmins, a multigene family thought to be exclusive to animals, are calcium (Ca2+) sensors that regulate synaptic vesicle exo/endocytosis. Our long term goal is to understand how synaptotagmins regulate virus movement and intercellular transport to control disease resistance, the spread of RNAi signals and cell fate. SYTA is 1 of 5 Arabidopsis synaptotagmins (SYT A-E). SYTA binds to the distinct movement proteins encoded by the Begomoviruses CaLCuV (MPCaLCuV) and SqLCV, and the Tobamovirus TMV (30K). We have shown that SYTA regulates both the formation of early endosomes and the cell-to-cell transport of MPCaLCuV and TMV 30K. We propose that SYTs regulate macromolecular trafficking to plasmodesmata via an endocytic recapture pathway, and potentially entry into and exit from the pore as well. We will use a combination of transgenic plant studies and cell-based expression assays to investigate if SYTs A, C and E are key regulators of plant virus movement and intercellular transport. Biochemical and infectivity studies, and a leaf-based cell trafficking assay will examine if SYTA alone regulates the cell-to-cell movement of most viruses, or whether SYTC and SYTE are also involved, either independent of or partnering with SYTA. The roles of SYTC and SYTE in vesicle trafficking will be defined by immune localization, and the use of cell- and leaf-based secretion assays. Multiphoton imaging of SYTA itself, or with MPCaLCuV and 30K, or SYTC and SYTE, will show how SYTA endosomes ferry movement proteins to plasmodesmata, and how SYTC or SYTE may cooperate in this process. This knowledge of SYT A, C and E action in plant cells will be connected to the regulation of development through promoter::GUS fusion studies to define their spatial and temporal expression, and analysis of transgenic lines in which the expression of each is inhibited by RNAi or T-DNA insertions. Our demonstration of a SYTA-regulated functional link between early endosome formation and intercellular transport provides novel insights into how plasmodesmal function is controlled. Beyond the agronomic impact of engineering disease-resistant plants, our studies are relevant to human health in that they can inform and accelerate studies of animal synaptotagmins in cell-cell communication and development, and of mechanisms by which retroviruses redirect the endosomal machinery for virus maturation and exit from an infected cell. This research investigates the regulation of what seems to be a common pathway for transport between plant and animal cells. As such, it can impact public health by improving nutrition, providing knowledge about growth defects, and identifying novel ways to limit the spread of HIV and other retroviruses.