Synaptotagmin function in virus movement and plant development

描述（由申请人提供）：植物病毒运动蛋白通过改变连接相邻植物细胞的胞间连丝和跨壁孔，将病毒基因组运输过细胞壁。Synaptotagmins是一个多基因家族，被认为是动物独有的，是钙（Ca2+）传感器，调节突触囊泡外/内吞作用。我们的长期目标是了解synaptotagmins如何调节病毒运动和细胞间运输，以控制疾病抵抗，RNAi信号的传播和细胞命运。SYTA是5个拟南芥突触tagmins （SYT A-E）中的1个。SYTA与begomovirus CaLCuV （MPCaLCuV）和SqLCV以及Tobamovirus TMV （30K）编码的不同运动蛋白结合。我们已经证明SYTA调节早期内体的形成和MPCaLCuV和TMV 30K的细胞间转运。我们认为SYTs通过内吞再捕获途径调节大分子运输到胞间连丝，并可能进入和退出孔。我们将结合转基因植物研究和基于细胞的表达分析来研究SYTs a、C和E是否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子的细胞运输试验将检验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，独立于SYTA或与SYTA合作。SYTC和SYTE在囊泡运输中的作用将通过免疫定位和基于细胞和叶片的分泌测定来确定。SYTA本身的多光子成像，或MPCaLCuV和30K，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如何在这一过程中合作。SYT A、C和E在植物细胞中的作用将通过启动子：：GUS融合研究来确定它们的空间和时间表达，并分析每一种基因的表达被RNAi或T-DNA插入抑制的转基因系，从而与发育调节有关。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，为胞浆间质功能的控制提供了新的见解。除了工程抗病植物的农艺影响外，我们的研究还与人类健康相关，因为它们可以为细胞-细胞通信和发育中的动物突触蛋白的研究提供信息并加速研究，以及逆转录病毒重定向内体机制以使病毒成熟并从感染细胞中退出的机制。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.