Proteins Involved in Transport Through Plasmodesmata

描述(申请人提供)：建议研究的目的是研究 蛋白质和核酸通过植物细胞间连接胞间连丝(PD)运输的调节机制。PD互连大多数电池 在成熟的植物中，对维持和调节 不同植物组织内部和之间的交流。为了研究 Pd运输的控制机制，该项目将开发植物 病毒，胞间连丝的海盗，在细胞之间和系统之间移动， 通过这些通道在宿主中传播。 烟草花叶病毒是研究最多的植物病毒之一，通过PD传播。 在其运动蛋白(MP)的帮助下。拟议的研究将利用 MP作为实现三个特定目标的分子工具，每个目标都寻求 研究PD调节的不同方面，这些方面将共同做出贡献 朝着了解控制的分子机制的单一目标前进 PD传输： (1)细胞间PD转运的负性调节作用的研究 磷酸化。一个与细胞壁相关的MP激酶被分离出来，它是MP活性的负调节因子。磷酸化对MP的影响 靶向PD和MP的U螺旋和蛋白酶抗性结构域， 被认为参与其PD门控活动的人，将受到检查。另外， 将确定MP激酶的细胞底物，并在最初 特征和反向遗传学将被用来研究功能作用 MP激酶在健康和病毒感染的植物中的表达。 (2)细胞间PD的正向调节和/或要求的研究 运输：MP运输所需基因中的植物突变体。转基因 产生的植物仅在其体内表达MP报告酶融合 毛状体(叶毛)；然而，由于MP在细胞之间移动，这种融合 蛋白质存在于每个毛状体周围的大片细胞中。跟随 对这些植物进行诱变，鉴定出一株突变株系，其中 MP的细胞间运动受阻。这种突变，据推测 受正向调节和/或细胞间所需的基因影响 PD运输，将被鉴定和克隆，并需要更多的基因 对于MP的运输途径，将在本章的后续部分中确定 基因筛查。 (3)全身性PD转运调控研究。我们已经确定了一个 可诱导的植物基因，控制病毒的全身性但不是局部运输 转录后基因沉默(PTGS)信号。的蛋白质产品 该基因将根据其在细胞内的定位来表征， 对PD通透性的影响，以及可能的细胞伙伴。

DESCRIPTION (provided by applicant): The proposed research aims to study regulatory mechanisms of transport of proteins and nucleic acids through plantintercellular connections, the plasmodesmata (PD). PD interconnect most cells within a mature plant and are critical for maintaining and regulating communication within and between different plant tissues. To study the controlling mechanisms of PD transport, this project will exploit plant viruses, pirates of plasmodesmata, that move cell-to-cell and systemically, throughout the host via these channels. Tobacco mosaic virus, one of the best studied plant viruses, moves through PD with the help of its movement protein (MP). The proposed research will utilize MP as a molecular tool to achieve three specific aims, each of which seeks to study a different aspect of PD regulation, and which together will contribute toward a single goal of understanding of the molecular mechanisms that control PD transport: (1) Study of negative regulation of cell-to-cell PD transport: the role of phosphorylation. A cell wall-associated MP kinase was isolated, that acts as a negative regulator of MP activity. The effects of phosphorylation on MP targeting to PD and on the u-helical and protease-resistant domains of MP, thought to be involved in its PD-gating activities, will be examined. Also, cellular substrates for the MP kinase will be identified and initially characterized, and reverse genetics will be used to study the functional role of MP kinase in healthy and virus-infected plants. (2) Study of positive regulation of and/or requirements for cell-to-cell PD transport: plant mutants in genes required for transport of MP. Transgenic plants were generated that express an MP-reporter enzyme fusion only in their trichomes (leaf hairs); because MP moves between cells, however, this fusion protein is found in large sectors of cells surrounding each trichome. Following mutagenesis of these plants, a mutant line was identified, in which cell-to-cell movement of MP is blocked. This mutation, which presumably affected a gene that positively regulates and/or is required for cell-to-cell PD transport, will be characterized and cloned and additional genes required for the MP transport pathway will be identified in the continuation of this genetic screen. (3) Study of regulation of systemic PD transport. We have identified an inducible plant gene that controls systemic but not local transport of viruses and post transcriptional gene silencing (PTGS) signals. The protein product of this gene will be characterized in respect to its intracellular localization, effects on PD permeability, and possible cellular partners.