Regulation of Macromolecular Transport Through Plasmodesmata

描述（由申请人提供）：拟议的研究旨在研究通过植物细胞间连接（胞间连丝（PD））的大分子运输的调节。PD连接成熟植物中的大多数细胞，并且对于维持和调节不同植物组织内和之间的通信至关重要。为了研究PD运输控制发生的机制，我们利用植物病毒，胞间连丝的海盗，通过这些通道在宿主细胞之间移动。为了广泛感染，植物病毒必须从最初感染的细胞转移到其周围的细胞。由于PD是相邻植物细胞之间的唯一连接，植物病毒使用这些通道作为它们从细胞到细胞的主要通道。烟草花叶病毒是研究最多的植物病毒之一，其PD转运是在病毒编码的运动蛋白（MP）的帮助下进行的。因此，MP代表了一个强大的分子工具，研究大分子通过PD的运输。在拟议的研究中，我们将继续利用这种实验方法，专注于最有趣的，但知之甚少，PD运输方面-其调节。在植物-病毒相互作用以及正常植物发育和形态发生过程中，需要严格控制PD运输是其核心作用所固有的。实现这种PD转运控制的分子机制在很大程度上仍然未知。在当前的项目中，我们已经分离出了参与这些调节途径的几种植物因子，它们可能充当PD运输不同阶段的“检查点”。计划中的实验将继续并扩展这一研究方向。具体来说，以下两个目标的每一个拟议的工作将寻求研究PD调节的不同方面，共同促进对控制PD运输的分子机制的理解的单一目标。I. MP的差异磷酸化作为PD转运的"开/关"开关。我们已经确定了ER相关蛋白激酶（ERPK），特异性磷酸化MP的Ser-37残基，激活其PD门控活性。早些时候，我们还鉴定了PD相关蛋白激酶（PDPK），其在其Ser-258、Thr-261和Ser-265残基磷酸化MP，并作为MP门控PD能力的负调节剂。因此，ERPK和PDPK代表MP通过PD转运的调节"检查点"。在这里，我们将进一步研究这两种酶对MP的影响（例如，本发明的目的在于研究对PD的识别和靶向以及MP的α-螺旋和蛋白酶抗性结构域中的改变（被认为参与其PD靶向和门控活性）和PD渗透性的发育调节的影响，鉴定并初步表征它们的细胞底物，并使用反向遗传学来确定ERPK和PDPK敲除/敲低对植物病毒和细胞蛋白的PD转运的表型效应。二.通过MP-葡聚糖酶和GrIP/pdGRP/葡聚糖酶系统控制PD转运。我们发现MP直接与？- 1，3葡聚糖酶，一种破坏位于PD颈部区域的胼胝质的酶，并且已知限制PD转运。我们推测MP-葡聚糖酶相互作用促进胼胝质括约肌松弛，导致PD门控。另一方面，我们发现了一个GrIP/pdGRP/葡聚糖酶系统，其中PD相关的富含甘氨酸的蛋白（pdGRP）与？1，3葡聚糖酶，潜在地抑制其活性并导致胼胝质括约肌的收紧。pdGRP本身的水平由其相互作用蛋白GrIP调节。因此，调制的？- 1，3葡聚糖酶的MP和细胞因子可能代表另一个调节"检查点"的PD运输途径。我们将研究MP-葡聚糖酶相互作用和GrIP/pdGRP/葡聚糖酶系统通过胼胝质积累控制PD渗透性的机制。我们将研究MP-葡聚糖酶和pdGRP-葡聚糖酶的相互作用以及它们对？1，3葡聚糖酶。我们将研究GrIP结合pdGRP如何调节pdGRP的积累，并探索GrIP/pdGRP/葡聚糖酶系统在PD通透性发育调节中的作用。

DESCRIPTION (provided by applicant): The proposed research aims to study regulation of macromolecular transport through plant intercellular 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 examine the mechanism(s) by which the control of PD transport occurs, we exploit plant viruses, pirates of plasmodesmata, that move between host cells through these channels. For widespread infection, plant viruses must move from the initially infected cell to its surrounding cells. Because PD are the only connections between adjoining plant cells, plant viruses use these channels as their major routes of passage from cell to cell. PD transport of Tobacco mosaic virus, one of the best studied plant viruses, occurs with the help of a single virally-coded factor, the movement protein (MP). MP, therefore, represents a powerful molecular tool to study macromolecular transport through PD. In the proposed research, we shall continue to utilize this experimental approach, focusing on one of the most intriguing, yet poorly understood, aspects of PD transport - its regulation. The need to tightly control PD transport is inherent in its central role during plant-virus interactions as well as during normal plant development and morphogenesis. The molecular mechanisms by which such PD transport control is achieved remain largely unknown. In the current project, we have isolated several plant factors that are involved in these regulatory pathways, likely functioning as "checkpoints" of distinct stages of PD transport. The planned experiments will continue and expand this research direction. Specifically, each of the following two aims of the proposed work will seek to study a different aspect of PD regulation, together contributing toward a single goal of understanding of the molecular mechanisms that control PD transport. I. Differential phosphorylation of MP as an "On/Off' switch of PD transport. We have identified an ER-associated protein kinase (ERPK) that specifically phosphorylates MP at the Ser-37 residue, activating its PD-gating activity. Earlier, we also identified a PD-associated protein kinase (PDPK) that phosphorylates MP at its Ser-258, Thr-261, and Ser-265 residues and acts as a negative regulator of the MP ability to gate PD. Thus, ERPK and PDPK represent regulatory "checkpoints" for MP transport through PD. Here, we shall further study the effects of these two enzymes on MP (e.g., recognition of and targeting to PD and alterations in the a-helical and protease-resistant domains of MP thought to be involved in its PD targeting and gating activities) and on developmental regulation of PD permeability, identify and initially characterize their cellular substrates, and use reverse genetics to determine the phenotypic effects of the ERPK and PDPK knockouts/knockdowns on PD transport of plant viruses and cellular proteins. II. Control of PD transport by the MP-glucanase and GrIP/pdGRP/glucanase systems. We showed that MP directly interacts with ?-1,3 glucanase, an enzyme that destroys callose located in the neck region of PD and known to restrict of PD transport. We hypothesize that the MP-glucanase interaction promotes relaxation of the callose sphincter, resulting in PD gating. On the other hand, we discovered a GrIP/pdGRP/glucanase system, in which a PD-associated glycine-rich protein (pdGRP) interacts with ?-1,3 glucanase, potentially inhibiting its activity and leading to tightening of the callose sphincter. The levels of pdGRP itself are modulated by its interacting protein, GrIP. Thus, modulation of the ?-1,3 glucanase by MP and cellular factors likely represents another regulatory "checkpoint" in the PD transport pathway. We shall examine the mechanisms by which MP-glucanase interaction and the GrIP/pdGRP/glucanase system control PD permeability via callose accumulation. We shall study the MP-glucanase and pdGRP-glucanase interactions and their effects on the enzymatic activity of ?-1,3 glucanase. We shall investigate how GrIP binding to pdGRP modulates accumulation of pdGRP, and explore the role of the GrIP/pdGRP/glucanase system in developmental regulation of PD permeability.