Regulation of Macromolecular Transport Through Plasmodesmata

描述（由申请人提供）：本研究旨在研究植物细胞间连接——胞间连丝（plasmodesmata， PD）对大分子运输的调控。PD连接了成熟植物中的大多数细胞，对于维持和调节不同植物组织内部和组织之间的通讯至关重要。为了研究控制PD转运发生的机制，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被感染的细胞转移到周围的细胞。由于PD是相邻植物细胞之间的唯一连接，植物病毒将这些通道作为它们在细胞间传播的主要途径。烟草花叶病毒是目前研究得最好的植物病毒之一，其PD转运是在一种病毒编码因子运动蛋白（MP）的帮助下发生的。因此，MP是研究PD中大分子转运的有力分子工具。在拟议的研究中，我们将继续利用这种实验方法，重点关注PD输运的一个最有趣但却知之甚少的方面——它的调控。严格控制PD转运的需要是其在植物与病毒相互作用以及正常植物发育和形态发生过程中的核心作用所固有的。实现PD转运控制的分子机制在很大程度上仍然未知。在目前的项目中，我们已经分离出了几个参与这些调控途径的植物因子，它们可能是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，以下两个目标将寻求研究PD调控的不同方面，共同促进理解控制PD转运的分子机制。1 . MP的差异磷酸化作为PD转运的“开/关”开关。我们已经确定了一种er相关蛋白激酶（ERPK），它特异性地磷酸化MP的Ser-37残基，激活其pd门控活性。在此之前，我们还发现了一种PD相关蛋白激酶（PDPK），它可以磷酸化MP的Ser-258、Thr-261和Ser-265残基，并作为MP抑制PD能力的负调节因子。因此，ERPK和PDPK代表了MP通过PD传输的监管“检查点”。在这里，我们将进一步研究这两种酶对PD的影响（例如，识别和靶向PD，以及被认为参与PD靶向和门控活动的MP的a-螺旋结构域和蛋白酶抗性结构域的改变）和PD通透性的发育调节，识别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PD运输的表型影响。2。mp -葡聚糖酶和GrIP/pdGRP/葡聚糖酶系统对PD转运的控制。我们发现MP直接与b -1,3葡聚糖酶相互作用，b -1,3葡聚糖酶是一种破坏PD颈部胼胝质的酶，已知限制PD运输。我们假设mp -葡聚糖酶的相互作用促进胼胝质括约肌的松弛，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b -1,3葡聚糖酶相互作用，可能抑制其活性并导致胼肌肌收紧。pdGRP本身的水平受其相互作用蛋白GrIP的调节。因此，MP和细胞因子对b -1,3葡聚糖酶的调节可能代表了PD转运途径中的另一个调节“检查点”。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通透性的机制。我们将研究mp -葡聚糖酶和pdgrp -葡聚糖酶的相互作用及其对b -1,3葡聚糖酶活性的影响。我们将研究GrIP结合pdGRP如何调节pdGRP的积累，并探讨GrIP/pdGRP/葡聚糖酶系统在PD通透性发育调控中的作用。

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, 1 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 1 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 2 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 2 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 B-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 B-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 B-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 B-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.