Regulation of Macromolecular Transport Through Plasmodesmata

描述(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)进行的大分子运输的调节。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之间的通讯至关重要。为了研究PD转运的控制机制(S)，我们利用了植物病毒，胞间连丝的海盗，通过这些通道在宿主细胞之间移动。对于广泛的感染，植物病毒必须从最初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，植物病毒利用这些通道作为它们从细胞到细胞的主要途径。烟草花叶病毒是研究最多的植物病毒之一，它的PD运输是在单一的病毒编码因子--运动蛋白(MP)的帮助下发生的。因此，MP是研究大分子通过PD转运的有力分子工具。在拟议的研究中，我们将继续利用这一实验方法，重点关注PD运输最有趣但却鲜为人知的一个方面--它的调节。严格控制PD运输的需要是其在植物-病毒相互作用以及正常植物发育和形态发生过程中的核心作用所固有的。实现这种PD转运控制的分子机制在很大程度上仍不清楚。在目前的项目中，我们已经分离出了参与这些调控途径的几个植物因子，很可能是PD运输不同阶段的“检查点”。计划中的实验将继续并扩大这一研究方向。具体地说，拟议工作的以下两个目标中的每一个都将寻求研究PD调节的不同方面，共同促进了解控制PD运输的分子机制的单一目标。I.作为PD转运的“开/关”开关的MP的差异磷酸化。我们已经确定了一种ER相关蛋白激酶(ERPK)，它能特异性地磷酸化Ser-37残基上的MP，激活其PD门控活性。早些时候，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门控PD能力的负调节因子。因此，ERPK和PDPK代表着MP通过PD运输的调节“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，识别和靶向PD，以及被认为参与其PD靶向和门控活动的MP的α螺旋和蛋白酶抗性结构域的变化)以及对PD通透性的发育调节的影响，鉴定和初步表征它们的细胞底物，并利用反向遗传学来确定ERPK和PDPK敲除/敲除对PD运输植物病毒和细胞蛋白的表型影响。2.MP-葡聚糖酶和GRIP/pdGRP/葡聚糖酶系统对Pd转运的调控。我们发现MP直接与β-1，3葡聚糖酶相互作用，β-1，3葡聚糖酶是一种破坏位于帕金森氏病颈部的、限制帕金森氏病转运的酶。我们假设，MP-葡聚糖酶的相互作用促进了穹隆括约肌的松弛，导致PD门控。另一方面，我们发现了一个GRIP/pdGRP/葡聚糖酶系统，在这个系统中，PD相关的富含甘氨酸的蛋白(PdGRP)与β-1，3葡聚糖酶相互作用，潜在地抑制了它的活性，并导致穹隆括约肌收缩。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β-1，3葡聚糖酶的调节很可能是PD转运途径中的另一个调节“关卡”。我们将研究MP-葡聚糖酶的相互作用和GRIP/pdGRP/葡聚糖酶系统通过痂积累控制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β-1，3-葡聚糖酶活性的影响。我们将研究GRIP与pdGRP结合如何调节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.