Control of Macromolecular Traffic Through Plasmodesmata

描述（由申请人提供）：从哺乳动物到植物，高等真核生物中细胞间转运的最有趣但研究最少的方面之一可能是大分子复合物通过细胞间细胞质桥的运输。这些连接在哺乳动物中被称为隧道纳米管（TNT），在植物中被称为胞间连丝（Pd），它们在宿主细胞之间的运动被入侵的病原体破坏。而哺乳动物和植物病原体，例如，朊病毒和（潜在的）HIV以及大多数植物病毒利用细胞到细胞的转运途径，第一个这样的能力被鉴定为植物病毒。因此，通过Pd的病毒运输代表了大分子细胞间运输的概念和机制范例。我们利用烟草花叶病毒（Tobacco mosaic virus，TMV）的运动蛋白（Movement protein，MP）介导Pd的转运，研究了TMV中Pd转运的调控机制，主要研究两个基本问题：（1）MP如何激活宿主途径，从而门控Pd通道？以及（ii）宿主如何调节MP的Pd门控活性？我们的数据表明，涉及MP的三个监管机制的存在：MP诱导的激活的细胞通路的多糖Pd括约肌的松弛，激活和失活的MP磷酸化，和下调MP的主机泛素/蛋白酶体系统（UPS）。这些研究结果将用于寻求三个目标：目标1。了解MP通过调节多糖括约肌来控制Pd的机制。我们的数据确定了一个主机的细胞质蛋白ANK，这是由MP识别，并表明MP-ANK复合物积累在Pd和ANK的存在下，需要MP诱导的门控Pd。我们还表明，ANK与β-1，3葡聚糖酶（BG），一种降解多糖Pd括约肌的酶相互作用。我们将检验MP将ANK从细胞质重定向到Pd的假设，其中ANK（或ANK-MP复合物）激活BG，BG松弛Pd括约肌并提高Pd渗透性。 目标2.了解MP磷酸化的调节功能。我们鉴定了特异性磷酸化MP的ER相关和Pd相关蛋白激酶（ERPK和PdPK），分别激活和失活其门控Pd的能力。我们将探讨的假设，这些PKs作为一个“开/关”开关MP运输通过Pd。 目标3.了解主机UPS在MP下调中的作用。我们的数据表明，与病原体的挑战诱导植物防御相关的F盒蛋白VBF的表达。在其病原体编码的底物中，VBF识别MP。我们将检验VBF通过SCFVBF途径将MP靶向蛋白酶体降解的假设。 总的来说，拟议的实验的预期成果将定义和表征基本概念和分子机制，其基础是激活和失活的细胞间运输的大分子复合物一般，特别是病原体。 公共卫生相关性：该建议侧重于病毒病原体调节宿主细胞间连接的渗透性以进行传播的机制，使用烟草花叶病毒的运动蛋白（MP）作为模型系统，其介导病毒通过胞间连丝（Pd）运输。具体而言，我们的目标是确定（i）的机制，MP门Pd，（ii）的MP激活和失活的磷酸化“开/关”开关的机制，和（iii）参与的主机泛素/蛋白酶体系统（UPS）在MP的下调。 在一般的生物学背景下，所提出的研究的意义在于（i）机械概念的定义-募集宿主因子以激活去除限制其渗透性的运输通道的结构元件的途径-病原体用于通过细胞质细胞间通道（例如Pd或TNT）在宿主细胞之间传播，和（ii）确定病原体和宿主用于控制这种细胞间运输破坏的分子机制。

DESCRIPTION (provided by applicant): Perhaps one of the most intriguing, yet least studied, aspects of intercellular transport in higher eukaryotes, from mammals to plants, is traffic of macromolecular complexes through intercellular cytoplasmic bridges between cells. These connections, termed tunneling nanotubes (TNTs) in mammals and plasmodesmata (Pd) in plants, are subverted by invading pathogens for their movement between the host cells. Whereas both mammalian and plant pathogens, e.g., prions and (potentially) HIV as well as most plant viruses, utilize cell-to-cell transport pathways, the first such capability was identified for plant viruses. Thus, viral transport via Pd represents a conceptual and mechanistic paradigm for intercellular traffic of macromolecules. We exploit Tobacco mosaic virus (TMV), whose Pd transport is mediated by its movement protein (MP), as a tool to study the regulatory mechanisms of Pd transport, focusing on two fundamental questions: (i) How does MP activate the host pathway for gating the Pd channel? And (ii) how does the host regulate this Pd-gating activity of MP? Our data suggest the presence of three regulatory mechanisms that involve MP: MP-induced activation of the cellular pathway for relaxation of a polysaccharide Pd sphincter, activation and deactivation of MP by phosphorylation, and down-regulation of MP by the host ubiquitin/proteasome system (UPS). These findings will be used to seek three objectives: Aim 1. Understand the mechanism by which MP gates Pd by modulating the polysaccharide sphincter. Our data identified a host cytoplasmic protein ANK that is recognized by MP and showed that the MP-ANK complexes accumulate at Pd and that the presence of ANK is required for MP-induced gating of Pd. We also showed that ANK interacts with ss-1, 3 glucanase (BG), an enzyme that degrades the polysaccharide Pd sphincter. We will test the hypothesis that MP redirects ANK from the cytoplasm to Pd, where ANK (or ANK-MP complexes) activates BG, that relaxes the Pd sphincter and elevates the Pd permeability. Aim 2. Understand the regulatory function of MP phosphorylation. We identified an ER- associated and Pd-associated protein kinases (ERPK and PdPK) that specifically phosphorylate MP, activating and deactivating its ability to gate Pd, respectively. We will explore the hypothesis that these PKs act as an "On/Off" switch of MP transport through Pd. Aim 3. Understand the role of the host UPS in down-regulation of MP. Our data show that challenge with pathogens induces expression of the plant defense-related F-box protein VBF. Among its pathogen-encoded substrates, VBF recognizes MP. We will test the hypothesis that VBF targets MP to proteasomal degradation via the SCFVBF pathway. Collectively, the expected outcomes of proposed experiments will define and characterize basic concepts and molecular mechanisms that underlies activation and deactivation of intercellular transport of macromolecular complexes in general, and pathogens in particular. PUBLIC HEALTH RELEVANCE: This proposal focuses on the mechanisms by which viral pathogens modulate permeability of the host intercellular connections for their spread, using as model system the movement protein (MP) of Tobacco mosaic virus which mediates viral transport through plasmodesmata (Pd). Specifically, we aim to determine (i) the mechanism by which MP gates Pd, (ii) the mechanism of MP activation and deactivation by phosphorylation "On/Off" switch, and (iii) the involvement of the host ubiquitin/proteasome system (UPS) in down-regulation of MP. In the general biological context, the significance of the proposed studies lies in (i) definition of the mechanistic concept-recruitment of the host factors to activate a pathway that removes the structural elements of the transport channel that restrict its permeability-employed by pathogens for spread between host cells via cytoplasmic intercellular channels, such as Pd or TNTs, and (ii) determination of molecular mechanisms that both the pathogen and the host use to control this subversion of intercellular transport.