Control of Macromolecular Traffic Through Plasmodesmata

描述(由申请人提供)： 在高等真核生物从哺乳动物到植物的细胞间运输中，最耐人寻味但研究最少的方面之一，可能是大分子复合体通过细胞间细胞质桥梁的运输。这些连接在哺乳动物中被称为隧道纳米管(TNTs)，在植物中被称为胞间连丝(PD)，它们被入侵的病原体颠覆，使它们在宿主细胞之间移动。虽然哺乳动物和植物病原体，例如普恩病毒和(潜在的)艾滋病毒以及大多数植物病毒都利用细胞到细胞的运输途径，但第一次发现了植物病毒的这种能力。因此，通过PD的病毒运输代表了一种大分子细胞间运输的概念性和机械性范例。我们利用烟草花叶病毒(TMV)作为研究PD转运调控机制的工具，它的PD运输是由其运动蛋白(MP)介导的，主要集中在两个基本问题上：(1)MP如何激活宿主途径来门控PD通道？以及(Ii)宿主如何调节MP的这种PD门控活性？我们的数据提示存在三种调节机制，包括：MP诱导细胞通路的激活，通过磷酸化激活和失活MP，以及通过宿主泛素/蛋白酶体系统(UPS)下调MP。这些发现将被用来寻求三个目标：目的1.了解MP通过调节多糖括约肌来开启PD的机制。我们的数据鉴定了一种被MP识别的宿主细胞质蛋白ANK，并表明MP-ANK复合体在PD处积累，ANK的存在是MP诱导PD门控所必需的。我们还发现ANK与SS-1，3葡聚糖酶(BG)相互作用，BG是一种降解多糖PD括约肌的酶。我们将验证这样一种假设，即MP将ANK从胞浆重定向到PD，在那里ANK(或ANK-MP复合体)激活BG，从而松弛PD括约肌，提高PD通透性。目的2.了解MP磷酸化的调节功能。我们鉴定了一种内质网相关和PD相关的蛋白激酶(ERPK和PdPK)，它们特异性地磷酸化MP，分别激活和失活其门控PD的能力。我们将探索这样一种假设，即这些PKs作为MP通过PD运输的“开/关”开关。目的3.了解东道主UPS在下调MP方面的作用。我们的数据表明，病原菌的攻击诱导了与植物防御相关的F-box蛋白VBF的表达。在病原体编码的底物中，VBF识别MP。我们将验证VBF通过SCFVBF途径将MP靶向蛋白酶体降解的假设。总的来说，拟议实验的预期结果将定义和表征基本概念和分子机制，这些基本概念和分子机制一般地激活和停用大分子复合体的细胞间运输，特别是病原体。

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 underly activation and deactivation of intercellular transport of macromolecular complexes in general, and pathogens in particular.