Department of Biochemistry and Cell Biology, State University of New York;
Department of Biochemistry and Cell Biology, State University of New York.
J Vis Exp. 2024 Nov 1(213). doi: 10.3791/67378.
Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, macromolecules, as well as invading viruses. Plasmodesmata play fundamental roles in the regulation of intercellular communication, contributing to plant development, environmental responses, and interactions with viral pathogens. Discovering plasmodesmal localization of plant or viral proteins could provide useful functional information about the protein and is important for understanding the mechanisms of plant-virus interactions. To facilitate these studies, we describe a protocol for confocal microscopy-based analysis of different plasmodesmal targeting proteins to select the best plasmodesmal marker for studying the virus-plasmodesmata interactions or plasmodesmal transport. Specifically, the analyses of these events are illustrated using the cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV), the Arabidopsis Plasmodesmata-Localized Protein 5 (PDLP5) and Plasmodesmata Callose-Binding Protein 1 (PDCB1). The protein plasmodesmal localization data are analyzed in parallel with the global visualization of plasmodesmata using aniline blue staining of the sampled tissues. These approaches can be easily adapted to analyze the plasmodesmal localization of any cellular or pathogen proteins in planta.
胞间连丝是连接相邻植物细胞细胞质的膜纳米孔，使营养物质、大分子以及入侵病毒在细胞间运输。胞间连丝在细胞间通讯的调节中发挥着基础性作用，有助于植物的发育、对环境的响应以及与病毒病原体的相互作用。发现植物或病毒蛋白在胞间连丝上的定位，可以为该蛋白提供有用的功能信息，对于理解植物-病毒相互作用的机制非常重要。为了促进这些研究，我们描述了一种基于共聚焦显微镜的分析不同胞间连丝靶向蛋白的方法，以选择研究病毒-胞间连丝相互作用或胞间连丝运输的最佳胞间连丝标记物。具体来说，使用芜菁叶脉清病毒（Turnip vein-clearing virus，TVCV）的细胞间移动蛋白（cell-to-cell movement protein，MP）、拟南芥胞间连丝定位蛋白 5（Plasmodesmata-Localized Protein 5，PDLP5）和胞间连丝结合的胼胝质结合蛋白 1（Plasmodesmata Callose-Binding Protein 1，PDCB1）对这些事件进行了分析。对蛋白的胞间连丝定位数据与使用苯胺蓝对采样组织进行的胞间连丝整体可视化平行进行分析。这些方法可以很容易地适应于分析任何植物细胞或病原体蛋白在植物体内的胞间连丝定位。
