TRANSPORT THROUGH PLASMODESMATA

在植物中，细胞通过细胞间相互连接。 胞间连丝(PD)。类似于细胞间 在动物的交流中，通过帕金森病的运输在 植物生理学与发育。最近的证据表明，帕金森病可以 主动运输大分子。其分子机制 主动局部放电传输的发生是未知的。有一种方法可以解释这些 机制是研究植物病原体如何通过宿主细胞传播。 一般来说，入侵的微生物不会发明新的新陈代谢 但根据其生命周期调整现有的细胞过程。这个 研究最多的通过帕金森病传播的病原体的例子是细胞间。 烟草花叶病毒(TMV)基因组RNA的迁移 病毒编码蛋白P30。在这里，P30将被用作学习的工具 参与PD转运的细胞蛋白。拟议中的研究已经 四个具体目标。 (I)纯化参与PD转运的P30胞浆受体。 通过模拟到核的进口，通过PD的主动运输可能需要 特定的细胞质受体。为了检验这一假设，潜在的 用抗P30抗体鉴定和纯化P30细胞质受体 抗独特型抗体以及与固定化P30的结合。基因 将通过免疫筛选克隆P30受体的编码基因 表达文库或纯化后的微测序 蛋白质。 (Ii)一种植物细胞壁相关蛋白激酶的纯化 特异性地使P30磷酸化。与许多其他交通工具类似 过程中，磷酸化可能参与了PD的运输。在这里， 细胞壁相关蛋白激酶，一种潜在的PD成分，能够 磷酸化P30，将被研究。这种蛋白质将通过以下方式提纯 细胞壁基质的去除和增溶，然后进行离子交换 凝胶过滤层析。纯化的蛋白激酶将是 用于克隆其编码基因。 (3)纯化P30的生物化学和细胞生物学研究 细胞质受体和细胞壁相关蛋白激酶。这个 纯化的受体的生物学作用将在体内进行研究 通过显微注射植物细胞，以及P30-受体的相互作用 将在体外利用酵母基因双杂交蛋白进行表征 检测系统、天然凝胶电泳法和斑点印迹法。 所有纯化蛋白的亚细胞定位将被确定 使用电子显微镜。潜在的发展规则 克隆的基因将通过Northern分析、原位杂交进行检测 以及利用转基因植物进行的基因实验。 (Iv)使用纯化的蛋白质开始鉴定细胞 大分子钯的运输途径。使用蛋白质-蛋白质 在该方案中开发的相互作用分析，纯化的蛋白质将 作为特定的探针来识别更多的细胞成分 帕金森病的转运途径。除了表征钯的传输之外， 拟议的研究结果可能具有普遍的生物学意义 与大分子跨膜运输的相关性。

In a plant, cells are linked with each other by intercellular connections, the plasmodesmata (PD). Similar to intercellular communication in animals, transport through PD plays a vital role in plant physiology and development. Recent evidence suggests that PD can actively transport macromolecules. The molecular mechanisms by which active PD transport occurs are unknown. One approach to elucidate these mechanisms is to study how plant pathogens spread through host cells. Generally, the invading microorganism does not invent novel metabolic pathways but adapts existing cellular processes for its life cycle. The best studied example of pathogen spread through PD is cell-to-cell movement to tobacco mosaic virus (TMV) genomic RNA which is mediated by a virus-encoded protein, P30. Here, P30 will be used as a tool to study cellular proteins involved in PD transport. The proposed research has four specific aims. (i) Purification of P30 cytoplasmic receptors involved in PD transport. By analog to nuclear import, active transport through PD may require specific cytoplasmic receptors. To test this hypothesis, the potential P30 cytoplasmic receptors will be identified and purified using anti-P30 antiidiotype antibodies as well as binding to immobilized P30. The genes coding for P30 receptors will be cloned by immunoscreening of cDNA expression libraries or following microsequencing of the purified proteins. (ii) Purification of a plant cell wall-associated protein kinase that specifically phosphorylates P30. Similar to many other transport processes, phosphorylation may be involved in PD transport. Here, the cell wall-associated protein kinase, a potential PD component capable of phosphorylating P30, will be studied. This protein will be purified by removal of cell wall matrix and solubilization followed by ion exchange and gel filtration chromatography. The purified protein kinase will be used to clone its encoding gene. (iii) Study of biochemistry and cell biology of the purified P30 cytoplasmic receptors and cell wall-associated protein kinase. The biological role of the purified receptors will be studied in vivo following their microinjection plant cells, and P30-receptor interaction will be characterized in vitro using yeast genetic two hybrid-protein detection system, native gel electrophoresis and dot blot assay. Subcellular localization of all purified proteins will be determined using electron microscopy. Potential development regulation of the cloned genes will be examined by northern analysis, in situ hybridization and genetic experiments using transgenic plants. (iv) Use of the purified proteins to begin characterization of cellular pathways for PD transport of macromolecules. Using protein-protein interaction assays developed in this proposal, the purified proteins will serve as specific probes to identify additional cellular components of the PD transport pathway. In addition to characterizing PD transport, the results of the proposed research may have a general biological relevance for transmembrane transport of macromolecules.