CELLULAR PROTEINS INVOLVED IN TRANSPORT THROUGH

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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.