Little is known about how plant cells interact and communicate with their biological environment, which includes their cell wall and neighboring cells. These short range interactions play a central role in plant cell morphogenesis, differentiation, and multicellular pattern formation. Many proteins that are involved in cell-cell and cell-wall interaction would be expected to localize to the cell periphery, and perhaps to domains of the cell periphery. As a means of identifying proteins of potential functional interest due to their localization pattern, we have screened a GFP::cDNA expression library in primary transformed seedlings of Arabidopsis thaliana. We identified a fusion protein (D41) that localizes to discreet sites of cell-cell contact. The localization pattern shows sensitivity to cell type, cell polarity, and peripheral microdomains. The cell-type and subcellular localization patterns are consistent with the cell-cell channels called plasmodesmata, organelles for which there is very little molecular or genetic information. The sequence fused to GFP is a syntaxin, a protein that mediates vesicle targeting and fusion. We propose to characterize this syntaxin and to exploit the fusion protein s a staring point for a functional and molecular characterization of the cell-cell contact sites and their possible role in cell-cell interactions. These studies will consist of the following: (A) Fine scale localization of D41 and the native protein. (B) Functional analysis of the native gene by (a) genetic disruption, and (b) global, tissue-specific, and mosaic expression of a dominant negative allele. Phenotypic analyses will be performed by cell injection studies, TEM, and multi-fluorescent protein confocal microscopy. (C) Identification of interacting proteins using the yeast 2-hybrid interaction system and by immunoprecipitation of protein complexes. Candidate proteins and genes will be analyzed by intracellular localization and genetic interaction with native and mutant syntaxin alleles. (D) Identification of genes required for the assembly and patterning of the cell-cell contact sites using D41 as an in vivo marker for a microscopic genetic screen.
