Collaborative Research: Arabidopsis 2010: In Vivo Genomics: Visualizing G Protein Interactions in Arabidopsis

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Heterotrimeric G proteins couple multiple and diverse signals (e.g. light, pathogens, hormones) recognized by receptors to downstream effectors in a cell-specific manner. In Arabidopsis, hypothesized G-protein involvement in multiple signal transduction and developmental processes can be assessed using the tools developed by this project. The gene set comprises 16 heptahelical membrane proteins (candidate G protein-coupled receptors), one canonical Ga subunit, one Gb, and two Gg subunits (of the heterotrimeric complex), and three 'extralarge' Ga-related proteins (XLGs). The resources/tools that will be developed are: 1) GUS constructs allowing localization of expression for the gene set and; 2) constructs allowing visualization of physical interactions between members of the gene set in vivo in real time and with spatial dimensions using fluorescence energy transfer (FRET) between cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged proteins. The GUS constructs will be available at the conclusion of the first year of funding, and the YFP and CFP translational fusion constructs will be available at the conclusion of the second year of funding. Vector maps, images of expression levels and patterns, protocol updates, and instructions for obtaining material will be posted weekly at the following URL, which will be TAIR-linked: www.plantbiology.unc.edu/ Constructs will be available for order via the webpage. For each translational fusion, three stable transgenic lines will be deposited in the stock center following validation.The significance of the proposed work in relation to the overall 2010 project objectives is twofold. Function of the members of the gene family that is the target of this project will be determined as follows. Expression patterns of the genes will be evaluated in plants. The role the gene projects have in root cell proliferation and guard cell signaling will be evaluated, and novel small molecules that modulate plant G protein signaling pathways, as assessed by the FRET 'readout', will be identified. Tools developed by the project will allow the community to perform direct in vivo tests of hypothesized G protein involvement in any signal transduction or developmental context. As insights are gained into use of plant cell in vivo FRET analysis for monitoring the interaction between G protein complexes and G protein-coupled receptors, this tool will be made available to other plant research groups for evaluation of in vivo protein:protein interactions in other systems. In addition, the YFP constructs and lines will be compatible with BRET analysis being developed by the von Arnheim/Johnson 2010 project (Univ. Tennessee).The broader impact of the proposed research includes pre- and postdoctoral training by direct mentorship of the two Principal investigators, and a hands-on tutorial on FRET measurements during a G protein workshop organized by the PIs. The workshop will be advertised in particular at small liberal arts colleges and minority institutions and undergraduate students will be eligible to apply for travel grants.