This project will provide research tools and resources for the plant science community to determine the in planta interactions of proteins. The research will develop Bimolecular Fluorescence Complementation (BMFC) methodology to investigate protein-protein interactions in planta. BMFC involves tagging proteins with complementary half-YFP molecules, neither of which fluoresces on its own. When half-YFP molecules are brought together as fusions with interacting proteins, fluorescence is restored. The BMFC design is a novel in planta "two hybrid" system. BMFC can also be utilized to identify the subcellular location of interacting proteins. The project will develop BMFC vectors to express half-YFP-tagged proteins for use both in transient transformation experiments, and for Agrobacterium-mediated transformation. The vectors will incorporate Gateway technology for high-throughput mobilization of amplified genes and cDNA libraries among the various vectors. Several Arabidopsis cDNA-coding regions will be cloned into the BMFC vectors; these genes are representative of large families of cytoskeleton, importin, chromatin, and F-box proteins. The project will also provide several Arabidopsis cDNA expression libraries in these vectors, which will be useful for comprehensive screens for protein interactions. As proof of concept, the project will test these tagged plant proteins for interaction with tagged Agrobacterium Virulence proteins in both transient and stable transformation assays. The tagged Virulence proteins include VirE2, VirD2, and VirF, as well as the Arabidopsis protein VIP1. The test Arabidopsis proteins will be selected based upon prior genetic and biochemical information. The limits of sensitivity of expression of the tagged proteins will be determined using both high-level constitutive as well as native promoters. All data derived from this project will be posted on the project web site: www.bio.purdue.edu/about/faculty/gelvin/gelvinweb/gelvin.html
Broader Impacts: This project will develop the first broad-based technology to investigate protein-protein interactions in planta, and will additionally identify plant proteins whose expression may be manipulated to increase the frequency of Agrobacterium-mediated transformation of recalcitrant plant species. This project also contains a substantial training component. In addition to training postdoctoral research scientists and technicians, the project will include a vigorous outreach program with local colleges to identify undergraduate students from under-represented minority groups and introduce them, through summer and academic year collaborations with our research groups, to the conduct of scientific research. Multi-year training of these students to solidify their interest in pursuing a career in science will be encouraged. When they return to their home institutions, the students will expose additional students to the techniques learned in our laboratories. The under-represented minority students trained in the project during the summer will continue their research at their home institutions, thus broadening the number of their peers who will come into contact with scientific research. We expect that these efforts will encourage undergraduate students to select a career in science.
