We shall 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. We shall 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. The vectors will additionally incorporate rare-cutting restriction endonuclease sites into which can be cloned RFP-tagged proteins of known sub-cellular localization ("marker proteins"). We shall construct several Arabidopsis cDNA expression libraries in these vectors, which will be useful for comprehensive screens for protein interactions. As proof of concept, we shall test these tagged plant proteins for interaction with tagged Agrobacterium Virulence proteins in both transient and stable transformation assays.
This project will develop a 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. All resources and deliverables of this project will be made public as soon as their quality control is completed.