A Host Protein Interaction and Localization Map for a Plant

Did you eat today? If yes, then it means, in part, that the viruses that threaten your food supply and national economy are being held in check...at least for now. Viruses pose constant threats to human health and welfare. In addition to maladies caused by pathogens such as the immunodeficiency, hepatitis and influenza viruses that are human-infecting viruses per se, diseases caused by plant viruses lead to diminished food quality or yield which, in turn, may have devastating economic, social, political or historical consequences. Defending against viruses is a continual challenge as new viral pathogens emerge or old ones evolve mechanisms to avoid methods to protect against them. The development of novel anti-viral strategies requires increasingly detailed insight into how viruses interact with their host cells to cause disease. In order to replicate or to be transported in host cells, viruses must divert, commandeer, or inhibit cellular functions to their advantage while concomitantly avoiding defense responses mounted by host cells. In these regards, proteins encoded by the genomes of host cells that associate physically with viral proteins are frequently found to be essential for the establishment of successful infections. In many cases these proteins are recruited away from their normal sites of accumulation to sub-cellular loci where critical viral processes, such as replication, occur. To prevent virus infections, defense-signaling cascades are often triggered following the physical interaction of a viral protein with a host-encoded receptor. Taken together, establishment of viral infection in host cells is a consequence of the complex integration of altered gene expression, protein:protein interactions and changes in protein localization. Integration of these processes into systems biology approaches for the development of novel anti-viral strategies requires as comprehensive a view of host-virus protein interaction and localization as possible. Additionally, the question arises whether genetically related viruses associate with the same host factors in infected cells. Therefore, in the proposed research, Sonchus yellow net virus (SYNV)and Potato yellow dwarf virus (PYDV), will be used to test the hypothesis that, despite both being members of the genus Nucleorhabdovirus, SYNV and PYDV utilize different subsets of host factors in order to establish compatible interactions in Nicotiana benthamiana. Construction of protein interaction maps for SYNVand PYDVare essential for the long-term goal of understanding how host and viral factors function in concert at the molecular level leading to disease. Additionally, given their unique structure, genetics and site of replication, nucleorhabdoviruses serve as novel probes for investigating fundamental aspects of nuclear import and export as well as macromolecular assembly and transport in plant cells. Therefore, the objectives of this proposal are, objective 1: To use high-resolution yeast two-hybrid screens to identify N. benthamiana proteins that interact with the SYNVnucleocapsid (N), phosphoprotein (P), putative movement (sc4) and matrix (M) proteins, objective 2: To produce an interaction matrix of nucleorhabdovirus and host nuclear proteins, objective 3: To determine the localization of up to fifty N. benthamiana proteins isolated in the two-hybrid screens and to confirm their interaction, in plania, using bimolecular fluorescence complementation (BiFC). Data from these three objectives will be integrated in objective 4 to produce the first protein interaction maps for plant-adapted rhabdoviruses. The intellelectual merit of the proposed research lies in the fact that it will contribute to an understanding of how pathogens evade host defense responses while establishing compatible interactions. With this knowledge in hand, novel control strategies may be developed. Additionally, these studies should provide novel insight into the adaptations made by rhabdoviruses that have evolved to replicate in nuclei instead of the cytoplasm of infected cells. The project will have a broader impact as novel resources will be generated that will benefit the plant biology community in general, and particularly those that use N. benthamiana, which has emerged as a critically important model host for plant pathogens, particularly viruses. Furthermore, it will provide multidisciplinary training for postdocs, graduate and undergraduate students, potentially recruited from underrepresented and minority populations, in microscopy-, molecular- and computer-based analyses. In collaborations with extension plant pathologists, materials will be generated to translate data from this project into teaching aids in biotechnology to educate county extension agents. An added benefit of this project is its potential to stimulate international collaborations (U.S.-Australia). Dr. Ralf Dietzgen, an internationally recognized leader in cytorhabdovirus research, would like to conduct a sabbatical in the Goodin lab during July of 2008. In addition to participating in this project, Dietzgen needs to learn advanced microscopy techniques. In this regard, Goodin, serving as Director of the University of Kentucky-Plant Science Imaging Facility, will continue to train members of the plant biology community in fluorescence microscopy techniques.