Intellectual merit: This project will investigate the formation, composition, and sub-cellular site(s) of localization of the Agrobacterium T-complex as it is assembled in and transported through the plant cell. Agrobacterium transfers single-strand DNA (T-strands), covalently linked to VirD2 protein, through a Type IV Secretion System (T4SS) into plant cells. Several other effector virulence proteins, including VirE2, are separately transferred to the plant via the T4SS. VirE2 is a single-strand DNA binding protein that, in vitro, can complex with and coat T-strands. A current favored hypothesis is that in plant cells, VirE2 coats also helps target T-strands to the nucleus, where T-strands become double-stranded and integrate into the plant genome. Targeting T-strands through the cytoplasm and into the nucleus is likely directed by plant proteins such as importin-alpha and VIP1. In vitro and in plant cells, importin-alpha interacts with VirD2, VirE2, and VIP1, and these proteins have been hypothesized to form a mature or super-T-complex with T-strands in planta. However, these studies have all investigated protein-protein interactions in plant cells in the absence of T-strands. In addition, expression of VirE2 has been from a strong plant promoter, resulting in high levels of protein that forms aggregates. Bimolecular Fluorescence Complementation (BiFC) technology can be used to track VirE2 as it exits Agrobacterium and interacts with proteins within the plant cell. BiFC fluorescence technology, along with a modified T-DNA immunoprecipitation assay, will be used to monitor the assembly and intra-cellular trafficking of T-complexes in living plant cells. Under these conditions, the various putative T-complex components are synthesized at natural levels, and in their native organisms, prior to assembly in the plant cell. The results of these studies are important to resolve much conflicting data in the literature regarding the roles of various putative T-complex components in T-DNA trafficking through the plant cell.
Broader impacts of the proposed research: Horizontal gene transfer has been recognized as a major component of evolution, and Agrobacterium represents one of the best studied examples of horizontal gene flow. Agrobacterium-mediated genetic transformation is also the major mechanism to generate transgenic plants for basic research and for agricultural biotechnology purposes. Understanding how T-DNA traffics through the plant cell is important for understanding how extra-cellular protein-nucleic acid complexes (including viral genomes) target the nucleus after entering a cell. Because many of the steps in nuclear targeting may be rate-limiting, understanding the process will be important for preventing disease (such as Crown Gall caused by Agrobacterium, or viral diseases), and for improving the transformation of recalcitrant crop species. In addition to training research scientists and graduate students, this project will be used to conduct a vigorous outreach program with Brooklyn College to identify undergraduate students from under-represented minority groups and introduce them, through summer and academic year collaborations, to the conduct of scientific research. Multi-year training of these students will be encouraged to solidify their interest in pursuing a career in science. When they return to their home institution, they will expose additional students to the techniques learned in our laboratory. The under-represented minority students trained during the summer will continue the projects at their home institutions, thus broadening the number of their peers who will come into contact with scientific research. These efforts will encourage undergraduate students to select a career in science.
