| The soil bacterium, Agrobacterium tumefaciens, causes crown gall disease on dicotyledonous plants and is the primary vector in genetic engineering of plants for research and commercial agriculture. Agrobacterium exports DNA and at least four protein substrates, VirD2, VirE2, VirE3, and VirF, into the plant cell via a transporter complex called the type IV secretion system. This system, which includes a pilus, is comprised of twelve proteins, named VirB1 to VirB11, and VirD4, and is similar to other secretion systems in human pathogenic bacteria and those used for DNA exchange during bacterial mating, or conjugation. While a general structural model of the transporter complex exists, detailed protein subassemblies and the process of protein and DNA export remains unknown. To better understand Agrobacterium transformation, a genetic screen using the yeast Saccharomyces cerevisiae as the host was developed to identify host proteins involved in transformation. While developing this screen, factors that alter Agrobacterium transformation of yeast were identified. Among these, nitrocellulose filters, recovery after heat-shock transformation, thick plates, and use of galactose as a carbon source, all increase yeast transformation efficiency. To study the first stage of substrate export during Agrobacterium transformation, protein interactions between the exported proteins and the type IV secretion system were identified using the yeast two-hybrid assay. Fluorescent protein fusions to exported proteins indicated that while VirD4 targets VirE2 to the type IV secretion system, VirF is targeted by either VirB4 or an assembled secretion system. These results suggest there are two distinct pathways for protein export in the Agrobacterium type IV secretion system. Additional studies also indicate the secreted proteins are targeted to the type IV secretion system via a conserved C-terminal transport sequence. Lastly, to identify protein subassemblies in the transporter complex, the yeast two-hybrid system was used to assay for interactions between proteins of the secretion system. The results support a new role of VirB1 in pilus assembly. Together, these studies provide insights into transporter complex protein subassemblies, the process of protein export during Agrobacterium transformation, and further contribute to understanding homologous systems in human pathogens and bacterial conjugation. |
