RUI: Protein-Protein Interactions Mediating Substrate Recognition by the VirB Complex of Agrobacterium Tumefaciens

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Banta99051261. Technical Agrobacterium tumefaciens causes crown gall tumors to form on susceptible plants by stably transforming the host organism with a 20-kb fragment of single-stranded DNA (the T-DNA) encoding plant growth hormone biosynthetic enzymes. The VirB proteins mediate export of this oncogenic DNA from the bacterium. The focus of this study is the protein-protein interactions mediating recognition by the transport machinery of the various substrates that utilize the VirB pore. These substrates include not only the T-DNA, but also a single-stranded DNA-binding protein (VirE2) involved in targeting the T-DNA to the nucleus of the host cell, and a DNA-protein complex derived from the mobilizable plasmid RSF1010, which appears to compete for access to the transport apparatus, inhibiting delivery of the other substrates. A novel chromosomally-encoded, 40 kD membrane protein, which the PI discovered earlier, interacts with VirE2. This protein will be purified, taking advantage of its affinity for VirE2, and its partial amino acid sequence determined. Degenerate primers will be designed accordingly and used to isolate a genomic clone for quantitative production and further characterization of this protein. Generation of alkaline phosphatase fusions and antisera will facilitate studies on the membrane topology and probing potential interactions with other components of the VirB transport machinery. Gene disruption will be used to assess directly the role of the 40 kD protein in virulence, specifically in the delivery of VirE2 to host plant cells. Strikingly, this protein appears to be destabilized in cells carrying RSF1010 or by over-production of one component of the VirB pore, VirB10; both of these conditions also result in an inhibition of VirE2 export. To pursue these observations, turnover of the 40 kD protein in response to production of an RSF1010-derived transfer intermediate will be measured. In a second set of experiments, a mutant screen will be initiated in the hopes of identifying one or more VirB proteins responsible for activating release of a substrate through the VirB pore in response to host-cell contact. Screening for plant cell-independent secretion of VirE2 will identify such mutants. Together, these studies should provide some insight into the process by which DNA and/or associated proteins are recognized and transported across the two apolar membranes that enclose the Agrobacterium cell. Molecular dissection of the mechanism mediating export of these virulence determinants will, in turn, enhance our understanding of pathogenesis in other organisms, including Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila, that utilize homologous transport systems to deliver their pathogenic substrates.2. Non-technical The bacterium Agrobacterium tumefaciens causes a disease in which tumors form on certain plants. The symptoms result from the transfer of a piece of DNA from the bacterium to the susceptible plant cell. Movement of a protein, VirE2, is also required for efficient tumor formation. The goal of this study is to elucidate the mechanism by which the disease-causing DNA and VirE2 are recognized and delivered through the membrane surrounding the bacterium into the host plant cell. The VirB proteins are known to mediate this transport; we have identified an additional novel, non-VirB, component that appears to interact with VirE2. The hypothesis to be tested is that this protein may allow the VirB transport apparatus to engage with VirE2, since the protein is no longer detectable under conditions that are known to block VirE2 movement from the bacterium. The gene encoding this protein will be cloned to generate this protein in quantity so that if and how the protein interacts with the VirB proteins can be determined. A second line of experimentation will also be initiated to investigate how contact with the plant cell triggers transport of the VirE2 from the bacterium. This will be done by searching for mutants that release VirE2 even in the absence of a host plant cell.