VirB Mediated DNA and Protein Transfer from A. tumefaciens

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Agrobacteria are bacteria that infect damaged plants. Bacterial DNA is transferred to the plant nucleus, and this process is used to move desired genes into plants in the process of genetic engineering. It is important to understand how this process occurs, as it represents a natural occurence of inter-kingdom DNA transfer, as well as of practical importance. The movement of DNA from Agrobacterium tumefaciens into plant cells depends upon the activities of the virulence (vir) genes of the Ti plasmid. Several of the Vir proteins are required for the production of a single-stranded DNA covalently attached to VirD2 at its 5' terminus. The long term objectives are to understand how the VirB complex assembles and functions. Towards this end, a variety of genetic and biochemical methods have been developed to study the interaction of VirB proteins with themselves and with transported substrates. Recent studies yielded the surprising result that the expression of a subset of the VirB proteins in the recipient vastly increased the efficiency of virB-mediated conjugal transfer of RSF1010 between agrobacteria. This result leads to several questions about the VirB proteins and recipient activity: a) what is the minimal complement of VirB proteins necessary for increased recipient activity, b) does the presence of RSF1010 in the recipient affect this activity and c) what is the generality of this phenomenon? The answer to these questions may allow the identification of a "core" VirB complex that could simplify genetic and biochemical studies, and may have important implications to plasmid transfer in general.In order to define the VirB complex and its activity, the interactions between the VirB proteins and themselves or the transported substrates must be understood.. In this project physical/biochemical analysis, utilizing chemical cross-linking and immunoprecipitation will be continued; yeast two hybrid system analysis will be carried out, looking specifically at the possibility that other VirB proteins - and possibly other unidentified proteins - interact with the VirB7-10 group; and saturation mutagenesis on the virB7-10 genes will be carried out to help define regions required for protein interaction and for virulence in general.