Agrobacterium tumefaciens mediates the movement of DNA from the Ti plasmid of the bacterium into plant cells, where the "transferred DNA" (T-DNA) is ultimately integrated into the host genome and expressed. Processes within the bacterium that result in the formation of "transfer intermediates" have been well studied and have led to the hypothesis that the DNA transfer process is similar to bacterial conjugation. The mechanism whereby the transfer intermediates (composed of protein and DNA) exit the bacterium and enter the plant cells is virtually uncharacterized. The virB genes of the Ti plasmid encode proteins that are hypothesized to form a membrane localized "transfer apparatus" through which the transferred intermediates move from the bacterium into the plant cell. In this renewal project, studies will be undertaken to continue the genetic, molecular, and biochemical analysis of the putative virB-encoded transfer apparatus. The following specific aims will be pursued. First, the genetic analysis of the virB operon will be continued, taking advantage of a recently constructed transposon that will make possible the construction of non-polar mutations. It should be possible to determine which of the virB genes are required for virulence, and to carry out extensive point mutagenesis on individual virB genes. These mutants will be used in conjunction with biochemical and molecular strategies to study VirB related activities. Second, the relationship between the putative virB encoded apparatus and the transported substrates will be characterized. Molecular genetic approaches will be used to test the hypothesis that pJW323 produces an intermediate in such abundance that it outcompetes the T-complex for access to the transport pore. The specificity of the virB encoded transfer apparatus for the various transferred molecules will be characterized. Further, it will be determined whether overexpression of other vir genes affects either the transfer of PJW323 into plants or its ability to inhibit T-DNA transfer. %%% This work focusses on basic issues concerning DNA transfer from Agrobacterium to plant cells. Historically, the field originated with studies of how Agrobacterium caused Crown Gall Tumors in tobacco plants. Today, the utilization of Agrobacterium as a vector to transfer DNA into plant cells, and, ultimately, the regeneration of transgenic plants from such cells, is a central technology in both basic and applied plant science. The advantages of the technique are that Agrobacterium-mediated transformation is technically simple to execute and, in many plants, occurs at remarkably high efficiency. A major problem with the technique is that, for many agriculturally important plants, it is either highly inefficient or hasn't been shown to transform at all. Understanding the cell biology involved in the efficient transformation of certain hosts may provide insight into means by which procedures can be modified so as to transform hosts currently outside the useful host range of Agrobacterium. This would be of major significance to agricultural research and new product development.
