Analysis of virB genes in Agrobacterium T-DNA transfer

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During plant transformation by Agrobacterium tumefaciens the virB gene products most likely mediate T-DNA transport across the bacterial and plant cell membranes and walls. The long-term objective of this proposal is to elucidate the role of the eleven virB gene products in the T-DNA transfer process. The specific aims will examine the following questions: 1) Which virB genes are needed for virulence ? 2) Do VirB proteins compose the T-DNA transport machinery? and 3) Is phosphorylation involved in controlling or activating VirB functions? These questions will be addressed using a combination of genetic and biochemical approaches. First, VirB-specific antibodies will be raised and A. tumefaciens mutants will be isolated that each lack a different single virB gene. These antisera and mutants will then be used in combination with cellular fractionation, immunoelectron microscopy, protein cross-linking, site specific mutagenesis, and other techniques to identify, localize, and functionally characterize the virB gene products. The results will have immediate significance in providing insight into the mechanism of T-DNA transport. On a practical level, characterization of virB gene function in Agrobacterium will likely contribute to understanding the molecular mechanism by which macromolecules are transported across membranes.