In vitro Reconstruction and Biochemical Characterization of the Agrobacterium T-DNA Transfer Process

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The proposed research will allow for a further understanding of cell transformation. Studies to data have used whole Agrobacterium cells to identify several (vir) protein products essential for the production of a transferable DNA copy, the T-strand. The proposed experiments will use predominantly biochemical approaches to analyze T-strand synthesis and processing into a T-strand-protein complex (T- complex) using purified components in vitro. Specifically, these studies will (1) reconstruct T-strand synthesis in vitro using a DNA substrate and purified vir proteins. If necessary, commercially available enzymes (DNA polymerases, topoisomerases) or whole cell extracts from Agrobacterium will be used to supplement the reaction. (2) Assays will be carried out to determine whether the T-complex characterized to date (and assembled in vitro) consisting of T-strands and VirE2 and VirD2 is capable of transforming plant protoplasts. As other proteins of the T-complex are identified they will be added to the T-strand-VirD2-VirE2 complex to test their role in enhancing plant cell transformation. (3) Additional proteins that potentiate T-complex transfer will be defined by their affinity to columns carrying covalently bound VirD2 or VirE2 and by a recently developed assay for proteins that mediate export of T-strands from whole Agrobacterium cells. (4) The functional domains of proteins required for T-strand synthesis and processing will be defined by assaying for activity subfragments of these proteins produced by deletion mutants or proteolytic digestion. (5) By using a sensitive in vitro assay for integration of exogenous DNA it will be determined if the T-complex has an integrase function. The proposed experiments are relevant to the biology of the Agrobacterium-plant cell interaction and to DNA transfer technology. In nature, Agrobacterium transfers a specific DNA segment, the T-DNA, into a plant nucleus where, following stable integration, the genetic material is expressed in the plant cell. This property of Agrobacterium has prompted an intense investigation of the requirements for transfer and has led to the design of vectors capable of transferring any DNA of interest to plant cells without interfering with normal plant growth and differentiation. While Agrobacterium can now be used routinely as a vector to transfer DNA to many plant cells without the need to understand the underlying mechanisms involved, it is important to understand the biology of the system so that advances in the use of the vector properties of the system can be made. This research will investigate the detailed steps involved during the DNA transfer process so that improved methods to allow directed DNA transfer to plant cells can be achieved.