9513662 Binns 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-binding protein, VirE2. This "T-complex" is thought to be the transferred intermediate. VirB proteins are proposed to form a membrane-localized apparatus responsible for T-complex transfer. Consistent with this hypothesis, VirB proteins are membrane associated and required for virulence. Others have shown, surprisingly, that a T-DNA containing a mutant virE2 (minus) strain could function to deliver its T-DNA to plant cells if, just prior to plant infection, it was mixed with a strain carrying wild type vir genes (VirE2 donor) but lacking the T-DNA. This "extracellular complementation" suggests that VirE2 can move into plant cells independent of T-DNA transfer. Because VirB genes are required for a bacterium to serve as a VirE2 donor, it appears that the VirB complex may also function to transfer proteins. In this project, three aims are designed to further characterize the interaction of VirB proteins amongst themselves and with transported substrates. First, continued genetic analysis of the virB operon is required in order to understand how the various VirB proteins interact to form a membrane bound complex capable of macromolecular transfer. These studies will focus on virB5, 7, 8, 9, and 10. Data suggest that virB7-10 interact in a fashion that is critical for the construction of a transfer complex, and the genetic basis of this interaction will be defined; virB5 is of interest because it has been proposed to encode a protein that interacts with the transported substrate, but has not been extensively characterized. Second, the phenomenon of extracellular complementation indicates that Agrobacterium may be capable of transporting proteins into plant cells independent of the T-DNA. Despite this intriguing observation, and the similarity o f the virB operon to genes encoding the protein secretion apparati of certain bacteria, there has been virtually no genetic or cellular characterization of this process. A quantitative analysis of extracellular complementation at the single plant cell level will be carried out, and the genetic and cellular basis of the proposed VirE2 movement into plant cells will be characterized. Methods to monitor the presence of VirE2 in plant cells will be developed and used to characterize VirE2 transfer after initiation of plant-bacterial interaction, and a genetic analysis of the regions of VirE2 necessary for transport will be carried out. Third, the inhibition of macromolecular transfer by the incQ RSF1010 derivative plasmid pjW323 will be analyzed. These experiments will increase our understanding of the mechanism whereby DNA is specifically transferred from Agrobacterium tumefaciens to plant cells. Agrobacterium-mediated DNA transfer is a significant gene transfer method in agricultural biotechnology. A better understanding of the molecular basis of this process may lead to improved gene transfer technologies. ***
