The goal of this research is to elucidate the mechanism of T-DNA transfer from bacteria to plants. Agrobacterium tumefaciens infects plants and stably transfers a segment (T-DNA) of its tumor inducing (Ti-) plasmid DNA into the plant nuclear genome. DNA transfer requires the virulence (vir) genes of the Ti-plasmid. The vir gene products catalyze processing of the Ti-plasmid to generate a transfer intermediate, a single stranded T-strand DNA. The T-strand DNA presumably travels through a membrane pore composed primarily of the VirB proteins. In addition to the T-DNA two proteins, VirE2 and VirF, are transferred to plants. For translocation the transport pore must recognize the substrates. The recognition may involve direct interaction of the substrate or it may be mediated by another protein. VirD4 is hypothesized to mediate the recognition process. This research will test this hypothesis and will determine the role of VirD4 in DNA transfer. Immunoelectron microscopy will be used to study whether VirD4 and the DNA transfer apparatus are proximal to each other in the bacterial cell. Functional domains of VirD4 will be identified by mutagenesis methods. Random mutations will be introduced in virD4 and mutations that fail to complement or poorly complement a mutation in virD4 will be isolated and characterized. The effect of a mutation on virD4 function will be investigated. VirD4 contains a nucleotide binding motif sequence. The role of ATP utilization in VirD4 function, if any, will be investigated by in vivo and in vitro methods. Recognition of a substrate by the transport pore is hypothesized to involve interactions among the various components. These studies will identify these interactions and elucidate the mechanism of substrate recognition. Genetic and biochemical methods will be used for these studies. Both pathogenic and non-pathogenic bacteria use a similar mechanism for macromolecule export. The T-DNA transport system is the first member of the type IV transport system. A similar mechanism is used by E. coli for plasmid transfer by conjugation, for example. An understanding of the Agrobacterium substrate recognition mechanism will have a major impact on understanding many biological processes.
