The goal of the proposed research is to understand the mechanism of DNA transfer from Agrobacterium to plant cells. The role of the virulence (vir) genes in the DNA transfer process and the mechanism of transcription regulation of the vir genes will be investigated. Two regulatory proteins, VirA and VirG, in conjunction with a plant phenolic acetosyringone control vir gene expression. The signal molecules (plant phenolics) generated by a susceptible host plant induce modification of the regulatory proteins presumably via phosphorylation leading to the activation of the pathogenic machinery of the bacterium. Mutational studies will be performed to identify the signal processing and transcriptional activator domains of VirA and VirG. The effect of these mutations on protein phosphorylation will be investigated. An in vitro transcription system will be developed to study the role of protein phosphorylation in regulation of vir gene expression and to elucidate the mechanism of transcription activation. An intermediate in DNA transfer from bacteria to plant cells is a single- stranded (ss) DNA complexed with proteins (T-complex). Formation of the ssDNA is catalyzed by the VirD2 endonuclease and requires the presence of VirD1. In vivo studies will be performed to determine the mechanism of VirD1-VirD2 endonuclease action. Deletion mutagenesis studies will be performed to define the function domains of the VirD2 endonuclease. Both VirD1 and VirD2 presumably form covalent protein-DNA complexes. The amino acids that participate in the formation of these complexes will be identified. The role of the other VirD proteins in DNA transfer and integration will be studied. Biochemical studies will be performed to determine the composition of the T-complex and to stud its interaction with bacterial membranes. Mutations in vir genes will be used to identify the gene products that participate in these reactions. An in vitro system will be developed to identify Vir and other proteins required for DNA transfer to plant cells. Using assays that can distinguish between DNA transfer and DNA integration, the role of different Vir proteins in these reactions will be investigated. The long term goal of this study is to understand the mechanism of agrobacterial pathogenicity. The vir genes play a central role in this process. Elucidation of the mechanism of expression of these genes and understanding the functions of the encoded gene products are vital towards a better understanding of mechanisms bacteria employ for their pathogenic action.
