The goal of this research is to understand the regulation of expression of the Agrobacterium tumefaciens virulence (vir) genes, and to define the roles of their gene products play in the overall process of transfer and integration of a bacterial DNA segment into the plant nuclear genome. In vivo and in vitro approaches will be used to address the regulatory aspects. Both in vitro transcription-translation and transcription systems will be developed. Extracts from wild-type and mutant strains of Agrobacterium will be used in the coupled transcription- translation system to define the regulatory elements in inducible expression of the vir genes. A fusion gene containing E. coli beta- galactosidase under the control of an inducible vir promoter will be used as template DNA. The regulatory sequences and their minimum length will be defined by deletion mutagenesis studies. Biochemical and genetic approaches will be used to identify and study repressor functions, if any. The regulatory proteins and RNA polymerase will be purified to develop an in vitro transcription system which will be used to define the minimum necessary components for vir gene expression and their regulation. The purified proteins will be used to study specific binding of RNA polymerase and/or of the regulatory proteins to template DNAs by restriction-site protection assays, foot-printing, and other methods. These studies will be instrumental in establishing a mechanism for the regulation of the vir gene expression. To determine the various activities associated with the vir gene products, the proteins will be overproduced in E. coli as well as in Agrobacterium. The proteins of the virB operon will be tested for beta-1 leads to 4 hydrolase activity because of significant homology between a segment of the derived amino acid sequence of virB1 and the active-site sequences of animal lysozymes. Gene fusion procedures will be used to determine if the site of action of virE gene products is within the plant cells. The long-term objective of this research is to understand the molecular mechanisms of microbial pathogenesis. The vir gene plays a central role in the confering pathogenicity to Agrobacterium. The knowledge of how these genes are regulated and their protein products function will greatly facilitate the understanding of pathogenicity and gene transfer processor
