We propose to study the two-way exchange of chemical signals between the pathogenic microbe Agrobacterium tumefaciens and its plant hosts. This organism causes crown gall tumors in higher plants by transferring a segment of oncogenic DNA into the plant genome. Our primary focus is on the transcriptional regulation of genes in response to chemical signals released from hosts. Virtually all pathogens induce pathogenesis-related genes in response to host-released signals, and A. tumefaciens is a valuable model for studying this phenomenon. At the outset of infection, wound-released compounds act through three proteins (the transmembrane sensory protein kinase VirA, the response regulator VirG, and the periplasmic sugar binding protein ChvE) to activate transcription of the vir regulon. Later, a compound called octopine acts through the OccR protein (a LysR-type protein) to induce other genes, including the conjugation regulator TraR. TraR and TraI are members of the LuxR/LuxI family of quorum-sensing proteins, and induce tra genes only at high donor cell densities. At least one other plant pathogen and one animal pathogen (Pseudomonas aeruginosa) use homologous proteins to regulate pathogenesis- related genes. A second focus of our work is to study the transfer of T- DNA to the host. We will determine whether three previously uncharacterized genes in the vir region are required for virulence, and whether they are regulated by VirG. We will also study a tra operon from a different plasmid as a model for the eleven genes of the virB operon, as the two operons are closely homologous. Understanding these conjugation- like processes may lead to new ways to prevent the transfer of antibiotic resistance genes and toxin determinants between bacteria. More generally this phenomenon is an example of export of macromolecular complexes from cells and transport of these complexes between cells.
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