Bacteria that live in association with eukaryotic hosts often have intricate mechanisms to detect host-released chemical signals. This theme is well-studied in the plant pathogen Agrobacterium tumefaciens, which detects several classes of plant-released signals. This project focuses on the perception of a class of phenolic compounds that are released from plant wound sites. These compounds are detected by a two-component system consisting of the transmembrane histidine kinase VirA and the cytoplasmic response regulator VirG, which direct the induction of the vir regulon. Products of the vir regulon mediate the transfer of oncogenic DNA fragments from the bacterium to the nuclei of host plant cells, resulting in crown gall tumors. VirA is a dimeric protein that has four domains: the kinase itself and three domains that modulate kinase activity. In the past, functions have been assigned to each domain by systematically ablating each one at the genetic level. In this project, genetic techniques will be used to create VirA heterodimers containing just one phosphodonor and one phosphorecipient and the role of each domain will be determined in vivo. Soluble VirA fragments will be tested for binding of phenolic inducers and phenolic-responsive autophosphorylation and phosphotransfer reactions. The Winans' laboratory recently determined that one of the operons in the vir regulon (virH) directs the destruction of phenolic vir-inducers. For example, wild type strains can demethylate ferulic acid (a strong vir gene inducer), creating caffeic acid (a noninducer), while virH2 mutants fail to carry out this conversion. Whether related phenolic compounds are substrates for VirH2 or for the closely related protein VirH1 will be addressed. They will also test whether the products of these reactions are further catabolized by other A. tumefaciens proteins. Finally, they will learn what biological role these enzymes play in plant colonization. All pathogens must detect their hosts and make appropriate responses. Detection is generally carried out using olfactory mechanisms (essentially, by sense of smell). This project will provide use genetic and biochemical techniques to learn how a particular plant pathogen (Agrobacterium tumefaciens) detects host plants. It was recently discovered that Agrobacterium can destroy these signal molecules, and this project will study whether all types of these molecules are destroyed, or only certain ones. It will also study the overall effects of these reactions on the ability of this bacterium to colonize its host. These studies will provide fundamental information about host-pathogen interactions that will be useful in combating this and other pathogens.
