MCB-94-19252 Abstract This is a proposal to examine the signal recognition steps in the expression of the virulence (vir) genes necessary for the transfer of DNA from Agrobacterium tumefaciens into susceptible, wounded plant cells. The vir gene system is encoded on the Ti plasmid and is in part comprised of the genes for the transmitter/receiver complex (virA and vir G). The ability of the vir A/vir G system to respond to a broad range of compounds, and yet provide sufficient interaction enrgy to transmit information, might be achieved by the catalysis of the transfer of a phenolic proton to the receptor, thereby activating the signal transduction cascade. This model makes several mechanistic predictions which have already given rise to the synthesis of the first known inhibitors of vir expression. The previous studies have been in vivo; in this study in vitro testing of these predictions is accomplished by examination of the autophosphorylation of VirA in a reconstituted system. Methods for the synthesis of the required inducer analogs and molecular genetic manipulation of VirA are in place. Affintiy labeling reagents based on the inhibitors have implicated two phenol-binding proteins, p10 and P21, as likely candidates for the phenol receptors. These, as well as the other proteins identified in screens for the requisite chemical reactivity, will be purified, sequenced and the sequence used to isolate the respective genes to test their relevance in vir expression. %%% The ability to obtain transgenic plants for basic research and for biotechnological enhancement of our nation's food supply has in large part been made possible through plant infection with transformed Agrobacterium tumifaciens, a bacterium which causes Crown Gall disease in nature. The mechanism of DNA transfer and how the bacterium can "sense" an appropriate site for transfer is unknown. This proposal deals with the latter point and will isolate and characterize the proteins which perceive the appropriate "signalling" cues provided by the plant. These cues are plant phenolics which, in most instances, serve as antibiotics. A. tumifaciens is sensitive to these phenols and "hones in" on them, perceiving them through an interesting signalling mechanism which may provide a model for plant recognition of hormones or bacterial recognition of small molecules. The work characterizes the phenol binding proteins and the proteins involved in relaying binding information to the cell. To do that, in vitro reconstitution of part of the signalling pathway will be done using purified "stabilized" proteins involved in this signaling cascade. The proteins are stabilized by synthesizing an active, but truncated version of the protein through genetic engineering. The outcome of the experiments will be to shed light on this biotechnologically important, yet fundamental signalling event. ***
