The goal of this project is to understand the distribution within plants of the molecules that induce expression of the virulence genes of Agrobacterium tumefaciens, which leads to transformation of susceptible plant tissues. The biochemical system that controls this process (the VirA, VirG and ChvE proteins) recognizes at least 3 different inducing signals that come from the plant: phenols, sugars and low pH. In this project plant tissues will be exposed to bacteria that contain a "reporter" gene controlled by the virulence inducing system. When this gene is expressed it yields a particular color (e.g. from the green fluorescence protein) significantly different than the plant tissues, allowing the investigators to determine where in the plant the signals are actually found. By altering the genetic properties of the VirA/VirG/ChvE system the investigators will be able, for the first time, to track the presence of individual classes of inducing molecules and relate this information to the capacity of the tissues and cells to be transformed. These experiments test the hypothesis that there are only a few regions in the plant in which all three signals are found at levels sufficient for vir induction and that these signify to the bacteria the presence of tissues capable of being transformed. Finally, the investigators will use genetic methodologies to broaden the range of chemical compounds that VirA recognizes as inducing signals and then determine whether such foreign compounds can be detected in the plant via the methodologies described above.
Broader impact: Understanding the relationship between the distribution of inducing signals in the plant and the capacity of those same tissues to be transformed will have implications for the use of Agrobacterium as a tool for genetic modification of plants that are presently resistant to transformation. This could have important consequences on agricultural biotechnology. Furthermore, the directed evolution of variants of VirA with altered chemical recognition properties has the potential to provide inexpensive, easy to use analytical tools with the capacity to indicate the presence in the plant of a variety of plant secondary metabolites with consequences for both nutrition and toxin studies. The project will involve undergraduate students as active participants in the research efforts, graduate students and postdoctoral researchers as collaborators in undergraduate course instruction, and partnerships with local public schools in which undergraduates develop and test new life science modules in classrooms.
