The long term objective of this proposal is to understand in molecular terms, the mechanism by which Agrobacterium tumefaciens causes crown gall tumors in a wide variety of higher plants. Previous studies in this laboratory have shown that this prokaryotic organism transfers a large piece of DNA (T-DNA) from its tumor inducing (Ti) plasmid into the plant cell where it becomes integrated into the nuclear DNA of the plant. The expression of this prokaryotic DNA gives rise to altered properties of the plant tissue. Thus, this bacterium behaves in a way that is similar to the retroviruses which cause tumors in animal cells by integrating a DNA copy of their genome into the host genome. The specific areas under investigation in this proposal relate to the early events in plant cell transformation. Specifically, we wish to understand the mechanism by which Agrobacterium recognizes environmental signals present in the plant wound site and transduces the signals to activate a set of genes required for the transfer and integration of T-DNA. These studies will be carried out using a variety of biochemical and genetical techniques. In particular, we will identify mutants lacking the ability to respond to environmental signals and identify the gene products altered in these cases. Further, radioactive signal molecules will be used to identify proteins to which the signal molecules bind. The other area of investigation relates to the mechanism of transfer of the T-DNA out of the bacterial cell into the plant cell. We will identify and characterize the components of the TDNA complex and T-DNA transport machinery by biochemical and genetical methods. Genetic characterization will involve the isolation of point mutants and generation of extragenic suppressor mutations to identify the proteins which interact with one another in the transport process. Finally, the role of the plant cell structural components in T-DNA movement into and within the plant cell will be investigated. These studies should lead to a clear understanding of the mechanism by which bacterial DNA is transferred into a plant cell and targeted to the nucleus.
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