This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Agrobacterium tumefaciens is a bacterium that also serves as the most widely utilized vector for transferring DNA to plant cells. However, exactly how DNA and proteins are transported out of the bacterial cell and into the plant cell remains unclear. Genes encoding the Agrobacterium T-DNA transporter protein belong to growing class of evolutionarily conserved transporters, called type IV secretion systems (T4SS). T4SS are involved in bacterial mating and protein toxin export, and are required for disease-provoking interactions between bacteria and plant and animal hosts. The PI's long-term goal is to understand the structure and function of the T4SS. This research project will use high-resolution microscopy techniques to determine where the T4SS localizes within the bacteria and whether this localization changes upon binding to plant cells. The T4SS has two major parts: a membrane spanning channel and an extracellular pilus (called the T-pilus) that facilitates bacterial attachment to host cells. New data reveal that protein components that form the T4SS membrane channel, as well as proteins that are transported by the T4SS, localize in a helical pattern around the circumference of the bacterial cell. Helical localization is detected by fusion of a fluorescent tracer to the above proteins. Notably such fusions are functional and do not interfere with the ability of Agrobacterium to transfer DNA to plant cells. Multiple T4SS around the bacterial cell likely increases the efficiency whereby Agrobacterium can make contact with plant host cells. Aim 1 of the research will determine a) what is the time course for the localization of the components of the T4SS? b) does the T4SS co-localize with T-pili? c) do components of the T4SS or its substrate proteins co-localize with the bacterial cytoskeleton (which is known to form helical arrays)? and d) does the localization of the T4SS alter during attachment to host cells? Aim 2 will characterize the T-pilus and its relation to T4SS function during T-DNA transfer and determine a) what is the composition of the T-pilus? b) do T-pili contain DNA or T4SS substrate proteins? and c) what happens to the T-pili during host cell attachment? Do pili extend or contract? The research will impact society by helping us understand how bacterial cells attach to and interact with non-bacterial cells.
Broader Impacts. In parellel with research activities pursued in this project, the Principal Investigator will develop a 15-week freshman course centered on the biology of Agrobacterium and its use as a tool for basic and applied research. Fluorescent probes and fluorescence microscopy will allow students to see biology first hand. Students will observe a) live bacteria carrying proteins that localize to the cytoskeleton, membranes or other specific cellular sites, b) transformation of plant cells with Agrobacterium carrying plant genes that when expressed can be detected in different parts of the cell or c) Agrobacterium-mediated inoculation of fluorescently tagged plant viruses that display movement throughout the entire plant. How scientific discoveries are made and long-term societal impacts, such as GMOs (genetically modified organisms) will be discussed. This course will stimulate freshmen to pursue further studies in the biological sciences.
