Project Summary:
The research proposed focuses on root hair infection by rhizobia, a poorly characterized and experimentally difficult step in the symbiosis to study. The research will make use of soybean whose large physical size allows the isolation of purified root hairs in gram quantities. This amount enables the use of both DNA microarray and proteomic analysis to study root hair infection. One objective will be to identify genes/ proteins that are likely critical to the early events of root hair infection by rhizobia. The function of these identified genes will be examined using high-through-put methods of RNAi silencing. Transgenic plants expressing various protein-GFP fusions will be utilized to examine the cell biology of root hair infection. Although soybean provides advantages for biochemical and cell biological studies, this plant is less ideal for genetic studies. Therefore, we will employ a comparative approach using the large and growing collection of symbiotically defective plant mutants in the model legumes, M. truncatula and L. japonicus.The research involved will impact education at all levels: high school, undergraduate, graduate and postgraduate. Specifically, we intend to apply a ?high energy physics? model of distance experimentation to bring DNA microarray resources to the development of undergraduate biology curriculum.

The work proposed has broader biological significance since it represents one of the few systems in which the cell biology of a single cell type, the root hair, can be studied in detail. We expect our research to address important basic questions relevant to plant cell biology; such as, polar cell growth, cytoskeleton dynamics, calcium signaling, and many others.  In order to translate plant genomic knowledge into crop improvement, genomic knowledge of crop plants, such as soybean, must be developed well enough to allow information transfer from models to occur. Our research will contribute to the development of soybean functional genomics and comparative legume genomics. The increasing knowledge base of soybean biology will allow plant scientists to respond to new threats and opportunities that may impact this agronomically important species in the future. The further development of the genomics of many plant species, including soybean, will accommodate comparative approaches and will provide synergistic opportunities to advance plant science. The training provided by the research described will help prepare the next generation of plant scientists to meet these challenges.

II. Utilize RNAi in transient and stable transformation to analyze the function of specific genes in the root hair infection process. We will utilize the rapid, facile, hairy-root transformation system for high-through-put analysis of soybean gene function using RNAi. This will make use of specific root hair promoters obtained through our functional genomic studies. Interesting genes can then be further analyzed using stable transformation of soybean. Construction of transgenic model legume plants with selected genes will allow us to make use of a wide range of plant mutants available in M. truncatula and L. japonicus.

III. Utilize molecular tools, developed through functional genomics, to microscopically investigate the cell biology of the root hair infection process.  We will study root hair cell biology during the infection process with a particular focus on cytoskeletal changes, formation of the infection thread, and root hair growth. A variety of gene-GFP fusions will be used to target specific cell organelles, as well as key genes identified by our functional genomic studies. This, coupled with the use of both bacterial and plant mutants blocked in the infection, should allow us to unravel the complexity of root hair infection.

IV. Provide education and outreach opportunities through research experiences and curriculum. Through interaction with existing programs at the Univ. of Illinois, Univ. of Missouri and Danforth Center, the research proposed will impact education at all levels: high school, undergraduate, graduate, and postgraduate. However, we also intend to apply a ?high energy physics? model to bring DNA microarray resources to the undergraduate biology curriculum.

In order to develop the curriculum and establish the program, we will initiate this effort in collaboration with Dr. Michael Torres, Professor, Warren Wilson College. In our proposed curriculum, students, working in teams, will plan their experiments during the last semester of their junior year. We will develop a workbook containing the essential background material, suggested experimental plans and worksheet for development of experiments. Representative students from these groups will then be given the opportunity to work over the summer. At the Univ. of Missouri and Danforth Center these students will also participate in the normal LSUROP programs. The data generated over the summer will be available to students at Warren Wilson via our website. All students can then use this information in their curriculum and to complete the research project.

We intend to test run this curriculum initially with Warren Wilson College then expand its coverage to other undergraduate institutions. We will host a one week workshop starting in the second summer of the program in which we bring faculty (~4) from undergraduate institutions (including HBCUs) to learn about the program and obtain basic training in the methods. This workshop will serve as a recruitment tool to identify future collaborators. We believe that this is an innovative way to extend modern functional genomics research to undergraduate students who would not normally have direct access to these resources.