Symbiotic associations between two organisms cost each organism resources in exchange for the benefits they receive from the association; thus they are tightly regulated by both partners. In the case of the relationship between the legume plant M. truncatula and its bacterial partner, a major plant regulator of the number of symbiotic nitrogen-fixing nodules that form is a protein called SUNN. SUNN regulates the interaction systemically (throughout the plant) and thus must communicate regulation through other proteins not yet identified. This project addresses the question of what proteins interact with SUNN by taking the critical first step of creating a tool to answer the question. The investigators will create a plant carrying a version of SUNN with a tag to allow retrieval of SUNN and interacting proteins from whole plants for analysis. The tag is a short piece of protein for which commercial antibodies are available, allowing the investigators to isolate SUNN through standard techniques and then identify the interacting partner proteins. The investigators will prove that this tagged protein behaves like the normal version of SUNN by a genetic technique called complementation, in which the tagged SUNN placed in a mutant plant with no SUNN protein restores the mutant plant to normality. Because legumes contribute to 33% of humankind's protein intake and serve as an important source of fodder and forage for animals as well as a source of edible and industrial oils, understanding the signaling involved in regulating legume symbiosis will allow scientists to develop strategies to maintain soil quality and sustainability while protecting the environment over the long term, reducing costs for food and biofuel production. Undergraduate students, including members of under-represented groups, will be trained as part of this project, and will be recruited every year through existing programs.
The goals of our one-year project were (1) to create a transgenic plant in which we have placed a fluorescent tag onto a molecule that regulates the formation of nitrogen fixing nodules in the roots of legume plants and (2) in the process train undergraduate and graduate science students in scientific research protocols. The project required the purchase and construction of some specialized equipment and a significant amount of trial and error to develop the technique, but we report the successful creation of the plant as well as the sharing of the technique with other laboratories. Our findings will be part of a research journal publication we are preparing using this and other funding, and have been presented at national and international scientific meetings. Four undergraduate researchers (three female and one male) worked on portions of this project. Funds from this project also supported the training of a technician and a graduate student. Among the outreach projects was a Plant DNA project with high school students for their science fair. The creation of the plant with the fluorescent tag allowed us to find where in the plant our molecule worked (the plasma membrane of the cell) and gives us a handle we can use to fish for other molecules that interact with the protein of interest. This will be a tool for understanding legume symbiosis, allowing scientists to develop strategies to maintain soil quality and sustainability while protecting the environment over the long term, reducing costs for food and biofuel production.
