Sara LeFevre

Identification of protein interactors in Medicago truncatula exocyst complex

Project Summary

Most vascular land plants participate in a beneficial relationship with arbuscular mycorrhizal (AM) fungi; in which the plants provide carbon to the fungi in exchange for phosphates and nitrogen. This exchange is mediated by a variety of mechanisms within the root and the fungal structures that enter the root cells, known as arbuscules. Arbuscules are surrounded by a plant-derived membrane, the periarbuscular membrane (PAM), which forms an interface where controlled exchange of nutrients takes place. Plant cells build the PAM by redirecting the exocytotic pathway. Vesicle fusion in exocytosis is executed by an octameric protein complex called an exocyst. Research in the Harrison lab showed that a knockout mutant of exo70i, a subunit of the exocyst complex, was unable to support normal arbuscule development (Zhang, et al., 2015). In order to confirm that MtEXO70I is a true subunit of the exocyst complex, split ubiquitin yeast two hybrid mating experiments were performed to visualize protein-protein interaction between EXO70 and other exocyst subunits. This experiment demonstrated that MtEXO70I is a true subunit of the exocyst complex. A second experiment explored the activation of MtEXO70I. Cell polarity and activation of the membrane exocyst complex in yeast and mammals is reliant on the small GTPases of the Rho family. In plants, these are called ROP (Rho of Plants) GTPases. To determine if the same mechanism of activation exists in plants, split ubiquitin yeast two hybrid analysis was performed between ROP GTPases and EXO70. These experiments provided evidence that MtEXO70I is regulated by ROP GTPases. Research that expands our knowledge about the mechanisms which cause exocytosis in plants can lead to a greater ability to control the beneficial symbiosis that occurs between plants and mycorrhizal fungi; for example, subunits of the exocyst complex may be manipulated to cause a longer symbiosis, or cause the plant to uptake a larger quantity of nutrients. This research is important because it will enhance the ability of molecular biologists to manipulate plants in order to improve/develop sustainable agriculture.

My Experience

My summer spent at Boyce Thompson Institute was as challenging as it was rewarding. I truly learned what it means to do science: asking questions, understanding not only how to do a technique but why, using both precision and accuracy, never taking anything at face value, never cutting corners, and not giving up when the first, second, or hundredth time was a failure. The core skillset I am taking away from this experience is not simply the techniques I learned, but the ways to learn new techniques in the future, such as how to read a protocol or glean important information from a dense scientific paper. In addition, I learned how to communicate with other scientists for their expertise on any subject. This was a brief, intense, and truly worthwhile internship. I feel more prepared now than ever to pursue a career in biological sciences.