Carin Ragland
Faculty Advisor: Maria Harrison
Year: 2014

Identifying AM symbiosis genes in Medicago truncatula through reverse genetics

Project Summary

Arbuscular mycorrhiza (AM) is an endosymbiotic association between biotrophic fungi and vascular plants in which the fungus supplies water, phosphorus, and other nutrients to the plant and receives carbon in return. AM is a potentially powerful fertilizing tool in agriculture because, in some cases, fungal interactions increase the amount of nutrients accessible to the plant without increased external nutrient applications to the soil. About 80% of angiosperms can form AM, and it is considered one of the most ubiquitous plant symbioses on earth.

In two experiments during this internship, two gene deactivation methods—RNA interference (RNAi) constructs and insertion lines—were used to test several genes proposed as AM participants by phylogenic and bioinformatic analyses. The RNAi constructs were prepared via molecular cloning and introduced into M. truncatula seedlings by root transformation. The RNAi constructs and insertion lines of the candidate genes were cultivated in “cone-tainers” with 200 fungal spores for three weeks and then harvested. Root samples were collected from the insertion lines and stained with fluorescent dye. RNAi construct samples were selected via a Red Root fluorescent marker before staining. Root colonization and arbuscule development were assessed in the insertion lines and RNAi transgenic roots to determine whether gene deactivation affected AM symbiosis.

Studies featuring gene deactivation methods (reverse genetics) have made significant progress in explaining the molecular mechanism behind AM. With continued AM research future discoveries could generate widespread use of AM symbiosis within agriculture to increase crop yields and address resource conservation concerns.

My Experience

For two years I worked with sustainable agriculture projects, informally utilizing and studying plant science concepts such as microbial and fungal symbiosis within urban agriculture initiatives. I was excited for the opportunity to participate in formal plant science research regarding AM symbiosis because it is such a fundamentally crucial ecological interaction. I enjoyed my experience at BTI, and truly appreciate the time my mentor, PI, and lab mates dedicated in contributing to my first research experience. This summer I have learned widely applicable lab techniques, implemented an independent project, and became acquainted with young scientists from a myriad of different fields. The program demystified the graduate school experience and application process and exposed us to useful resources and knowledgeable researchers and educators. In the future I will definitely continue plant research and am considering graduate studies in Agronomy.