Cell-type marker gene expression in Medicago truncatula

Arbuscular mycorrhizal (AM) fungi participate in a mutualistic endosymbiosis with most land plants, exchanging mineral nutrients for carbon compounds through structures called arbuscules. Decoding this symbiosis can contribute to improvements in fertilization, phosphorus conservation, and plant growth. One approach being adapted to AM symbiosis research is single-cell transcriptomics, where RNA-seq data from cells are clustered into cell types using cell-type marker genes. Gene expression in roots, such as in response to AM colonization, can be analyzed by cell type using this method. This project aims to validate putative cell-type marker genes in Medicago truncatula, a model legume for AM symbiosis. Hairy root transformation, GUS staining, and optical microscopy were used to determine if cell-type marker gene expression occurred in the expected cell types. Expression was evaluated in AM-colonized/uncolonized plants (for a putative AM-colonized cortex marker) and across multiple genotypes (wild type R108, Tnt1 mutant della1,2-wtseg [wild type segregant], and Tnt1 mutant GRAS transcription factor knockout lines rad1 and della1,2). Cell-type marker gene expression generally aligned with the expected cell types, aside from two markers where expression primarily occurred in the vasculature instead of the cortex. Expression patterns were similar across genotypes, but with downregulation in the rad1 and della1,2 mutants. Additionally, a root length colonization assay was conducted for the roots transformed with the putative AM-colonized cortex marker promoter::GUS construct. The rad1 and della1,2 mutants showed lower AM colonization than the wild type plants, consistent with the downregulated expression of the AM-colonized cortex marker.

Through this program, I had the opportunity to explore the field of host-microbe interactions by studying a key plant-fungal symbiosis. I enjoyed my research very much, and am grateful to have worked in a supportive environment at the Harrison lab with Trevor Tivey as my mentor. I also became familiar with a variety of techniques, including plant transformation, histological methods, and colonization assays. Outside of the lab, I participated in insightful discussions with graduate admissions personnel, faculty, research/social mentors, and other REU students. These conversations helped me define my goals for the coming years and gain a more nuanced perspective of graduate school and career paths. I liked the variety of events built into the program as well, such as the seminar series and the science communication activity. I will be sure to draw on my experiences from this program at my home institution and in my future research.