Characterization of CP43 and PIF1 GWAS Selected Candidate Genes in Medicago truncatula and their Contribution to Rhizobia Symbiosis and Phenotypes of Interest Using an hpRNA Based RNAi Platform

Medicago truncatula is a model organism used to study the symbiotic relationship between plants and nitrogen fixing soil bacteria of the genus rhizobia. By studying the genes that are involved in this symbiosis we can accomplish the goal of transplanting the symbiotic relationship into agriculture staples (e.g. corn, wheat, etc.) that do not utilize similar symbiotic relationships with nitrogen fixers. This would help to reduce our reliance on nitrogen-based fertilizers. In order to identify plant genes that might be important for establishing and regulating optimal nitrogen-fixing symbioses, a large-scale Genome Wide Association Study (GWAS) was performed. This study identified the top 50 candidate genes that were hypothesized to be relevant to Medicago-Sinorhizobium symbiosis. The purpose of the work presented as part of this thesis was to characterize two candidate genes, PIF1 and CP43 and investigate their role in symbiosis. These genes were identified by GWAS and encode a DNA helicase and a chlorophyll apoprotein, respectively. Hairpin RNA constructs (hpRNA) were created and inserted into Medicago truncatula to reduce expression of the PIF1 and CP43. These constructs reduced the expression of the two candidate genes by 80-85%. However, reduced gene expression of the candidate genes did not lead to significant phenotypic changes in the height of the plants or number of nodules on the plant roots in comparison to unmodified control plants. Interestingly, preliminary results indicate that reduction of CP43 gene expression alters the strain of rhizobia that nodulates the Medicago truncatula roots. This observation suggests that the CP43 gene may play a role in host-partner specificity in the symbiotic relationship between Medicago truncatula and rhizobia