Feeding your guests: The role of putative amino acid transporters UMAMIT in symbiotic root nodulation in the model legume Medicago truncatula

Abstract

This thesis investigates the role of UMAMIT Nodulins (UTNs) in the symbiotic relationship between legumes and rhizobia, which are necessary for symbiotic nitrogen fixation. This symbiotic relationship involves the plant providing nutrients to the bacteria, with amino acids being a potential source of nitrogen. However, the specific genes responsible for transporting these amino acids were not known. I identified eight UTN genes specifically expressed during nodulation of Medicago truncatula. Phylogenetic analysis grouped UTNs into two clades within the UMAMIT family, with UTN1 and UTN2 falling in clade I and UTNs3-8 clustering in a legume-sequence rich clade III with additional nodulin like expression, suggesting their function is conserved across different legume species. To investigate the role of these UTNs, I generated triple mutant knockout lines, targeting UTNs 1, 2, and 6. These mutant plants did not show significant differences in nodule number compared to wild-type plants, suggesting that these UTNs are not essential for the initial formation of nodules. However, the mutants displayed a reduced amount of pink leghemoglobin containing nodules at 14 days post inoculation, and showed changes in starch accumulation patterns, indicating impaired nitrogen fixation in the mature nodules. Promoter:GUS fusion analysis revealed distinct and overlapping expression patterns for different UTNs, suggesting a potential role in supporting rhizobia in the infection zones, and, later, the nitrogen fixation zone. Specifically, UTN1 expression was localized to nodulation zone II and the interzone. UTN2 was expressed in multiple tissues, including zone II, the interzone, zone III and the nodule vascular traces. UTN6 was primarily expressed in infected cells in zone III and the nodule endoderm. Antibodies were raised against a 13 amino acid epitope from the C-terminal region of UTN2. Western blotting studies verified that UTN2 was a nodulin. Immunofluorescence studies on nodule tissue sections revealed that the UTN2 protein localised to multiple membranes within the mature nodule, specifically the symbiosome membrane of infected cells, the cell periphery of non-infected cells, and the periphery of multiple cell types in root vascular bundles of nodules, including those immediately adjacent to the xylem vessels. The localization pattern of UTN2 to the symbiosome membrane suggests a direct role for this protein in facilitating nutrient exchange between the plant and rhizobia at the symbiotic interface. The presence of UTN2 in nodule vascular bundles supported a role for this protein in amino acid transport from, and, potentially, to the nodule. This is the first investigation of a symbiosome localised UTN. The stains and materials generated in this thesis represent new tools to investigate host-symbiont dependencies. The results also indicate that the substrates transported by UTNs1 2 and 6 are likely to be necessary for rhizobia to establish a normal metabolism in root nodules. Show more