Symbiosis‐associated <scp>UMAMIT</scp> transporters required for establishing efficient nitrogen fixation in <i>Medicago truncatula</i>

To address a critical gap in understanding amino acid transport in legume-Rhizobium symbiosis, we investigated the role of symbiosis-associated USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs) in Medicago truncatula nodulation. Transcript profiling, phylogenetic analysis and promoter-reporter fusions identified five symbiosis-associated MtUMAMIT genes. CRISPR-Cas9 genome editing generated two triple mutant lines lacking MtUMAMIT14, -17 and -36. Physiological assays, amino acid quantification, and immunolocalisation using a MtUMAMIT17-specific antibody were performed to assess symbiotic function and protein localisation. The induction of MtUMAMIT14, -17 and -36 required Nod factor perception. Triple mutant nodules exhibited reduced nitrogen fixation, leading to nitrogen starvation symptoms, lower leghaemoglobin and amino acid levels, as well as increased starch accumulation. Immunolocalisation revealed MtUMAMIT17 at symbiosome and infection thread membranes, and vascular and uninfected zone III nodule cells. MtUMAMIT17 localised in the cell periphery in zone II cells, while it colocalised with the symbiosomes in infected zone III cells. We conclude that MtUMAMIT14, -17 and -36 are essential for efficient nitrogen fixation, functioning in amino acid transport across symbiotic interfaces and vascular tissues. We propose that their recruitment into nodulation programs represents a key evolutionary adaptation facilitating nutrient exchange critical for symbiotic success.