Identification of symbiotic nitrogen fixation‐modulating factors in alfalfa and mechanism elucidation of <scp>MsHHO3</scp>

Symbiotic nitrogen fixation (SNF), a unique nitrogen acquisition mechanism formed through the interaction between leguminous plants and rhizobia, plays a critical role in reducing dependence on chemical fertilizers. However, in alfalfa (Medicago sativa L.), the mechanisms underlying nitrate-responsive SNF remain poorly understood. In this study, we elucidate a nitrate-responsive regulatory network governing SNF in alfalfa and identify MsHHO3 as a key regulator. qRT-PCR analysis revealed that MsHHO3 expression is significantly higher in the LAPIOSZELEI (LA) variety, which exhibits fewer nodules and lower nitrogenase activity, than in the TING SI (TS) variety, which shows a greater number of nodules and higher nitrogenase activity. Morphological characterization demonstrated that MsHHO3-overexpressing lines exhibited significantly reduced nodule number, nodule fresh weight, and nitrogenase activity, whereas suppression of MsHHO3 by RNA interference (RNAi) in alfalfa resulted in an opposite phenotype. The CRISPR/Cas9 mutant of MtHHO3, a Medicago truncatula homolog of MsHHO3, displayed the similar phenotype as MsHHO3-Ri. RNA-seq, ChIP-seq, and qRT-PCR analysis showed that a set of nodulation-associated genes were altered in MsHHO3-overexpressing plants, as well as in MsHHO3-Ri and mthho3 mutants. Among these genes, several hormone-related TF-encoding genes were directly regulated, including the JA signaling pathway master gene MsMYC2. EMSA and dual-luciferase reporter assay further demonstrated that MsHHO3 can directly bind to the MsMYC2 promoter. We propose that MsHHO3 regulates SNF by modulating MsMYC2 and other intermediate TFs, orchestrating a transcriptional cascade that ensures precise fine-tuning of the nodulation process. These findings provide novel mechanistic insights into nitrate-responsive regulation of SNF in alfalfa.