The root nodule symbiosis regulator NIN exhibits broad DNA binding specificity conferred by an NLP-inherited motif

Nitrogen-fixing root nodule symbiosis (RNS) occurs in some eudicots, including legumes, and is regulated by the transcription factor NODULE INCEPTION (NIN), derived from the NIN-LIKE PROTEIN (NLP) family. However, how the NIN protein acquired RNS-specific functions remains unclear. We identify a previously undescribed motif in <i>Lotus japonicus</i> NIN, located downstream of the RWP-RK domain, which we term the FR. This motif broadens NIN's DNA binding specificity by stabilizing the RWP-RK dimer interface. <i>nin</i> mutants lacking the FR motif show defective nodulation and impaired nitrogen fixation. <i>Arabidopsis</i> NLP2 carries a NIN-type FR and shares key features with NIN. Furthermore, the NIN-type FR had already emerged before the divergence of gymnosperm and angiosperm lineages, suggesting that a specific molecular feature of NIN involved in RNS regulation was inherited from ancestral NLPs prior to the emergence of RNS.