Distinct domain regions of NIN and NLP1 mediate symbiotic and nitrate signalling in Medicago truncatula

Nodule Inception (NIN) and NIN-like protein 1 (NLP1), both belonging to the RWP-RK type transcription factors, play critical roles in plant development. Specifically, NIN is pivotal in facilitating root nodule symbiosis in nitrogen-starved conditions, while NLP1 coordinates nodulation in response to nitrate level. In this study, we conducted domain swapping experiments between NIN and NLP1 in Medicago truncatula to elucidate the functional significance of their respective domains. The findings reveal that the C-terminal regions, including the RWP-RK and PB1 domains of NIN, can substitute for those of NLP1, whereas reciprocal substitution does not yield equivalent outcomes. Moreover, our results emphasize the critical role of PB1-mediated interactions in the activity of NLP1, a feature not essential for NIN. Additionally, the N-terminal segment, conserved in NLPs but containing deletions or mutations in NIN, is essential for the proper functioning of both NIN and NLP1. Collectively, our research indicates evolutionary divergence of NIN from ancestral NLPs, suggesting specific adaptations that have enabled NIN as a central regulator in root nodulation processes. A domain-swapping approach was employed to elucidate the essential functions of specific domain regions of NIN and NLP1, contributing to the advancement of nitrogen use efficiency and symbiotic nitrogen fixation in leguminous plants.