Ancestral functionality and symbiotic refinement of NIN in root nodule symbiosis

Abstract

Abstract Nitrogen-fixing nodule symbiosis is an ecologically and economically important trait in legumes and some related species. A critical step in the evolution of nodulation is the recruitment of NODULE INCEPTION (NIN); a homolog of the nitrate-sensing NIN-LIKE PROTEIN (NLP) transcription factors. However, whether adaptations have occurred in the NIN protein upon its recruitment in symbiosis remains elusive. Here we show that non-symbiotic NIN orthologs can function in intracellular infection and even nodule initiation, indicating that these properties of NIN predate the evolution of nodulation. Concurrent with the evolution of nodulation, symbiotic NIN proteins were optimized for their role in symbiosis by acquiring nitrate independent functionality, including constitutive nuclear localization. A single amino acid substitution in the non-symbiotic Arabidopsis AtNLP2 enhances its nuclear localization under low nitrate conditions, making it functionally comparable to the symbiotic Parasponia PanNIN. Our study provides insight in the evolutionary trajectory and molecular adaptation that allowed NIN to function as the central regulator of nitrogen-fixing nodule symbiosis.