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.
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Acknowledgements
We thank Tom Peeters, Cloé Villard, Robin van Velzen, Joël Klein, Joan Wellink, Ella Köbben, and Stan van Wijk for their contribution and suggestions to this project. This project is supported by funding from the project Enabling Nutrient Symbioses in Agriculture (ENSA) that is funded by Bill and Melinda Gates Agricultural Innovations (INV-57461), China Scholarship Council (201506300062 to J.L., 201906170085 to S.Y., 202008150090 to M.L.), and the Dutch Science Organization (Nederlandse Organisatie voor Wetenschappelijk Onderzoek VI.Veni.212.132) to R.H.
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J.L. conceived and designed the work, performed the experiments, analyzed the data, and wrote the manuscript. S.Y. conceived and designed the work, performed the experiments, analyzed the data, and wrote the manuscript. M.L. performed experiments and analyzed the data. D.S. performed experiments. M.T. performed experiments. R.B. performed experiments and analyzed the data. K.R.A analyzed the data. F.V. performed experiments. O.K. analyzed the data. R.G. conceived and designed the work, analyzed the data and wrote the manuscript. T.B. conceived and designed the work, analyzed the data and wrote the manuscript. R.H. conceived and designed the work, performed the experiments, analyzed the data, and wrote the manuscript.
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The use of chimeric proteins and amino acid substitutions reported in this manuscript to increase symbiotic functionality of nonsymbiotic NIN orthologs is considered for patent application. 63/863,186 (New U.S. Provisional Application based on U.S. Provisional Application No. 63/677,618). Patent filed on 13-08-2025. Applicant: Wageningen Universiteit. Inventors; Jieyu Liu, Siqi Yan, Min Li, Rik Huisman, Ton Bisseling, and Rene Geurts. The other authors declare no competing interests.
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Liu, J., Yan, S., Li, M. et al. Ancestral functionality and symbiotic refinement of NIN in root nodule symbiosis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71330-1
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DOI: https://doi.org/10.1038/s41467-026-71330-1
