Abstract

The evolution of root nodule symbiosis (RNS), a key innovation for plant nitrogen acquisition, has long been studied but lacks a mechanistic, gene-level evolutionary framework. Here, we reconstruct the gene regulatory network underlying RNS (GRN-RNS) at single-gene resolution using comparative genomic and phylogenomic analyses of 10 newly sequenced and published genomes across all RNS families. We discover that symbiosis-related gene families originated from γ paleohexaploidy in core eudicots, fueling the molecular foundation for network assembly. The initial GRN-RNS emerged at the crown node of the nitrogen-fixing clade through the recruitment and rewiring of genes from three pathways: arbuscular mycorrhizal symbiosis, nitrate response, and stress response. In legumes, GRN-RNS was further refined to enable symbiosome formation via convergent recruitment of modules for cell wall remodeling and kinase signaling. Our work resolves the temporal and regulatory architecture of RNS, providing a unifying framework to understand the evolution of this complex trait.