A NSP2-MYB module orchestrates flavonoid biosynthesis and root symbiosis

Flavonoids are crucial metabolites that enhance plant adaptation to nutrient stress and, through specific subsets, initiate legume-rhizobium symbiosis by inducing bacterial Nod factor production. Notwithstanding the central role of flavonoids in nitrogen-fixing symbiosis, their transcriptional regulation in legume and the manner of their recruitment to nodulation remain poorly understood. Here, we show that the Nodulation Signaling Pathway 2 (NSP2) is required for transcriptional activation of flavonoid biosynthesis genes during nodulation. We further identify a MYB transcription factor MYB40 from a legume-expanded clade, which is induced by nutrient limitation and rhizobia in the root epidermis of Medicago truncatula. MYB40 directly binds to flavonoid synthetic gene promoters and is required for nodule symbiosis. Biochemical and genetic evidence reveals that NSP2 interacts with MYB40 to form a regulatory complex that upregulates the symbiotic flavonoid genes in a manner dependent on MYB40 binding sites. Furthermore, this NSP2-MYB module appears to be functionally conserved between Medicago and barley under nutrient starvation conditions. Our findings establish the NSP2-MYB40 module that integrates symbiotic signaling with metabolic reprogramming, representing an evolutionary innovation for optimizing nitrogen acquisition in dynamic environments.