Discovery of pseudobaptigenin synthase in fungal‐infected <i>Trifolium pratense</i> roots

SUMMARY Pterocarpans are structurally complex defense compounds produced by legumes (Fabaceae). They are commonly associated with antimicrobial activity and thought to be synthesized de novo or accumulated in response to microbial pathogens. (−)‐Maackiain is a lineage‐specific pterocarpan detected in some legumes, including red clover ( Trifolium pratense ). The biosynthesis of (−)‐maackiain involves the formation of a distinctive methylenedioxy bridge to convert calycosin to pseudobaptigenin, which is predicted to be catalyzed by a cytochrome P450. We integrated metabolomic and transcriptomic datasets of T. pratense roots treated with the fungal pathogens, Fusarium oxysporum (hemibiotroph) and Phoma medicaginis (necrotroph), to identify candidate P450 genes associated with pterocarpan accumulation. Fungal elicitation initiated a robust defense response via upregulation of core metabolism and over 30 isoflavonoid‐associated genes, including both early (chalcone‐flavanone) and late (pterocarpan) biosynthetic modules. These transcriptomic changes were accompanied by drastic pathogen‐specific divergence in metabolic profiles, featuring over 40 differentially accumulated (iso)flavonoids. F. oxysporum infection resulted in severe necrosis and markedly greater transcriptional reprogramming relative to P. medicaginis . However, the defense response to P. medicaginis was characterized by the strong accumulation of pterocarpans, including (−)‐maackiain. Using a combination of strategies, including weighted gene co‐expression network analysis, candidate cytochrome P450s were selected for putative pseudobaptigenin synthase (PbS) activity. Functional characterization using biotransformation and microsomal fraction assays identified T. pratense pseudobaptigenin synthase (TpPbS/CYP76F319), which catalyzes the conversion of calycosin to pseudobaptigenin. This discovery facilitates the reconstitution of (−)‐maackiain biosynthesis and its future heterologous production at scale for agricultural and health applications.