Discovery of the rosalexin pathway expands the modular network of maize diterpenoid phytoalexins.

The evolutionary expansion of specialized metabolism has shaped the ability of plants to adapt to combined pest, disease, and environmental pressures. In maize (Zea mays), the duplication and divergence of ancestral gibberellin pathway genes have given rise to specialized kauralexin and dolabralexin diterpenoids that serve core functions in stress resilience. Here we identify rosalexins as a previously unrecognized branch of the maize diterpenoid defense network and show how duplication of hormone-metabolic genes generated a new biosynthetic pathway with distinct chemical and biological functions. By integrating genomics-enabled gene discovery, combinatorial enzyme assays, metabolomics, and AI-assisted enzyme mechanistic studies we show that rosalexin biosynthesis proceeds via an unusual 5-rosanol scaffold formed from ent-copalyl diphosphate through the sequential activity of ZmTPS38/CPS2/AN2 and ZmTPS42/KSL1. Further oxygenation by the promiscuous P450 enzyme, ZmCYP71Z18, yields epoxyrosanol. Unexpectedly, only epoxyrosanol strongly inhibited fungal pathogen growth, whereas neither its immediate precursor nor its downstream hydrolysis product showed comparable activity, identifying epoxidation as a key determinant of antibiotic efficacy. Large variation in rosalexin abundance exists across maize genotypes due to ZmTPS42/KSL1 gene sequence variation and pseudogenization. Although no dominant pathogen resistance phenotype associated with rosalexin abundance was observed in maize plants, transcriptomics and metabolomics studies demonstrated the pathogen-elicited accumulation of rosalexins in maize lines featuring functional ZmTPS42/KSL1 genes. Together, these findings reveal how duplication and functional divergence of hormone-metabolic genes generated modular diterpenoid defense layers, illustrating how plants expand chemical immunity through reuse of existing metabolic scaffolds.