Genetic elucidation of complex biochemical traits mediating maize innate immunity

Specialized metabolites constitute key layers of immunity underlying crop resistance; however, challenges in resolving complex pathways limit our understanding of their functions and applications. In maize () the inducible accumulation of acidic terpenoids is increasingly considered as a defense regulating disease resistance. To understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies, and targeted mutagenesis. We now define ten genes in three zealexin (Zx) gene clusters comprised of four sesquiterpene synthases and six cytochrome P450s that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants blocked in the production of β-macrocarpene exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate-promiscuity creating a biosynthetic hourglass pathway utilizing diverse substrates and combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes underlying disease resistance demonstrates a predominant maize defense pathway and informs innovative strategies for transferring chemical immunity between crops.