A functional atlas of secondary metabolite biosynthetic gene clusters governing growth, stress adaptation, and pathogenicity in Fusarium graminearum

Filamentous fungi harbor a vast potential for secondary metabolite (SM) biosynthesis, yet the biological functions of numerous biosynthetic gene clusters (BGCs) remain obscure. In Fusarium graminearum, a devastating cereal pathogen, SMs are best known as virulence factors, but their broader contributions to fungal physiology are poorly defined. Here, we present a genome-scale functional dissection of 53 predicted SM-BGCs by constructing a knockout library targeting cluster backbone genes and systematically quantifying 24 phenotypic traits, generating 1,272 phenotypic measurements. This dataset reveals that secondary metabolism is not a dispensable metabolic burden; instead, SM-BGCs are broadly integrated into vegetative growth, asexual development, and abiotic stress adaptation. Transcriptome analyses further uncover pronounced spatiotemporal regulation and tissue-dependent requirements of SM-BGCs during infection of wheat heads versus coleoptiles, thereby revealing an ecological dimension of pathogenesis. Mechanistic investigation identified two previously uncharacterized clusters, PKS-type BGC36 and NRPS-type BGC47, as critical for full virulence. BGC36 positively regulates deoxynivalenol (DON) biosynthesis, whereas disruption of BGC47 compromises cell wall/membrane stress tolerance and is associated with reduced phosphorylation of the kinase Mgv1, impaired DON-toxisome formation, and reduced DON production. Together, our findings establish fungal secondary metabolism as a core physiological buffer against environmental fluctuations that supports homeostasis and virulence, and they provide a comprehensive genetic resource for dissecting the chemical biology of Fusarium.