Mitochondrial complex II as a self-resistance probe uncovers antifungal 2-pyridones from Penicillium oxalicum

: Mitochondrial complex II (SDH), a key enzyme in the tricarboxylic acid cycle and oxidative phosphorylation, represents a validated target for agricultural fungicide development. Capitalizing on filamentous fungi as valuable sources of bioactive metabolites, we employed the subunit B of SDH as a probe in the self-resistance gene-directed strategy to rapidly discover antifungal natural products. The apn biosynthetic gene cluster (BGC), harboring the SDHB homolog gene apnS , was identified in Penicillium oxalicum . Overexpression of the pathway-specific transcription factor gene apnH successfully activated the silent apn BGC in a marine-derived P. oxalicum strain, yielding four new 2-pyridones ( 1 - 3 and 9 ) and five known analogues ( 4 - 8 ). Antifungal profiling revealed broad-spectrum activity of 2-pyridones against phytopathogenic fungi. Importantly, compound 6 showed strong in vivo activity against B. cinerea -induced gray mold in tomatoes, with inhibition levels similar to those of carbendazim. Concurrently, gene knockout-complementation and heterologous expression confirmed apnS as a self-resistance gene. Leveraging this mechanism, we established a selectable system for fungal genetic transformation by combining apnS expression with 2-pyridone exposure. This work enables efficient discovery of antifungal 2-pyridones by targeting energy metabolism pathways, providing valuable lead compounds for agrochemical and food preservative development.