Raw data used in this study.

The excessive and irrational use of commercial fungicides has led to escalating drug resistance in phytopathogens, necessitating the discovery of novel antifungal targets and strategies. Plant secondary metabolites, serving as natural chemical defenses against pathogen invasion, offer promising scaffolds and potential targets for developing innovative crop protection approaches. This study elucidates the antifungal mechanism of the natural sesquiterpene lactone carabrone against Gaeumannomyces tritici through integrated multi-omics analyses. Time-series transcriptomic profiling revealed that carabrone significantly suppresses the oxidative phosphorylation (OXPHOS) pathway and disrupts nicotinate/nicotinamide metabolism, resulting in a reduced NAD⁺/NADH (NAD+, Oxidized nicotinamide adenine dinucleotide; NADH, Reduced nicotinamide adenine dinucleotide) ratio. Orthogonal elevation of NAD⁺ levels through exogenous supplementation diminished fungal susceptibility to carabrone, establishing a direct link between NAD⁺/NADH homeostasis and its antifungal activity. Activity-based protein profiling (ABPP), gene silencing screens, and physiological-biochemical validations collectively demonstrated that carabrone specifically inhibits the electron transport chain (ETC) rather than ATP synthase to regulate NAD⁺/NADH balance. Further evidence from pyruvate supplementation, expression of the yeast non-proton-pumping NADH dehydrogenase Scndi1, and enzymatic assays confirmed that carabrone directly targets mitochondrial respiratory chain complex I, thereby destabilizing NAD⁺/NADH homeostasis and suppressing G. tritici growth. This work first establishes complex I as the direct antifungal target of carabrone, revealing its lethal mechanism involving complex I inhibition-mediated blockade of NADH oxidation, followed by oxidative stress induction and energy metabolism collapse. Additionally, we demonstrate that Scndi1 serves as a critical tool for screening and validating complex I-targeted fungicides. These findings provide both a lead scaffold for developing novel complex I inhibitors and a systematic framework for antifungal agent validation, offering theoretical support to combat emerging fungal resistance challenges.