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.商用杀菌剂的过度非理性使用导致植物病原真菌的耐药性持续攀升，亟需开发新型抗真菌靶点与防控策略。植物次生代谢产物（plant secondary metabolites）作为抵御病原菌侵染的天然化学防御物质，为创新型作物保护手段的研发提供了极具潜力的骨架分子与潜在靶点。本研究通过整合多组学分析，阐明了天然倍半萜内酯（sesquiterpene lactone）卡拉布隆（carabrone）对小麦全蚀病菌（Gaeumannomyces tritici）的抗真菌作用机制。时序转录组分析显示，卡拉布隆可显著抑制氧化磷酸化（oxidative phosphorylation, OXPHOS）通路并干扰烟酸/烟酰胺代谢，导致烟酰胺腺嘌呤二核苷酸氧化态（NAD⁺, Oxidized nicotinamide adenine dinucleotide）与还原态（NADH, Reduced nicotinamide adenine dinucleotide）的比值降低。通过外源补充手段正向提升NAD⁺水平后，真菌对卡拉布隆的敏感性显著降低，直接证实了NAD⁺/NADH稳态与该化合物抗真菌活性之间的关联。基于活性的蛋白质谱分析（Activity-based protein profiling, ABPP）、基因沉默筛选以及生理生化验证实验共同证实，卡拉布隆可特异性抑制电子传递链（electron transport chain, ETC）而非ATP合酶，以此调控NAD⁺/NADH平衡。进一步通过丙酮酸补充实验、酵母非质子泵NADH脱氢酶Scndi1的异源表达实验以及酶活测定，证实卡拉布隆可直接靶向线粒体呼吸链复合物I，进而破坏NAD⁺/NADH稳态并抑制小麦全蚀病菌的生长。本研究首次明确复合物I为卡拉布隆的直接抗真菌靶点，揭示其致死机制为：通过抑制复合物I阻断NADH氧化，进而诱导氧化应激并引发能量代谢崩溃。此外，本研究证实Scndi1可作为筛选与验证靶向复合物I的杀菌剂的关键工具。本研究成果不仅为新型复合物I抑制剂的开发提供了先导骨架，也为抗真菌药剂的验证提供了系统性研究框架，可为应对日益严峻的真菌耐药性挑战提供理论支撑。