Identification of the Isocitrate Lyase SrICL as a Potential Target Protein of Butylidenephthalide in <i>Sclerotium rolfsii</i> via Drug Affinity Responsive Target Stability and Metabolomics

Butylidenephthalide (NBP), an active compound derived from <i>Ligusticum chuanxiong</i> essential oil, exhibits potent antifungal activity against <i>Sclerotium rolfsii</i>, a destructive plant pathogen that causes stem rot in peanuts. However, the target protein of NBP in <i>S. rolfsii</i> is unclear. This study elucidates that NBP mainly inhibits the glyoxylate cycle and pyruvate metabolism and also promotes ROS accumulation by affecting the activities of catalase (CAT) and superoxide dismutase (SOD) in the mycelium, thereby inhibiting the mycelial growth of <i>S. rolfsii</i>. The isocitrate lyase (SrICL) was identified as a potential target protein of NBP using drug affinity responsive target stability (DARTS) combined with transcriptomics and metabolomics. NBP significantly inhibited the SrICL activity, disrupted the succinate dehydrogenase (SDH) function, and inhibited energy metabolism, ultimately resulting in the inhibition of fungal growth. Furthermore, deletion of <i>FgICL</i> led to a significantly decreased sensitivity to NBP in <i>Fusarium graminearum</i>, which indicated that ICL might be a potential target of NBP. These findings demonstrate NBP's potential as an ecofriendly fungicide with a novel mode of action.