Indoloquinoline alkaloid neocryptolepine derivative inhibits <i>Botrytis cinerea</i> by targeting thiamine thiazole synthase

The emergence and rapid spread of multidrug-resistant <i>Botrytis cinerea</i> strains pose a great challenge to the quality and safety of agricultural products and the efficient use of pesticides. Previously unidentified fungicides and targets are urgently needed to combat <i>B. cinerea</i>-associated infections as alternative therapeutic options. In this study, the promising compound Z24 demonstrated efficacy against all tested plant pathogenic fungi. Thiamine thiazole synthase (Bcthi4) was identified as a target protein of Z24 by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) assays. Molecular docking and enzyme activity experiments have demonstrated that Z24 can affect the function of Bcthi4. Last, mechanistic studies show that Z24 inhibits thiamine biosynthesis by binding to Bcthi4 and induces up-regulation of alternative splicing [alternative 5' splice site (A5SS)] of the <i>Bcthi4</i> gene. In conclusion, by targeting Bcthi4, Z24 has the potential to be developed as a previously unidentified anti-<i>B. cinerea</i> candidate.