Genetic variation of drug target CYP51 conferring resistance to ergosterol biosynthesis inhibitors in Botrytis cinerea, causing lily gray mould

Gray mould caused by Botrytis cinerea poses a threat to Lily (Lilium spp.), an important plant with both medicinal and ornamental value, during pre-and post-harvest stages. Ergosterol biosynthesis inhibitors (EBIs) have been widely registered to control gray mould caused by B. cinerea in various plants over a prolonged period of time, yet no instances of resistance of B. cinerea to EBIs have been detected in the field. In this study, the field B. cinerea EBIs-resistant populations were detected from the diseased lily flowers. The CYP51 protein, which is the target of EBIs, was found to have G461S or R464K substitutions in these resistant strains. To further verify whether the G461S or R464K substitutions of CYP51 confer the resistance to EBIs in B. cinerea, the mutants carrying the G461S or R464K substitutions of CYP51 were generated from EBIs-sensitive wild type strains via site-directed mutagenesis. The G461S or R464K substitutions indeed resulted in decreased sensitivity of B. cinerea to EBIs. Meanwhile, the mutants conferring the S461G or K464R substitutions of CYP51 were generated from the field EBIs-resistant strains, and the significant increase in sensitivity to EBIs were observed in these mutants. Furthermore, the G461S or R464K substitutions suffered to fitness penalty in mycelial growth and virulence, but improved sporulation capability. Molecular docking analysis furtherly demonstrated that the CYP51 protein, carrying the G461S or R464K substitutions, exhibited reduced binding affinity with tebuconazole. To the best of our knowledge, it is the first report that G461S or R464K substitutions in CYP51 leading to field resistance to EBIs in B. cinerea. These findings not only provided valuable insights for managing lily gray mould caused B. cinerea, but also enhance our understanding of molecular mechanism underlying plant pathogen resistance to EBIs.