Mutations across Diverse Domains of CjXDR1 Lead to Multidrug Resistance in Clarireedia jacksonii

Recently, Clarireedia jacksonii has emerged as a significant pathogen threatening turfgrass, and its escalating resistance to multiple drugs often undermines field interventions. This study highlighted the critical role of the fungus-specific transcription factor CjXDR1 (formerly ShXDR1) in regulating multidrug resistance (MDR) in C. jacksonii. This was demonstrated through experiments involving CjXDR1-knockout and CjXDR1-complemented strains. Our sequence analysis revealed five mutations in CjXDR1: G445D, K453E, S607F, D676H, and V690A. All five gain-of-function (GOF) mutations were confirmed to directly contribute to MDR against three different classes of fungicides (propiconazole: demethylation inhibitor, boscalid: succinate dehydrogenase inhibitor, and iprodione: dicarboximide) using the genetic transformation system and in vitro fungicide-sensitivity assay. Comparative transcriptome analysis revealed that CjXDR1 and its GOF mutations led to the overexpression of downstream genes encoding a Phase I metabolizing enzyme (CYP68) and two Phase III transporters (CjPDR1 and CjAtrD) previously reported. Knockout mutants of CYP68, CjPDR1, CjAtrD, and double-knockout mutants of CjPDR1 and CjAtrD exhibited increased sensitivity to all three fungicides tested. Among these, the CYP68-knockout mutants displayed the highest sensitivity to propiconazole, while the CjPDR1 knockout mutant exhibited significantly increased sensitivity to all three fungicides. Double-knockout mutants of CjPDR1 and CjAtrD displayed greater sensitivity than the single knockouts. In conclusion, multiple GOF mutants in CjXDR1 contribute to MDR by upregulating the expression of CjPDR1, CjAtrD, and CYP68. This study enhances our understanding of the molecular mechanisms underlying MDR in plant pathogenic fungi, providing valuable insights into GOF mutation structures and advancing the development of antifungal drugs.