ABCG transporter gene PstABCG2 contributes to multidrug resistance in Puccinia striiformis

The emergence of triadimefon resistance in Puccinia striiformis f. sp. tritici (Pst) , the causal agent of wheat stripe rust, poses a growing threat to global food security. While the novel SDHI fungicide flubeneteram exhibits high efficacy against Pst , the molecular mechanisms governing resistance to both fungicides remain largely unknown. Here, we identify the ABC transporter PstABCG2 as a key mediator of multidrug resistance (MDR) through combined transcriptomic, genetic, and biochemical analyses. First, time-series transcriptomics revealed sustained upregulation of PstABCG2 under fungicide stress, with silencing via in planta RNAi and HIGS significantly enhancing Pst sensitivity to both triadimefon and flubeneteram. Heterologous expression of PstABCG2 in a Fusarium graminearum ΔFgABCG2 mutant restored fungicide tolerance, confirming its efflux function. Crucially, saturation mutagenesis and structural modeling pinpointed E1184 as a critical residue for fungicide binding: the E1184Y mutation disrupted triadimefon affinity (validated by MST/ITC, ΔG reduced from -7.1 to -6.5 kcal/mol) but minimally impacted flubeneteram binding due to adaptive halogen-bond networks. Furthermore, we discovered that the GATA-family transcription factor PstGATA directly activates PstABCG2 expression by binding to promoter cis-elements, and silencing PstGATA phenocopied the hypersensitivity of PstABCG2-silenced isolates. The results may provide a theoretical foundation for elucidating multidrug resistance mechanisms in Pst , field resistance management, and novel fungicide target development.