ABCG Transporter Gene <i>PstABCG2</i> Contributes to Multidrug Resistance in <i>Puccinia striiformis</i>

The emergence of triadimefon resistance in <i>Puccinia striiformis</i> f. sp. <i>tritici</i> (<i>Pst</i>), the causal agent of wheat stripe rust, threatens global food security. Although the novel SDHI fungicide flubeneteram shows high efficacy, the molecular basis of resistance remains unclear. Here, we identify the ABC transporter gene <i>PstABCG2</i> as a key multidrug resistance (MDR) determinant in <i>Pst</i>. Transcriptomics revealed its sustained upregulation under fungicide stress. Silencing <i>PstABCG2</i> via RNAi and HIGS enhanced fungal sensitivity to both fungicides, while heterologous expression in <i>Fusarium graminearum</i> restored resistance. Structural and biophysical analyses identified E1184 as a critical binding residue-its mutation (E1184Y) impaired triadimefon affinity but preserved flubeneteram binding via adaptive halogen bonding. Furthermore, the GATA-family transcription factor PstGATA directly activated PstABCG2 expression, and its silencing phenocopied the hypersensitivity of <i>PstABCG2</i>-silenced isolates. The results provide a theoretical foundation for elucidating multidrug resistance mechanisms in <i>Pst</i>, field resistance management, and novel fungicide target development.