Transcriptional activation of PbrFLOT1 by PbrWRKY33 contributes to pear resistance against Botryosphaeria dothidea

Pear ring rot, caused by the necrotrophic fungus Botryosphaeria dothidea , is a major disease affecting pear production and fruit quality. Plasma membrane nanodomains are important for immune signaling, but the roles of stomatin–prohibitin–flotillin–HflK/C (SPFH) proteins in pear disease resistance remain largely unknown. In this study, 18 PbrSPFH genes were identified in ‘Dangshan Suli’ pear and classified into five subfamilies: flotillin, prohibitin, stomatin-like, hypersensitive-induced reaction, and erlin. Analyses of chromosomal distribution, gene structure, conserved motifs, and collinearity indicated duplication-driven expansion of the family. Promoter analysis revealed abundant hormone- and stress-responsive cis-elements, especially in PbrFLOT and PbrHIR genes. Following B. dothidea inoculation, most PbrHIR genes were rapidly induced, whereas PbrFLOT1 and PbrFLOT3 showed delayed but pronounced upregulation. Subcellular localization showed that PbrFLOT1 was mainly localized to the plasma membrane, while PbrFLOT3 was detected in the plasma membrane, cytoplasm, and nucleus. Silencing of PbrFLOT1 or PbrFLOT3 increased lesion size in pear seedlings, whereas overexpression in pear calli enhanced resistance to B. dothidea . At 4 dpi, PbrFLOT1/3 silencing reduced the expression of several redox- and defense-related genes, particularly PbrPR1 , whereas infected overexpression calli showed largely opposite transcriptional patterns. In addition, PbrWRKY33 directly bound the PbrFLOT1 promoter and activated its transcription, and silencing of PbrWRKY33 reduced pear resistance and suppressed PbrFLOT1 expression. These findings identify PbrFLOT1 and PbrFLOT3 as positive regulators of pear resistance to B. dothidea and demonstrate that PbrWRKY33 directly activates PbrFLOT1 transcription. • Eighteen SPFH genes were identified in the pear genome and classified into five subfamilies. • PbrFLOT1 and PbrFLOT3 positively regulate pear resistance to Botryosphaeria dothidea. • Silencing of PbrFLOT1 or PbrFLOT3 increased pear susceptibility to ring rot. • Overexpression of PbrFLOT1 or PbrFLOT3 enhanced resistance and affected redox/SA-related markers. • PbrWRKY33 directly bound the PbrFLOT1 promoter and activated its transcription.