The mitogen-activated protein kinase PbrMAPK5 functions as a positive regulator of resistance to Cytospora pyri in pear

• A pathogenic strain isolated from blighted pear branches was highly pathogenic to pear tissues and identified as Cytospora pyri based on genetic and host analyses. • RNA-Seq analysis of C. pyri -inoculated pear calli at 72 hours post-inoculation (hpi) showed significant activation of the MAPK signaling pathway. • Based on a telomere-to-telomere (T2T) haplotype-resolved genome assembly, we precisely identified 23 PbrMAPK genes, among which PbrMAPK5 exhibited the most pronounced upregulation under C. pyri induction. • Functional studies confirmed that PbrMAPK5 plays a pivotal role in resistance to Cytospora pyri . Overexpression of PbrMAPK5 in pear calli via Agrobacterium -mediated transformation significantly enhanced disease resistance, whereas its silencing through virus-induced gene silencing (VIGS) in tissue-cultured seedlings increased susceptibility. • Yeast two-hybrid assays revealed that PbrMAPK5 interacts with PbrWRKY1, PbrWRKY5, and PbrWRKY9, suggesting that PbrMAPK5 may phosphorylate these transcription factors to regulate disease resistance responses. Cytospora pyri -induced pear Valsa canker represents a destructive stem disease causing substantial economic losses in pear cultivation, yet its molecular pathogenesis remains poorly characterized. Transcriptomic profiling of C. pyri -inoculated pear calli in this study revealed significant activation of the MAPK signaling pathway following pathogen challenge. To identify pivotal MAPK components mediating this response, we systematically identified 23 PbrMAPK genes using a telomere-to-telomere (T2T) haplotype-resolved genome of the susceptible cultivar 'Dangshansuli' ( Pyrus bretschneideri ). Phylogenetic classification organized these genes into four distinct clades, while collinearity analysis demonstrated that family expansion was primarily driven by tandem duplication (TD) and whole-genome duplication (WGD) events, promoting functional diversification through subfunctionalization. Expression dynamics identified PbrMAPK5 as the most strongly upregulated gene during infection. Functional validation via Agrobacterium -mediated overexpression in pear calli and virus-induced gene silencing (VIGS) in seedlings confirmed that PbrMAPK5 acts as a positive regulator of disease resistance. Further investigation using yeast two-hybrid assays revealed that PbrMAPK5 interacts with PbrWRKY1, PbrWRKY5, and PbrWRKY9, suggesting that PbrMAPK5 may phosphorylate these transcription factors to regulate resistance responses. These findings provide mechanistic insights into pear's defense mechanisms against C. pyri and offer valuable molecular targets for breeding resistant cultivars.