Screening and Elucidating Functional Mechanisms of <scp>NAC</scp> Transcription Factors Regulating Valsa Canker Resistance in <i>Pyrus betulifolia</i>

Valsa canker, caused by Valsa pyri, threatens the pear industry significantly and hampers molecular breeding for disease resistance due to the scarce knowledge of resistance genes. The NAC (NAM, ATAF1/2, CUC2) transcription factor family plays a crucial role in plant responses to biotic stresses, yet its involvement in pear Valsa canker resistance remains largely underexplored. In this study, we performed a genome-wide identification and bioinformatics analysis of the NAC gene family across the plant kingdom. Our analysis covered 19 diverse species, identified 2501 NAC genes, and classified them into 10 distinct subfamilies. A high member size and expansion rate were discovered in subfamilies E and H, respectively. The subfamily- and species-specific expansion of NAC genes is primarily driven by whole-genome duplication (WGD). Using Weighted Gene Co-expression Network Analysis (WGCNA) combined with experimental functional validation, we identified two NAC genes, PbeNAC32 and PbeNAC105, as crucial positive regulators of Valsa canker resistance. These genes conferred enhanced resistance when overexpressed in both pear and apple fruits, as well as in suspension cells of 'Duli-G03' (Pyrus betulifolia). Overexpression of PbeNAC32 activated multiple immune pathways. Through transcriptome analysis of samples collected at 1 h (T1), 3 h (T2), and 6 h (T3) after Valsa pyri metabolites (VpM) treatment, 3198 shared differentially expressed genes (DEGs) were enriched in multiple immune-related processes. Based on the binding motif, 38 genes were identified, mainly resistance genes related to the PR10 family, as potential targets for PbeNAC32. These findings provide novel insights into the molecular mechanisms of disease resistance in P. betulifolia and offer valuable genetic resources for breeding apple and pear varieties with improved resistance to V. pyri.