Evolutional, expressional and functional analysis of WRKY gene family reveals that PbeWRKY16 and PbeWRKY31 contribute to the Valsa canker resistance in Pyrus betulifolia.

The WRKY transcription factor family plays a crucial role in regulating plant growth and stress responses. However, there are few studies on the regulation of resistance to Valsa canker. In this study, a comprehensive analysis of WRKY genes across 19 plant species was conducted. The potential members of Valsa canker resistance regulation were identified via functional validation. A total of 1641 WRKY genes could be categorized into seven groups. WRKY family members show subfamily- and species-specific expansions. In Rosaceae, Group II-d and II-e were rapidly expanded, which mainly originated based from whole genome duplication (WGD). Cis-element analysis and protein interaction network prediction underscored that most WRKYs respond to stress signals. Based on expressional investigation and Weighted Gene Co-expression Network Analysis (WGCNA), 9 WRKY genes in Pyrus betulaefolia were screened as candidates for Valsa canker resistance regulation. Functional analysis further demonstrated that PbeWRKY16 and PbeWRKY31 regulate the expression of genes involved in salicylic acid (SA) biosynthesis and transport, thereby enhancing resistance to Valsa canker and activating immune responses. Our results provide a foundation for understanding the evolutionary mechanisms of the WRKY gene family and screened potential family members on Valsa canker resistance regulation.