CRLK (calcium/calmodulin-regulated receptor-like kinase) plays an important role in low-temperature stress and starch biosynthesis, but its function in plant immunity remains unclear. This study aimed to investigate the role of PbeCRLK2 in the immune response of Pyrus betulifolia to pear Valsa canker, and to provide theoretical basis and genetic resources for the breeding of pear trees resistant to Valsa canker.

The basic characteristics of PbeCRLK2 were identified through bioinformatics and subcellular localization analysis. The role of PbeCRLK2 in disease resistance was verified using transient expression in fruits and stable expression in suspension cells. Downstream immune pathways were explored by measuring reactive oxygen species (ROS) and the expression of immune-related genes. To further investigate the mechanism by which PbeCRLK2 regulates resistance to pear Valsa canker, transcriptomic analysis and qRT-PCR verification were conducted on PbeCRLK2 overexpressing cells at different time points after treatment with metabolites of V. pyri.

PbeCRLK2 was a conserved cytoplasmic receptor-like kinase localized to the cell membrane. Its expression was significantly induced by metabolites of V. pyri. Both transient and stable expression of PbeCRLK2 significantly inhibited V. pyri infection and enhanced the resistance of P. betulaefolia to pear Valsa canker. Transient expression for 72 h reduced lesion diameter by 11.59% and stable expression by 44.06% on average. PbeCRLK2 enhanced pattern-triggered immunity (PTI) responses by activating WRKY22 expression. It also induced ROS bursts and up-regulated the expression of RBOHD and OXI1. Meanwhile, the expression of the key salicylic acid pathway gene ChiV was significantly up-regulated. All of these phenomena contributed to enhanced P. betulaefolia defense capabilities. GO and KEGG enrichment analyses were conducted separately for up- and down-regulated DEGs from four groups (DC_vs_YC, DT1_vs_YT1, DT2_vs_YT2 and DT3_vs_YT3) in the transcriptome data. DEGs were mainly enriched in pathways such as protein dephosphorylation, chitin catabolic processes, lipid metabolism, secondary metabolite and terpenoid biosynthesis, etc. qRT-PCR results showed that the expression trends of the selected genes were consistent with the transcriptome data, validating the transcriptomic findings.

PbeCRLK2 may be involved in chitin signal transduction. PbeCRLK2 enhances the resistance of P. betulaefolia to Valsa canker through the coordinated activation of PTI responses, ROS burst, and the salicylic acid signaling pathway. At the same time, it may maintain cellular immune homeostasis by dynamically regulating the expression of dephosphorylation-related genes.