Wenhui Wang

Valsa canker, caused by necrotrophic fungi in genus Valsa, is a devastating disease that threatens apple and pear production in China and other regions of East Asia. Receptor-like kinases (RLKs) play crucial roles in pathogen signal perception and the initiation of immune responses. However, key members of RLKs contribute to Valsa canker resistance and potential functions remain largely elusive. In this study, we identified a Malus domestica lysin-motif RLK gene, MdLYK7, whose overexpression substantially enhanced the Valsa canker resistance in apple and pear fruits and in suspension cells of Pyrus betulifolia “Duli-G03”. Heterologous overexpression of MdLYK7 in P. betulifolia significantly induced PbeBGLU12, which encodes the β-glucosidase 12-like protein. Among them, yeast two-hybrid (Y2H) library screening, bimolecular fluorescence complementation, luciferase complementation imaging, and co-immunoprecipitation assays showed that MdLYK7 interacted with PbeBGLU12. Functional analysis further demonstrated that PbeBGLU12 is required for MdLYK7-mediated immune response, promotes salicylic acid (SA) accumulation and SA-responsive gene expression, and increases fungal cell wall stress in Valsa pyri. Transcriptome analysis indicated that overexpression of MdLYK7 resulted in differential expression of genes associated with phenylpropanoid biosynthesis and starch and sucrose metabolism in “Duli-G03” cells in response to Valsa pyri signals. Collectively, these results reveal an MdLYK7–PbeBGLU12 module that enhances Valsa canker resistance by activating SA-associated defense responses and imposing stress on the fungal cell wall. This study provides new insight into the molecular basis of Valsa canker resistance and identifies candidate targets for resistance breeding in apple and pear.

The calcium ion-transporting ATPase (Ca²⁺-ATPase) gene family maintains plant intracellular Ca²⁺ homeostasis and regulates growth, development and stress immunity; however, its functions remain poorly characterized in Malus domestica. Here, we performed a genome-wide identification of apple Ca²⁺-ATPase genes and obtained 45 members, which were classified into MdACA (39) and MdECA (6) subfamilies and unevenly distributed on 14 chromosomes. Phylogenetic analysis of Ca²⁺-ATPase genes from Malus domestica, Arabidopsis thaliana, and Oryza sativa classified these proteins into five subgroups. The ACA and ECA subfamilies were highly conserved across species, whereas Group D was apple-specific. Collinearity and Ka/Ks analyses indicated that segmental duplication and purifying selection dominated the evolution of apple Ca²⁺-ATPase genes. Promoter cis-element prediction uncovered numerous regulatory elements related to phytohormone signaling, growth, development and stress defense. Codon usage bias analysis indicated that AUG (methionine) was the dominant codon. Tissue expression profiles showed differential expression of apple Ca²⁺-ATPase genes in various organs. Quantitative real-time PCR (qRT-PCR) assays demonstrated widespread responses of Ca²⁺-ATPase genes to Alternaria alternata infection, exogenous CaCl₂, salicylic acid (SA) and methyl jasmonate (MeJA), among which MdACA39 was strongly induced under all treatments. Subcellular localization verified that MdACA39 resides on the plasma membrane. Moreover, transient overexpression of MdACA39 significantly enhanced apple resistance to A. alternata, likely due to the activation of SA, MeJA and Ca²⁺ signaling-mediated immune pathways, the induction of disease resistance-related genes, and elevated antioxidant enzyme activity. Collectively, this study systematically characterizes the apple Ca²⁺-ATPase family and identifies MdACA39 as a key regulator of fungal resistance, providing valuable gene resources for dissecting Ca²⁺ signaling-mediated disease resistance in apple.

Background: Apples and pears, as important economic fruit crops, are frequently threatened by various diseases, including Valsa canker. Given the numerous advantages of disease resistance breeding, the identification of key resistance genes is particularly important. This study aimed to identify the “Duli-G03” (Pyrus betulifolia) resistance gene PbeZFP3 and clarify its regulatory mechanism in disease resistance via JA/SA pathways, providing a theoretical basis for resistant breeding. Results: In this study, we identified a C2H2-type transcription factor, PbeZFP3, in the Valsa canker-resistant rootstock “Duli-G03”. Expression analysis revealed that PbeZFP3 is induced by both Valsa pyri (Vp) and Vp metabolites (VpM). Transient expression in pear and apple fruits and stable expression in suspension cells confirmed that PbeZFP3 positively regulates Valsa canker resistance. Meanwhile, PbeZFP3 not only enhances the resistance of “Duli-G03” cells to Botrytis cinerea infection, but may also act as a negative regulator against Colletotrichum fructicola. The overexpression of PbeZFP3 in “Duli-G03” significantly upregulated the expression of genes related to jasmonic acid (JA) and salicylic acid (SA) signaling. Conclusions: These findings demonstrate that the jasmonic acid (JA) and salicylic acid (SA) signaling pathways are involved in the enhanced Valsa canker resistance conferred by PbeZFP3 overexpression. A deeper understanding of this host resistance mechanism will provide theoretical support for breeding strategies aimed at developing disease-resistant fruit trees.

Valsa canker, caused by Valsa pyri (Vp), severely threatens global pear production. The VAMP72 family modulates plant immunity, but its role in Valsa canker resistance remains unclear. In this study, it was found that PbeVAMP724, encoding a membrane-localized SNARE protein, was significantly induced by Vp infection and Abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in the resistant pear rootstock Pyrus betulaefolia. Transient overexpression of PbeVAMP724 in ‘Huangguan’ fruits reduced Vp-induced lesions, and the lesion diameter was reduced by 23.1% at 48 h and 20.0% at 72 h compared to the empty vector control (pFGC-5941), whereas VIGS silencing compromised the resistance. Stable overexpression in suspension cells ‘Duli-G03’ (P. betulifolia) enhanced tolerance to Vp metabolites (VpM), alleviated cell death, and induced ROS bursts and defense responses. Weighted gene co-expression network analysis (WGCNA) revealed that phloem/xylem histogenesis-related genes (GWHGAAYT028948, GWHGAAYT039435) are co-expressed with PbeVAMP724. In conclusion, we demonstrate that PbeVAMP724 integrates hormone signaling, triggers ROS bursts, and activates defense responses to positively regulate resistance to Valsa canker in pear, representing a promising candidate for breeding Valsa canker-resistant pear varieties.

Valsa canker, caused by Valsa mali (Cytospora mali) or V. pyri, is a destructive fungal disease affecting apple and pear production. NAC transcription factors have been shown to participate in multiple immune signaling pathways. However, the key members of this family involved in Valsa canker resistance remain largely unknown. In this study, we demonstrate that PbeNAC72, an NAC transcription factor responsive to Valsa canker signals in Pyrus betulifolia, positively regulates resistance in apple and pear fruits. In ‘Duli-G03’ (P. betulifolia) suspension cells, overexpression of PbeNAC72 significantly enhanced resistance to Valsa canker and induced reactive oxygen species bursts, as well as the expression of salicylic acid and jasmonic acid-related genes. Subsequent weighted gene co-expression network analysis revealed that PbeNAC72 is primarily linked to genes associated with “DNA replication” and “microtubule-based movement.” We therefore suggest that PbeNAC72 activates genes related to core signaling pathways and fundamental cellular processes, which further contribute to the positive regulation of Valsa canker resistance. Our results provide novel insights into the resistance mechanism and identify a candidate gene for future molecular breeding efforts.