Genome-Wide Analysis of AGPase Identifies CsAGP4 as a Regulator of Watermelon Mosaic Virus Resistance in Cucumber

The ADP-glucose pyrophosphorylase (AGPase) gene family plays an essential role in starch metabolism and stress adaptation. However, its function in antiviral defense remains largely uncharacterized. Cucumber (Cucumis sativus L.), a globally important vegetable crop, frequently experiences severe yield losses due to viral infections. In this study, we systematically identified five AGPase genes in cucumber, categorizing them into large and small subunits. Analysis of conserved motifs revealed ten conserved sequences, with the NTP (Nucleoside Triphosphate) transferase domain representing a signature feature of the AGPase family. Promoter regions contained multiple cis-regulatory elements associated with stress responses and hormone signaling. Transcriptomic profiling revealed tissue-specific expression patterns of CsAGP genes, with pronounced enrichment in leaves. Notably, CsAGP2, CsAGP4, and CsAGP5 were strongly induced under biotic and abiotic stresses. Of these, CsAGP4 exhibited rapid, transient induction specifically in the virus-resistant line ‘228’, but not in the susceptible line ‘65G’. Hormonal treatments showed that abscisic acid (ABA) rapidly activated most CsAGP genes and acted synergistically with viral infection to amplify CsAGP4 expression. Functional analysis via CRISPR/Cas9-mediated knockout of CsAGP4 revealed that the mutation disrupted starch granule formation and significantly altered resistance to watermelon mosaic virus (WMV) in ‘Poinsett 97’. Our work provides a systematic characterization of the AGPase gene family in cucumber and establishes its role in defense responses. Importantly, we identify CsAGP4 as a positive regulator of antiviral immunity, highlighting its potential as a target for breeding virus-resistant cucumber varieties.