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

Abstract

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.

1. Introduction

Cucumber (Cucumis sativus L.) is cultivated extensively across the world and represents a major vegetable commodity in both economic and agricultural terms [1,2], accounting for 59.5% of the global cultivation area and 80.8% of the total yield (http://faostat.fao.org, accessed on 10 April 2025). However, during production, cucumber plants face escalating challenges from viral pathogens, including watermelon mosaic virus (WMV), cucumber mosaic virus (CMV), and zucchini yellow mosaic virus (ZYMV). Infection by these viruses commonly causes chlorosis, mosaic symptoms, and stunted growth, leading to substantial yield losses [3,4].

Starch is a primary carbon storage polysaccharide in plants that plays a dual role in energy homeostasis and stress adaptation. Its synthesis depends on the coordinated action of several enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (SBE), and debranching enzyme (DBE) [5]. Among them, AGPase functions as the key rate-limiting enzyme because it converts glucose-1-phosphate and ATP into ADP-glucose, thereby exerting a strong influence on the rate of starch production and final starch accumulation.

Plant ADP-glucose pyrophosphorylase (ADP-Glc PPase) is a heterotetrameric enzyme consisting of two large subunits (APL) and two small subunits (APS), and each subunit contains the characteristic NTP-transferase (PF00483) domain. Enzymatic activity is stimulated by 3-phosphoglycerate and suppressed by inorganic phosphate, whereas additional metabolites such as fructose-6-phosphate may further influence activity in certain species or tissues [6,7]. The small (APS) and large (APL) subunits of ADP-Glc PPase are homologous proteins. In plants, the small subunit is typically encoded by one or two genes, whereas the large subunit is often represented by multiple paralogs that show tissue- or plastid-specific expression [8]. Therefore, compared to small subunits, the specific spatio-temporal expression pattern of AGPase genes is mainly determined by large subunits [9,10]. The AGPase family has already been described in a range of monocot and dicot species, including Arabidopsis thaliana (four APLs and two APSs), Zea mays (four APLs and two APSs), Oryza sativa (four APLs and three APSs), Solanum lycopersicum (three APLs and one APS), Manihot esculenta Crantz (four APLs and three APSs), and Musa acuminata L. (six APLs and two APSs) [11].

Beyond their well-known functions in plant growth and development, many AGPase genes are responsive to diverse biotic and abiotic stresses, including heat, cold, salinity, drought, and infection by phytopathogens [12,13,14]. During the maturation stage, heat stress on the AGPase enzyme seriously affects the biosynthesis of starch in rice [15]. In downy mildew-infected grapevine leaves, increased AGPase activity leads to starch accumulation [16]. Moreover, in melon (Cucumis melo), CMV infection downregulates the expression level of AGPase, which leads to decreased starch content and compromised antiviral defense [17]. However, whether and how AGPase responds to viral infection in cucumber remains largely unknown.

The plant defense system is coordinately regulated by the interplay between sugar metabolism and phytohormone signaling pathways [18,19]. Increasing evidence suggests that AGPase-dependent starch metabolism is embedded in hormone-mediated immune regulation, indicating that AGPase may connect metabolic adjustment with defense activation. In Arabidopsis, ABA not only induces AGPase expression under drought stress to stabilize carbon reserves [20] but also enhances starch accumulation by phosphorylating AGPase subunits via SnRK2 kinases, thereby optimizing energy allocation for survival [8,21]. Exogenous jasmonic acid (JA) treatment greatly activates the defense of Rosa chinensis against Botrytis cinerea [22]. Exogenous addition of methyl jasmonate (MeJA) to citrus fruits significantly enhances the activities of PEROX and polyphenol oxidase (PPO), thereby effectively inhibiting the occurrence of Penicillium digitatum and Alternaria alternata [23]. In addition, silencing of MeJA- and methyl salicylate (MeSA)-related genes led to significantly increased susceptibility to tobacco mosaic virus (TMV) in Nicotiana benthamiana [24].

Although extensive studies have revealed the importance of AGPase in plant stress responses, the functional mechanism of the CsAGP gene family in antiviral defense is still unknown. Here, we performed a comprehensive study of the AGPase family in cucumber, defining its membership, structure, and regulatory landscapes. Through comprehensive analyses involving transcriptomics, hormone application, and pathogen infection, we observed that CsAGP4 undergoes dynamic regulation. CRISPR/Cas9-mediated knockout of CsAGP4 confirmed its necessity for starch granule formation and virus resistance. These results indicate that CsAGP4-mediated starch metabolism is a positive facet of the plant antiviral response.

2. Results

2.1. Identification of the AGPase Gene Family in Cucumber

Using a combination of HMM and BlastP-based tools, we identified five AGPase genes in the cucumber genome (Table 1). Predicted subcellular localization placed all five proteins in the chloroplast. To further understand the structural and functional attributes of the CsAGP gene family in cucumber, we conducted a comprehensive analysis of their encoded proteins. The encoded proteins ranged from 521 to 536 amino acids in length, with a mean length of 527.8 amino acids. Their predicted molecular masses spanned 56,917.85 to 59,906.89 Da, and their theoretical isoelectric points ranged from 5.86 to 9.09. Overall, these parameters indicate that members of the CsAGP family share broadly conserved biochemical characteristics.

Table 1.

Characteristics of CsAGP gene members in cucumber.

Proposed Gene Name	Gene ID	Subcellular Localization of Predicted Protein	Isoelectric Point (pI) of Predicted Protein	Molecular Weight (Da) of Predicted Protein	Number of Amino Acids of Predicted Protein
CsAGP1	CsaV4_2G001288	Chloroplast	8.21	58,630.29	527
CsAGP2	CsaV4_3G001409	Chloroplast	8.8	57,640.16	523
CsAGP3	CsaV4_3G004518	Chloroplast	5.86	59,208.46	532
CsAGP4	CsaV4_6G001323	Chloroplast	9.09	59,906.89	536
CsAGP5	CsaV4_7G000260	Chloroplast	7.59	56,917.85	521

2.2. Phylogenetic and Structural Analysis of AGPase Proteins in Cucumber

To clarify the evolutionary relationships of cucumber AGPases, we constructed a phylogenetic tree based on five CsAGP (Cucumis sativus L.), six AtAGP (Arabidopsis thaliana), seven MaAGP (Musa acuminata L.), eight OsAGP (Oryza sativa L.), nine SlAGP (Solanum lycopersicum L.), eighteen TaAGP (Triticum aestivum L.) and five ClAGP (Citrullus lanatus L.) proteins. As shown in Figure 1, the proteins separated into two major branches corresponding to the large and small subunits; group II contained 35 large-subunit members, whereas group I comprised 20 small-subunit members. It is clear that the five AGPase genes in cucumber are closely related to the five AGPase genes in watermelon, and four of the five CsAGP genes belong to the large subunit. In comparison, their phylogenetic relationships with Arabidopsis thaliana and tomato AGPase genes are relatively more distant. In order to further investigate the structural features and evolutionary mechanisms of AGPase gene families, the conserved protein motifs (Figure 2a) and gene structures (Figure 2b) were comparatively analyzed in the genomes of cucumber, Arabidopsis, and tomato. MEME analysis identified 10 motifs that were retained in most AGPase proteins. Most of the conserved motifs were evenly distributed across genes in three different species. The distribution of conserved motifs diverged significantly among the different species. Furthermore, the AGPase gene family was identified by the presence of an NTP transferase domain (PLN02241, Figure 2c), which is characteristic of the AGPase protein family. These findings provide additional evidence for specific evolutionary characteristics of AGPase genes across plant species.

Figure 1.

Phylogenetic analysis of AGPases from cucumber, Arabidopsis, rice, watermelon, tomato, wheat, and banana. The neighbor-joining tree was constructed using MEGA X with 1000 bootstraps. Two subgroups were identified and classified according to large-subunit and small-subunit type. The red, light green, orange, light blue, pink, yellow and green represent AGPase proteins from cucumber, Arabidopsis, watermelon, tomato, wheat, rice, and banana, respectively.

Figure 2.

Analysis of conserved motifs and gene structure of AGPases. (a) Conserved motifs in different species (Arabidopsis, tomato, cucumber). The motifs, numbered 1–10, are indicated in different colors. (b) The yellow boxes indicate exons. Black lines represent introns. Green boxes represent the untranslated regions (UTRs). (c) NTP transferase (PLN02241) domains of AGPase in different species.

2.3. Genomic Organization and Evolutionary Analysis of CsAGP Genes

As illustrated in Figure 3a, five CsAGP genes were distributed across four chromosomes. There was one CsAGP gene on chromosome 2, one on chromosome 6, and one on chromosome 7, while two CsAGP genes were located on chromosome 3. The two CsAGP genes on chromosome 3 may indicate a gene duplication event or a potential hotspot for genes involved in starch biosynthesis and stress responses, which could provide insight into the functional diversity of the CsAGP family. Because gene duplication often promotes adaptation to changing environments, including responses to biotic challenges, this arrangement may be biologically meaningful. Collinearity analysis of the AGPase gene family in cucumber identified three pairs between cucumber and tomato and two pairs between cucumber and Arabidopsis (Figure 3b). This pattern suggests that these CsAGP genes may have been evolutionarily conserved across species.

Figure 3.

Chromosomal distribution and collinearity analysis of CsAGP genes. (a) Chromosomal distribution of CsAGP genes in cucumber. Light green lines represent different chromosomes, and marks on them indicate the corresponding genes and their locations. (b) Collinearity analysis of CsAGP genes in cucumber. Homologous gene pairs in Arabidopsis (blue), cucumber (green), and tomato (red). The red and blue lines represent the homologous pairs of the AGPase gene family.

2.4. Cis-Regulatory Element Profiling of CsAGP Gene Promoter

To further investigate the regulatory mechanisms of CsAGP genes and their modulation by phytohormones, defense signaling, and stress responses, we analyzed the 2000 base pair upstream promoter regions of CsAGP genes to identify cis-acting regulatory elements (CAREs). Numerous CAREs were detected, including motifs associated with light responsiveness, MeJA signaling, ABA responsiveness, salicylic acid signaling, and general defense or stress responses. Among these, light-, MeJA-, and ABA-related elements were the most abundant, underscoring their potential role in regulating CsAGP function in plant growth and development. Additionally, we identified numerous stress-responsive elements (Figure 4), including those responsive to low temperature and drought. This suggests that CsAGP genes not only regulate essential metabolic pathways but also contribute to enhancing plant resilience to both biotic and abiotic stresses.

Figure 4.

Putative cis-acting regulatory elements (CAREs) of the AGPase gene family. The CARE analysis was conducted on the 2 kb upstream region using the PlantCARE online server. The green CARE represents the binding site of the AT-rich DNA-binding protein (ATBP-1). The yellow CARE represents a cis-acting element involved in defense and stress responsiveness. The pink CARE represents a cis-acting element involved in abscisic acid responsiveness. The dark green CARE represents a cis-acting regulatory element involved in MeJA responsiveness. The red CARE represents a gibberellin-responsive element. The gray CARE represents an MYB binding site involved in the regulation of flavonoid biosynthetic genes. The light green CARE represents a cis-acting element involved in low-temperature responsiveness. The orange CARE represents a cis-acting element involved in gibberellin responsiveness.

Promoter analysis reveals that CsAGP genes are orchestrated by a network of phytohormones and stress-related signaling pathways. Together, these results indicate that CsAGP genes function as key regulators in coordinating plant responses to environmental challenges, a process critical for maintaining metabolic balance and improving overall plant fitness.

2.5. Expression Profiling of CsAGP Genes During Development and Under Diverse Stress Conditions

To explore the relationship between CsAGP genes and biotic/abiotic stress responses, we analyzed expression changes in the cucumber variety ‘9930’ under multiple stress conditions, including Cladosporium cucumerium (CC), gray mold (GM), powdery mildew (PM), angular leaf spot (ALS), salt stress, cold stress, and heat stress (Figure 5a–g). Based on heatmap analysis of transcriptome data, we found that CsAGP2, CsAGP4, and CsAGP5 were significantly induced when the plants experienced biotic and abiotic stresses, showing notable changes in expression levels, whereas CsAGP1 and CsAGP3 showed no significant changes. These analyses revealed dynamic CsAGP expression patterns in response to pathogenic infections and environmental stressors, highlighting their potential roles in plant defense and metabolic adaptation.

Figure 5.

Expression profiling of CsAGP genes during development and under diverse stress conditions. (a) The heatmap of cucumber CsAGP gene family expression at 0, 1, 2, and 4 days post-inoculation with bacterial angular leaf spot. (b) The heatmap of cucumber CsAGP gene family expression at 0, 6, 24, and 72 h post-inoculation with Cladosporium cucumerinum. (c) The heatmap of cucumber CsAGP gene family expression at 0, 6, 12, and 48 h post-inoculation with gray mold. (d) The heatmap of cucumber CsAGP gene family expression at 0, 1, 3, and 5 days post-inoculation with powdery mildew. (e) The heatmap of cucumber CsAGP gene family expression at 0, 6, 12, and 24 h under cold stress. (f) The heatmap of cucumber CsAGP gene family expression at 0 h, 20 min, 3 h, and 12 h under heat stress. (g) The heatmap of cucumber CsAGP gene family expression at 0, 10, 48, and 96 h under salt stress. (h) The heatmap of cucumber CsAGP gene family expression in different tissues, including radicle, juvenile root, adult root, cotyledon, first leaf blade, second leaf blade, fourth leaf blade, adult leaf, hypocotyl, tendril, juvenile stem, adult stem, and shoot apical meristem.

Furthermore, we characterized expression patterns across cucumber developmental stages and tissues. CsAGP genes were expressed predominantly in leaves, with particularly strong transcript accumulation for CsAGP2, CsAGP4 and CsAGP5 (Figure 5h). By mapping these expression changes across different growth stages, we elucidated their functional significance in starch biosynthesis, disease resistance, and stress adaptation mechanisms.

2.6. Expression Profiling of CsAGP Genes in Resistant and Susceptible Cucumbers in Response to Pathogen Infection

To compare the behavior of CsAGP genes in resistant and susceptible backgrounds under pathogen stress, we analyzed their expression levels in leaves following infection by several severe pathogens, including gummy stem blight (GSB), angular leaf spot (ALS), and watermelon mosaic virus (WMV).

For GSB, the susceptible line ‘CG25’ developed obvious lesions at 3 to 5 days post-inoculation (dpi), whereas the resistant line ‘CG64’ displayed no obvious symptoms. Upon GSB infection, CsAGP2 expression gradually decreased in both resistant and susceptible lines, while CsAGP3 expression increased progressively. CsAGP5 showed increased expression at 0 h and 12 h post-inoculation (hpi), and then gradually declined (Figure 6a).

Figure 6.

The expression levels of CsAGP genes under different disease stresses. (a) Expression analysis of CsAGP genes in ‘CG64’ and ‘CG25’ under GSB infection. (b) Expression analysis of CsAGP genes in ‘3259–154’ and ‘3259–167’ under ALS infection. (c) Expression analysis of CsAGP genes in ‘228’ and ‘65G’ under WMV infection. A one-way analysis of variance (ANOVA) was conducted, followed by Tukey’s HSD test. Data represent mean ± SD of three biological replicates. *, ** and *** represent data that differ significantly using a two-tailed Student’s t-test at p < 0.05, p < 0.01 and p < 0.001, respectively.

For ALS infection, the susceptible line ‘3259–167’ exhibited symptoms at 1 dpi. All four analyzed CsAGP genes displayed similar expression patterns in both susceptible and resistant materials. Notably, CsAGP3 expression in both lines increased continuously from 0 h to 6 hpi before gradually returning to basal levels, a pattern potentially associated with the observed dryness symptoms (Figure 6b).

Following WMV inoculation, the resistant line ‘228’ showed continuous and significant upregulation of CsAGP1 and CsAGP5 expression. CsAGP4 expression increased from 0 to 40 hpi and then decreased. In contrast, the susceptible line ‘65G’ exhibited no significant change in CsAGP4 expression. These findings suggest that CsAGP4 may play a positive role in antiviral mechanisms (Figure 6c).

2.7. Modulation of CsAGP Expression by ABA and JA Under Viral Infection

Given that abscisic acid (ABA) and jasmonic acid (JA) are well-established key regulators of plant antiviral responses—mediating defense signaling and enhancing resistance—we investigated whether exogenous applications of these hormones could modulate the expression of the CsAGP gene family. Specifically, we designed experiments to assess changes in CsAGP gene expression patterns following hormone treatment, aiming to elucidate potential functional linkages between these phytohormones and the CsAGP genes within plant antiviral mechanisms.

The expression levels of CsAGP genes exhibited dynamic changes in response to ABA and JA treatments. Exogenous spraying experiments were conducted on leaves of the cucumber line ‘228’, both with and without WMV inoculation. The results are summarized as follows. Group I (ABA Treatment, Figure 7a): Compared to the untreated control, CsAGP1, CsAGP2, CsAGP3, and CsAGP5 genes showed significantly upregulated expression upon ABA treatment. In contrast, the expression of CsAGP4 increased gradually from 0 to 72 hpi before rapidly declining to levels below those of the control. Group II (ABA and WMV Treatment, Figure 7b): Following WMV inoculation, exogenous ABA application resulted in a consistent expression pattern for CsAGP1, CsAGP2, CsAGP4, and CsAGP5, with expression levels at each time point being higher than those in Group I. Notably, the expression of CsAGP3 remained unchanged. This suggests that ABA induces the upregulation of this gene family in response to WMV infection, further indicating a potential interaction between viral infection and the ABA signaling pathway. Group III (JA Treatment, Figure 7c): The regulatory pattern of CsAGP genes was largely consistent with that observed in the ABA treatment group, differing primarily in response timing. This suggests that the gene family exhibits a similar response mechanism to both hormones. Group IV (JA and WMV Treatment, Figure 7d): Similar to the pattern in Group II, gene expression was altered under JA treatment in the presence of viral infection. While the overall expression trend for all genes was similar to that in Group III, CsAGP4 showed a slightly higher expression level at 24 hpi, whereas the overall expression levels of the other four genes were significantly lower than in Group III. This marked the most distinct difference compared to Group II. The differential expression patterns of CsAGP genes under ABA and JA treatments highlight the intricate regulatory mechanisms plants employ to cope with stress. These findings provide valuable insights into how ABA and JA regulate CsAGP gene expression and starch metabolism in cucumber, as well as their roles in stress responses.

Figure 7.

Expression pattern of five CsAGP genes in response to abscisic acid (ABA) or jasmonic acid (JA) treatment of WMV-infected cucumber seedlings. (a) Analysis of the changes in expression levels of CsAGP genes upon ABA application in the absence of WMV stress. (b) Analysis of the changes in expression levels of CsAGP genes under WMV stress with concurrent ABA treatment. (c) Analysis of the changes in expression levels of CsAGP genes upon JA application in the absence of WMV stress. (d) Analysis of the changes in expression levels of CsAGP genes under WMV stress with concurrent JA treatment. The response is indicated by vertical bars across five time points for ABA (0.1 mM), WMV + ABA (0.1 mM), JA (0.1 mM) and WMV + JA (0.1 mM) treated samples and is labeled on the horizontal axis. A one-way analysis of variance (ANOVA) was conducted, followed by Tukey’s HSD test. Data represent mean ± SD of three biological replicates. *, ** and *** represent data that differ significantly using a two-tailed Student’s t-test at p < 0.05, p < 0.01 and p < 0.001, respectively.

2.8. Functional Validation of CsAGP4 in Starch Biosynthesis and Antiviral Defense

We selected the ‘Poinsett 97’ accession for subsequent gene editing. Sequence analysis showed that the coding sequence of CsAGP4 in this accession is identical to that of the reference genome ‘9930’ (Figure S1). Moreover, CsAGP4 exhibited similar transcriptional levels to those in ‘9930’ (Figure 8a) and retained AGPase activity in this accession (Figure 8c), confirming that CsAGP4 is functionally competent in ‘Poinsett 97’. Using this accession as the background, we generated two homozygous mutant lines (CsAGP4^CR-1 and CsAGP4^CR-2) via CRISPR/Cas9-mediated genome editing (Figure 8b).

Figure 8.

Functional validation of the CsAGP4 gene. (a) CsAGP4 expression analysis in ‘Poinsett 97’ and ‘9930’. Not significant (ns, p ≥ 0.05). (b) CRISPR/Cas9 technology was used to edit the target sequence of the CsAGP4 gene and the sequence of two homozygous mutants. (c) ADPGase enzyme activity in wild type and knockout lines (CsAGP4^CR-1 and CsAGP4^CR-2) was measured at the seedling stage using an enzyme activity assay kit. (d) Transmission electron microscopy (TEM) analysis of starch granule accumulation in wild type and two CsAGP4 knockout lines. Scale bar: 20 μm, 5 μm, and 2 μm, respectively. (e) Observations of WMV symptoms in wild type and two independent CsAGP4 knockout lines (CsAGP4^CR-1 and CsAGP4^CR-2) at 9 days post−inoculation (dpi). Scale bar: 4 cm. (f) Wild−type and mutant plants were inoculated, and the disease index was assessed at 9 days post−inoculation (dpi). A one−way analysis of variance (ANOVA) was conducted, followed by Tukey’s HSD test. Data represent mean ± SD of three biological replicates. *, **, *** and **** represent data that differ significantly using a two−tailed Student’s t−test at p < 0.05, p < 0.01 and p < 0.001, p < 0.0001, respectively.

Since CsAGP4 encodes a key rate-limiting enzyme in starch biosynthesis, we next examined whether its disruption affects starch accumulation. Transmission electron microscopy (TEM) revealed that starch granules were nearly absent in the leaves of both mutants compared with the wild type (Figure 8d). Consistently, AGPase activity was significantly reduced in the mutants (Figure 8c), confirming that CsAGP4 mutation impairs starch biosynthesis. We next assessed the susceptibility of wild-type and mutant plants to WMV infection. At 9 dpi, both mutant lines developed more severe disease symptoms than the wild type, with disease indices of 60 and 58 in CsAGP4^CR-1 and CsAGP4^CR-2, respectively, both significantly higher than those of the wild type (Figure 8e,f). Collectively, these results demonstrate that CsAGP4 plays a positive role in plant resistance against WMV.

3. Discussion

In this work, we performed a systematic analysis of the AGPase gene family in cucumber and identified five CsAGP members distributed across four chromosomes. Phylogenetic clustering divided CsAGP genes into two clades corresponding to large and small subunits, consistent with previous classifications in Arabidopsis, rice, and banana [5,11]. Notably, CsAGP genes showed closer evolutionary relationships with watermelon homologs. The uneven chromosomal distribution of CsAGP genes, especially the tandem duplication on chromosome 3, points to lineage-specific expansion that may have contributed to functional redundancy and diversification. Such duplications are common in stress-responsive gene families and enable plants to adapt to environmental challenges [14]. The conserved NTP transferase domains and motif architectures across species further underscore the indispensable role of AGPase in starch biosynthesis. However, the divergence in exon-intron structures and cis-regulatory elements implies species-specific regulatory mechanisms, potentially fine-tuning AGPase activity in response to cucumber-specific stressors like WMV. This lays a foundation for further functional studies of CsAGP in cucumber.

The preferential expression of CsAGP genes in photosynthetically active tissues (leaves) (Figure 5h) underscores their role in balancing growth and defense. High AGPase activity in leaves ensures rapid starch synthesis during the day, while elevated expression in flowers may support nectar production or pollen development, as seen in maize [15]. The low expression in roots contrasts with findings in cassava, where root-specific AGPases dominate starch storage [25], suggesting tissue-specific subfunctionalization within the family. This divergence may reflect evolutionary adaptations to different sink–source relationships in annual (cucumber) versus perennial (cassava) species.

The interplay between ABA and JA signaling pathways and CsAGP expression provides critical insights into hormonal regulation of starch metabolism during viral infection. ABA treatment markedly induced the expression of CsAGP1, CsAGP2, CsAGP3, and CsAGP5 (Figure 7), in line with reports that ABA promotes AGPase activity in Arabidopsis to preserve starch reserves under drought stress [8]. Notably, CsAGP4 expression (Group II) was synergistically induced by ABA and WMV infection, suggesting a potential link between stress hormone signaling and metabolism. This aligns with emerging evidence that ABA not only regulates stomatal immunity but also reprograms carbohydrate metabolism to limit pathogen resource acquisition [26,27]. Similarly, the JA responsiveness of CsAGP may be linked to glycosylation-mediated defense processes, with the CsAGP promoter potentially participating in the regulation of defense metabolite accumulation [28,29].

One of the important findings of this study is the differential expression of CsAGP genes between virus-resistant (‘228’) and susceptible (‘65G’) genotypes following WMV inoculation. The significant upregulation of CsAGP4 in resistant plants (Figure 6) aligns with recent reports linking AGPase activity to pathogen defense. For instance, in grapevine, AGPase activity increases during downy mildew infection to bolster starch accumulation, which may restrict pathogen spread by altering carbon partitioning [16]. Similarly, wheat AGPase isoforms are modulated during fungal attacks to redirect resources toward defense metabolites [14]. The combined evidence from enzymatic assays, transmission electron microscopy (TEM), and pathogen inoculation demonstrates that the complete knockout of CsAGP4 leads to compromised AGPase activity, a reduction in starch granule accumulation, and a significant increase in disease susceptibility (Figure 8). These findings are also consistent with previous studies in grapevine [16] and tomato [30], where starch metabolism was found to enhance plant resistance to pathogens. These results suggest that CsAGP4 induction in cucumber may promote starch synthesis, which could restrict viral replication by reducing sugar availability or potentially generating soluble glucans that could function as immune-priming signals [31]. Regarding the potential mechanisms by which CsAGP4-mediated starch metabolism contributes to antiviral defense, two hypotheses are proposed. On the one hand, starch biosynthesis may act as a carbon sequestration mechanism, restricting the availability of sugars that viruses require for replication. Similar studies have shown that viruses hijack host sugar transporters to sustain their proliferation [32]. On the other hand, because most pathogens draw hexoses, primarily glucose, from the host cytoplasm [33], starch degradation may support host energy metabolism by supplying these sugars, thereby strengthening immune responses to viral invasion [34]. The high starch reserves ensure an adequate energy supply, strengthening the plant’s immunity and overall resistance to viral infection.

Despite providing strong evidence for the role of CsAGP4-mediated starch metabolism in antiviral defense, several key questions remain to be explored. First, it is necessary to determine whether CsAGP4 also confers resistance to other viruses, such as Cucumber mosaic virus (CMV) and Zucchini yellow mosaic virus (ZYMV), to evaluate whether it plays a broader role in plant disease resistance. Furthermore, future studies should investigate whether CsAGP4 regulates the metabolism of starch to influence viral replication, providing deeper insights into how carbon metabolism modulates virus–host interactions. Furthermore, metabolomic and transcriptomic analyses of wild-type and CsAGP4 mutant plants during viral infection will be performed to identify key disease-resistance pathways and interacting genes. Additionally, molecular and biochemical assays will be conducted to validate these key interactions, thereby deepening our understanding of plant–virus interaction mechanisms.

4. Materials and Methods

4.1. Plant Materials and Experimental Design

Eight cucumber inbred lines were used in this study: ‘CG64’ (resistant to Gummy Stem Blight), ‘CG25’ (susceptible to Gummy Stem Blight), ‘3259–154’ (highly resistant to Angular Leaf Spot), ‘3259–167’ (highly susceptible to Angular Leaf Spot), ‘9930’ (a North China-type line), ‘65G’ (sensitive to WMV), ‘228’ (resistant to WMV) and ‘Poinsett 97’ (picked type). All materials were obtained from the Cucumber Research Group, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China, and grown under controlled chamber conditions at Nankou, Beijing. For pathogen inoculation, seedlings were challenged with fungal (GSB), bacterial (ALS), or viral (WMV) pathogens.

Plants were grown under the same controlled chamber conditions, and seedlings were randomly assigned to different treatment groups. For pathogen inoculation and hormone treatment experiments, 21 plants were used per genotype with three biological replicates. At each sampling time point, fully expanded leaves from three plants were pooled as one biological replicate. For qRT–PCR analysis, three biological replicates were used for each time point, and each biological replicate was analyzed with three technical replicates. For AGPase activity assays, three biological replicates were collected for each genotype or treatment, with three plants included in each biological replicate.

4.2. Hormone Treatment and Pathogen Inoculation

For hormone treatments, fully expanded first leaves of ‘228’ seedlings were sprayed with 0.1 mM abscisic acid (ABA) or 0.1 mM jasmonic acid (JA) (Solarbio, Beijing, China). Four treatment groups were established: (I) ABA alone, (II) ABA after WMV inoculation, (III) JA alone, and (IV) JA after WMV inoculation. Inoculation of WMV was conducted according to a previous study [35]. All methods for inoculating pathogens mentioned in the text are based on the methods described in reference [36].

4.3. Genome-Wide Identification of the CsAGP Genes and Phylogenetic Tree Construction

Cucumber CsAGP protein sequences were retrieved from the ‘9930’ reference genome database (http://60.30.67.245:8070/#/download, accessed on 11 April 2025). AGPase family members were identified using two complementary strategies. First, the conserved NTP transferase domain (PF00483, https://pfam.xfam.org/, accessed on 11 April 2025) was used to search predicted CsAGP proteins with the Hidden Markov Model approach (version 3.4) (http://hmmer.org, accessed on 11 April 2025). Second, BLAST v2.441 searches were performed using AGP protein sequences from A. thaliana, O. sativa, M. acuminata, Solanaceae and Maize as queries. The AGP amino acid sequences were obtained from the TAIR database for A. thaliana (http://www.arabidopsis.org, accessed on 11 April 2025), the RGAP database for O. sativa (http://rice.plantbiology.msu.edu, accessed on 11 April 2025), the DH-Pahang genome database for M. acuminata (https://banana-genome-hub.southgreen.fr/downloads, accessed on 11 April 2025), the Solanaceae genomics database for Solanaceae (https://solgenomics.net/, accessed on 11 April 2025), and the Maize genomics database for Maize (https://maizegdb.org/, accessed on 11 April 2025). A phylogenetic tree for AGPs was constructed with 1000 bootstrap tests based on unrooted neighbor-joining using MEGA X software (version 10.2) [37].

4.4. Protein Property and Gene Structure Analysis

Theoretical molecular weight (MW) and isoelectric point (pI) values of AGP proteins were calculated using ExPAsy (version 3.0) [38]. Subcellular localization was predicted with the UniProt database (https://www.uniprot.org/, accessed on 12 April 2025) and Plant-mPLoc in Cell-PLoc (version 2.0) (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, accessed on 12 April 2025). Exon-intron structures were determined from genomic sequences using the Gene Structure Display Server (version 2.0) (http://gsds.gao-lab.org/, accessed on 12 April 2025) [39] and visualized with TBtools-II (version 2.371) [40]. Conserved protein motifs were identified with MEME (version 5.5.9) (http://meme-suite.org, accessed on 12 April 2025). A phylogenetic tree based on unrooted neighbor-joining was constructed with 1000 bootstrap tests using MEGA X software, and CsAGP genes were classified according to the topology of the phylogenetic trees [37].

4.5. Chromosomal Localization and Gene Duplication in CsAGP Genes

Chromosomal positions of AGP genes were visualized with TBtools based on genome annotation files [40]. MCscanX was used to perform tandem duplication analysis of AGPs in Cucumis sativus, Arabidopsis thaliana, O. sativa, M. acuminata, Solanaceae and Maize. Ka/Ks ratios were calculated with KaKs_calculator (version 2.0) using the YN model [41].

4.6. Prediction of Cis-Acting Elements in the CsAGP Gene Family

For promoter analysis, the 2000 bp upstream sequences of all five CsAGP genes were obtained from the cucumber genome database. Conserved cis-acting regulatory elements present in these regions were predicted with PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 13 April 2025) [42].

4.7. Expression Profiling of CsAGP Genes

To investigate CsAGP transcriptional patterns, we retrieved relevant expression datasets from the cucumber genome database (http://60.30.67.245:8070/#/home, accessed on 20 April 2025) [36]. Furthermore, we used TBtools to analyze the expression patterns of CsAGP genes in spatial-temporal development, stress resistance, and disease resistance.

4.8. Expression Analysis of CsAGP Genes by qRT-PCR

To investigate whether the response of CsAGP genes to different pathogens is specific, we conducted expression analysis at various time points after inoculation with the three pathogens. For each time point, three biological replicates were harvested, with three leaves per replicate. Collected leaves were immediately frozen in liquid nitrogen and stored at −80 °C until use. Gene-specific primers were designed with Primer 3.0 according to CsAGP sequences (Table S1). Total RNA was extracted with the Plant RNA Extraction Kit (TaKaRa MiniBEST, Beijing, China), and first-strand cDNA was synthesized using HiScript III RT SuperMix for qPCR (Vazyme Biotech, Beijing, China). Real-time PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Beijing, China) under the following program: 95 °C for 3 s, followed by 39 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s. Each treatment was performed with three biological and technical replicates. Actin1 (CsaV4_3G003833) was used as a reference gene, and the 2^−ΔΔCt algorithm was used to calculate gene expression levels. Statistical analyses were performed with GraphPad Prism (version 9.0) and Microsoft Excel 2021.

4.9. AGPase Enzyme Activity and Transmission Electron Microscopy (TEM)

When the first true leaf of seedlings was fully expanded, three biological replicates were taken separately, with three plants per replicate. A total of 0.1 g of leaves was collected and added to 1 mL of extraction solution, then ground into a homogenate in an ice bath. The measurement was then carried out according to the method described in kit BC0430 (Solarbio, Beijing, China).

When the first true leaf of seedlings was fully expanded, fresh tissue samples were immediately placed in electron microscopy fixative and cut into 1 mm³ pieces. After vacuum pumping to submerge the samples, they were fixed at room temperature for 2 h and stored protected from light at 4 °C. Following fixation, the samples were rinsed with 0.1 M phosphate buffer (pH 7.4), fixed with 1% osmium tetroxide protected from light for 7 h, and rinsed again with buffer. Subsequently, the samples were dehydrated stepwise using gradient ethanol (30% to 100%) and acetone, followed by gradient infiltration and embedding with 812 epoxy resin, and polymerized at 60 °C for 48 h. Ultrathin sections of 60–80 nm were cut using an ultramicrotome, collected on 150-mesh copper grids, and double-stained with uranyl acetate and lead citrate. The sections were then observed and imaged under a transmission electron microscope [43].

4.10. Plasmid Construction and Plant Transformation

For CRISPR/Cas9-mediated cucumber transformation of CsAGP4, sgRNA sequences targeting CsAGP4 were computationally designed through the CRISPR-GE platform [44]. Complementary oligonucleotides corresponding to selected guide sequences were annealed and directionally cloned into the BsaI-digested pKSE402 binary vector using T4 DNA ligase (New England Biolabs, Beijing, China). The recombinant plasmid was subsequently electroporated into Agrobacterium tumefaciens strain EHA105 for cucumber transformation. Primary transformants were screened through PCR amplification of the target region followed by bidirectional Sanger sequencing to verify editing events. Confirmed heterozygous mutants were advanced through self-fertilization cycles to isolate T2-generation homozygous lines, which were subsequently subjected to comprehensive phenotypic and molecular analyses. Positive clones were verified by colony PCR and Sanger sequencing (Sangon Biotech, Beijing, China). Primer sequences are provided in Supplementary Table S1.

5. Conclusions

In this study, we systematically characterized the AGPase gene family and identified CsAGP4 as a WMV-responsive isoform whose expression is preferentially activated in the resistant genotype. Hormone assays showed that ABA and JA rapidly remodel the transcriptional landscape of CsAGP genes during WMV infection and synergistically enhance CsAGP4 induction, indicating that CsAGP4 is not merely a constitutive metabolic enzyme but is integrated into classical antiviral hormone-signaling pathways. CRISPR/Cas9-generated knockout mutants demonstrated that CsAGP4 is indispensable for starch granule formation, ADP-glucose pyrophosphorylase activity and effective restriction of WMV accumulation at the seedling stage. Loss of CsAGP4 not only blocked starch biosynthesis but also markedly increased disease severity, showing that AGPase-mediated carbon allocation is an important component of antiviral immunity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27114764/s1.

Author Contributions

Conceptualization, X.S. and J.G.; methodology, S.D.; software, X.S.; validation, X.S., X.L. and S.D.; formal analysis, X.S. and H.M.; investigation, X.S.; resources, X.G.; data curation, X.L.; writing—original draft preparation, X.S.; writing—review and editing, J.G.; visualization, X.S.; supervision, S.Z.; project administration, H.M. and S.Z.; funding acquisition, S.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All relevant data are within this article and its Supplementary Materials. Further inquiries can be directed to the first authors.

Conflicts of Interest

The authors declare no competing interests.

Funding Statement

This research was supported by the National Key Research and Development Program of China (2023YFD1201503), the National Natural Science Foundation of China (No. 31872104), the State Joint Research Project on Cucumber Breeding, the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-23), the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-IVF), the State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China, and the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, China.