MdCDPK24 Encoding Calcium-Dependent Protein Kinase Enhances Apple Resistance to Colletotrichum gloeosporioides

Abstract

Calcium-dependent protein kinases (CDPKs) are unique serine/threonine kinases that play significant roles in response to environmental stresses in plants. In this study, we comprehensively characterized the CDPK gene family in the apple cultivar ‘Hanfu’ at the genome-wide level, and 38 MdCDPKs were identified. They were unevenly distributed across 14 chromosomes. Based on phylogenetic analysis, the MdCDPKs were classified into four subfamilies. Conserved domain analysis indicated that MdCDPKs contain the catalytic kinase domain and the Ca²⁺ binding domain. During Colletotrichum gloeosporioides infection, the expression level of MdCDPK24 was significantly upregulated. Subsequently, MdCDPK24 was fused to GFP to generate the MdCDPK24-GFP construct, and confocal microscopy imaging confirmed its cytoplasmic localization in Nicotiana benthamiana leaves. Using agrobacterium-mediated transformation, we generated the overexpression of MdCDPK24 transgenic calli. MdCDPK24-overexpressing calli demonstrated significantly reduced disease severity against C. gloeosporioides infection, indicating its positive role in apple bitter rot resistance. The analysis of the CDPK gene family in the apple cultivar ‘Hanfu’ provides a new insight into the identification of CDPK genes involved in biotic stress. MdCDPK24 represents a promising candidate for genetic manipulation to enhance apple bitter rot resistance.

1. Introduction

Apple (Malus domestica) is a globally significant economic crop, valued for its favorable organoleptic properties and nutritional composition [1]. China maintains the world’s largest apple cultivation area and production output. Fungal pathogen infections during cultivation have substantially reduced per-unit-area yield and compromised fruit quality, emerging as primary constraints to sustainable apple production in China [2]. Consequently, effective disease management remains a critical agricultural priority. Apple bitter rot (caused by Colletotrichum gloeosporioides) is among the most devastating fungal diseases due to its rapid spread. Conventional chemical controls have limited efficacy, while prolonged pesticide application raises environmental contamination concerns [3]. Therefore, elucidating the molecular mechanisms of apple bitter rot resistance and identifying associated disease resistance genes presents significant theoretical and practical value for sustainable agriculture.

Environmental stresses are perceived by diverse sensors and transduced through distinct signaling networks, inducing downstream defense responses such as stress-related protein and metabolite synthesis [4,5,6]. Calcium ions (Ca²⁺), as a universal second messenger, play a critical role in plant signal transduction pathways [7]. In plants, various stresses trigger a rapid transient increase in intercellular Ca²⁺ concentration [8,9]. Multiple Ca²⁺ sensors detect and interpret Ca²⁺ signatures, subsequently transducing them into diverse downstream effects [10]. Calcium-dependent protein kinases (CDPKs/CPKs) represent a major class of Ca²⁺-sensitive serine/threonine (Ser/Thr) kinases, rapidly detect transient intracellular Ca²⁺ signals, recognize specific substrates, phosphorylate them, and thereby propagate and amplify the signals through downstream signaling cascades [11]. The CDPK gene family has been identified in many plants. Arabidopsis thaliana contains 34 CDPK genes by analysis of the genome sequence. CDPK genes were also identified in Oryza sativa (29 rice CDPKs), Zea mays (40 maize CDPKs), Solanum tuberosum (25 potato CDPKs), Pyrus bretschneideri (30 pear CDPKs), and Populus trichocarpa (30 polar CDPKs) [12,13,14,15,16]. CDPK proteins have four conserved domains: an N-terminal variable region, a Ser/Thr kinase catalytic domain, an autoinhibitory domain, and a calmodulin-like domain containing EF-hands for Ca²⁺ binding [17]. The N-terminal variable domain exhibits intra-species length and sequence heterogeneity while undergoing constitutive and stimulus-induced in vivo phosphorylation mediated by autophosphorylation or upstream kinases [18]. The catalytic domain contains a conserved Ser/Thr kinase domain with high sequence homology. Mutations within this domain abolish catalytic activity, impairing the phosphorylation of substrate proteins [19]. Crucially, CDPK activation by Ca²⁺ binding is a prerequisite for kinase functionality [17]. At the basal Ca²⁺ level, the binding between the autoinhibitory domain and the catalytic domain suppresses protein kinase activity. Under Ca²⁺ influx conditions, Ca²⁺ binds to the EF-hand domain, inducing conformational changes that relieve autoinhibition and initiate autophosphorylation, thereby activating CDPKs [20,21].

Studies across multiple plant species demonstrate that CDPKs mediate plant responses to diverse stimuli, including drought, salinity, and pathogen stress. Transgenic lines overexpressing PtrCDPK10 increase ascorbate peroxidase activity and decrease ROS levels, elevating trifoliate orange drought stress tolerance [22]. In Phyllostachys edulis, PheCPK1 functions as a negative regulator of drought stress responses by repressing stress-responsive gene expression and impairing ROS scavenging capability [23]. Under salt stress, the AtCPK12-RNAi mutant accelerates salt-induced damage, including Na⁺ accumulation and ROS production in roots [24]. ZmCPK11 enhances maize salt tolerance by maintaining foliar Na⁺/K⁺ homeostasis, which mitigates salt-induced chlorophyll degradation and photosystem II impairment [25]. Under abiotic stress, CDPKs enhance plant stress tolerance by modulating ROS levels and ion homeostasis. Accumulating evidence shows that CDPK plays an essential role in plant–pathogen interactions. AcCDPK1 and AcCDPK5 from onion function as positive regulators of Phytophthora nicotianae resistance [26]. OsCPK17 stabilizes the receptor-like cytoplasmic kinase OsRLCK176, thereby directly enhancing rice immunity signaling [27]. During pepper’s response to Ralstonia solanacearum infection, CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 to promote plant immune responses [28]. During biotic stress, plants strengthen disease resistance via stabilizing disease-related proteins and upregulating resistance-gene expression. However, the role of apple CDPK genes in C. gloeosporioides resistance remains largely unclear.

In this study, we identified CDPK genes in ’Hanfu’ apple and performed bioinformatics analysis of the MdCDPK family. Furthermore, we analyzed the expression levels of MdCDPKs during C. gloeosporioides infection using RT-qPCR to identify C. gloeosporioides-responsive MdCDPK genes. The selected MdCDPK gene induced by apple bitter rot was overexpressed in apple to assess disease resistance. Our findings provide a valuable CDPK candidate for the targeted enhancement of apple bitter rot resilience.

2. Materials and Methods

2.1. Plant Materials and Microbial Strains

Apple cultivar ‘Orin’ calli were cultured in Murashige and Skoog medium with 0.4 mg/L 6-Benzylaminopurine and 1.5 mg/L 2,4-Dichlorophenoxyacetic acid at 24 °C. After two weeks in the dark, calli were subcultured onto fresh medium. N. benthamiana was grown in a controlled-environment chamber under a 16 h light/8 h dark photoperiod at 26 °C. C. gloeosporioides was grown in potato dextrose agar (PDA) medium in the dark at 28 °C. After a one-week incubation, fungal mycelium was subcultured onto newly prepared PDA plates.

2.2. Identification of CDPK Genes in Apple

Protein sequences of AtCDPK were obtained from the TAIR database (https://www.arabidopsis.org/, accessed on 10 August 2025). All protein sequences in ‘Hanfu’ apple were downloaded from GDR (https://www.rosaceae.org/, accessed on 10 August 2025) [29]. Local blast was performed using all AtCDPK proteins as queries for the identification of CDPK proteins from apple. All putative candidates were manually verified with the Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/cdd/, accessed on 10 August 2025) to confirm the presence of the protein kinase domain and the calmodulin-like domain [30]. The amino acid number, isoelectric point, and molecular weight of apple CDPK proteins were analyzed using the ExPASy tool (https://web.expasy.org/protparam/, accessed on 10 August 2025) [31].

2.3. Chromosomal Locations of CDPKs in Apple

Chromosome location information of MdCDPK genes was obtained from GDR. Then, the mapping of MdCDPK genes to the chromosomes was performed using the Gene Location Visualize tool with TBtools II software v2.326 [32]. Input parameters included chromosome ID, chromosome length, gene ID, gene start position, and gene end position. Processing these inputs generated a visualization of MdCDPK gene locations on the chromosome.

2.4. Phylogenetic Analysis

Protein sequences of AtCDPK and MdCDPK were used for phylogenetic tree analysis. A phylogenetic tree was constructed using MEGA X software utilizing the Neighbor-Joining method and the Poisson model [33]. All protein sequences were aligned using MUSCLE in MEGA X software with UPGMA clustering and default gap penalties. Following alignment, divergent sequences were trimmed from the termini. The curated dataset was subjected to phylogenetic reconstruction in MEGA X via the Neighbor-Joining method under the following parameters: bootstrap analysis (1000 replicates), uniform substitution rates, and pairwise deletion for gap/missing data treatment.

2.5. Conserved Motif and Domain Analysis

Multiple conserved motifs of MdCDPK proteins were identified by the MEME Suite online tool [34]. MdCDPK protein sequences were submitted through the online portal, followed by selection of the ‘Classic Motif’ discovery model, while retaining all other parameters at default settings. MdCDPK motif analysis results are available for download upon completion through the web interface. The conserved domain of MdCDPK proteins was analyzed by the CDD from the NCBI databases. Consistent with the motif identification protocol, MdCPK protein sequences were submitted to the batch CD-search web platform using default parameters. Conserved domain analysis results were subsequently retrieved from the specified output directory.

2.6. Gene Expression Analysis

Total RNA was isolated from ‘Hanfu’ leaves or apple calli using a CTAB protocol [35]. Gene expression with RT-qPCR was performed as previously described with minor modifications [36]. RT-qPCR reactions were conducted with UltraSYBR Mixture (CWBIO) on an ABI QuantStudio™ 6 Flex system. Each 10 μL reaction contained 10 μL UltraSYBR mix, 1 μL gene-specific primers (10 μM), 0.5 μL cDNA template, and 3.5 μL nuclease-free water. The thermal cycling conditions were as follows: initial denaturation, 95 °C for 10 min; 40 cycles, 95 °C for 15 s, 60 °C for 1 min; melt curve analysis, 60–95 °C. The results were calculated by normalization to MdEF-1α (GenBank: DQ341381 in the NCBI). Specific primers are listed in Table S2. Each experiment was performed with 3 biological replicates.

2.7. Promoter Cis-Acting Element Analysis

The promoter region (2000 bp) of the MdCDPK24 gene was extracted from the ‘Hanfu’ apple genome. The MdCDPK24 promoter sequence was analyzed for cis-acting elements using the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 August 2025). The MdCDPK24 promoter sequence was inserted into the designated input field of the bioinformatics tool PlantCARE. The analysis request was submitted after providing a valid email address for automated result delivery. Cis-acting elements in the MdCDPK24 promoter were integrated from PlantCARE online tool analysis. The results are listed in Table S3.

2.8. Subcellular Localization Analysis

The CDS sequence of MdCDPK24 without the stop codon was cloned into the pRI101-GFP. Then, the pRI101-MdCDPK24-GFP construct was introduced into Agrobacterium tumefaciens EHA105. MdCDPK24-GFP and NF-YA4-mCherry (a nuclear marker) were expressed in 4-week-old N. benthamiana using a previously described protocol [37]. Briefly, EHA105 was cultured overnight at 28 °C in Luria–Bertani medium supplemented with 50 μg/mL kanamycin and 25 μg/mL rifampicin. Then, 1 mL of the culture was transferred to 50 mL of fresh Luria–Bertani medium and incubated with shaking until an optical density was reached (OD₆₀₀ = 0.5). The culture was harvested by centrifugation at 5000 rpm for 5 min and resuspended in infiltration buffer to an OD₆₀₀ of 0.5. Suspensions were infiltrated into the abaxial surface of N. benthamiana leaves using a blunt syringe. The fluorescence signal was obtained using a confocal microscope (Leica DMi8, Wetzlar, Germany).

2.9. Generation of Transgenic Apple Calli and Fungal Treatment

For overexpressing MdCDPK24 in ‘Orin’ calli, the full-length coding region of MdCDPK24 was amplified from ‘Hanfu’ leaves. Then, MdCDPK24 was cloned into the pRI101-AN to generate pRI101-MdCDPK24. The construct was transferred into A. tumefaciens EHA105 and then transformed into calli using the previously described method [37]. For C. gloeosporioides inoculation treatment, mycelia were inoculated onto wild-type (WT, ‘Orin’) and MdCDPK24-overexpressing transgenic calli. Following inoculation, calli were maintained in darkness at 25 °C. Disease symptoms were photographed at 10 days.

2.10. Transient Overexpression of Genes in Fruit

‘Hanfu’ fruits were brought from the market. The transiently transformed apple fruit was performed using a previously established method [37]. EHA105 culture refers to subcellular localization analysis. The culture was resuspended in infiltration buffer to an OD₆₀₀ of 0.3. Suspensions were infiltrated into apple fruit using a blunt syringe. Then, C. gloeosporioides mycelia were inoculated on the surface of apple fruits. Disease symptoms were photographed at 5 d.

2.11. Statistical Analysis

Statistical analysis in this study was conducted with SPSS software 26. Data are shown as means ± standard deviations. All datasets underwent normality testing and satisfied the criteria for normal distribution. Data appropriate for one-way ANOVA were evaluated using homogeneity tests. Significance of differences was determined using one-way ANOVA and Duncan’s test (RT-qPCR analysis of MdCDPKs during C. gloeosporioides infection) or Student’s t-test (RT-qPCR analysis of transgenic calli and analysis of apple bitter rot incidence following inoculation in apple). Different lower-case letters or asterisks indicate statistically significant differences (p < 0.05).

3. Results

3.1. Identification and Chromosomal Distribution of CDPK Gene Family in Apple

To identify CDPK genes in apple, an apple genome database (HFTH1 Whole Genome v1.0) was searched using 34 AtCDPK proteins as a query sequence. A total of 38 MdCDPK genes were systematically designated as MdCDPK1 to MdCDPK38 based on their chromosomal positions (Table S1). These genes exhibited open reading frame lengths ranging from 1488 to 5109 bp, corresponding to proteins of 495–1702 amino acids. The encoded proteins showed molecular masses of 55.63–190.26 kDa and predicted isoelectric points of 5.19–9.31. Chromosomal mapping revealed uneven distribution of all 38 MdCDPKs across 14 chromosomes (Figure 1). Chromosome 5 harbored the highest gene number (five), followed by chromosomes 10/12/14 (four each), chromosomes 2/3/7/15 (three each), chromosomes 6/9/11 (two each), and chromosomes 1/4/17 (one each).

3.2. Phylogenetic Analysis of MdCDPKs

To analyze the evolutionary relationship, we constructed a phylogenetic tree using 72 CDPK sequences from apple and A. thaliana (Figure 2). Based on branch topology alignment and conserved domains of AtCDPK proteins, the 38 MdCDPKs were phylogenetically classified into four subfamilies (I-IV). Subfamily I contained 11 CDPKs from apple (MdCDPK4, MdCDPK9, MdCDPK11, MdCDPK12, MdCDPK16, MdCDPK20, MdCDPK21, MdCDPK23, MdCDPK27, MdCDPK28, and MdCDPK31). Subfamily IV consisted of 10 apple CDPKs (MdCDPK1, MdCDPK2, MdCDPK3, MdCDPK10, MdCDPK17, MdCDPK18, MdCDPK22, MdCDPK32, MdCDPK36, and MdCDPK37). Subfamily III contained nine CDPKs from apple (MdCDPK8, MdCDPK6, MdCDPK15, MdCDPK19, MdCDPK26, MdCDPK29, MdCDPK30, MdCDPK34, and MdCDPK38). Subfamily II was the smallest group and comprised eight apple CDPKs (MdCDPK5, MdCDPK7, MdCDPK13, MdCDPK14, MdCDPK24, MdCDPK25, MdCDPK33, and MdCDPK35).

3.3. Conserved Motif and Domain Analysis of MdCDPKs

The conserved motif of 38 full-length MdCDPKs was analyzed using the MEME program (Figure 3a). All MdCDPK proteins shared conserved motifs 1 to 3. The conserved domain of 38 MdCDPKs was analyzed based on the Conserved Domain Database using the National Center for Biotechnology Information (NCBI) online website (Figure 3a). All MdCDPK proteins contained two conserved domains, including a STKc_CAMK domain (calcium- and calmodulin-stimulated Ser/Thr kinases domain). Most MdCDPKs possessed the PTZ00184 superfamily domain. MdCDPK2, MdCDPK31, and MdCDPK36 contained the FRQ1 domain. The PTZ00183 superfamily domain only existed in the MdCDPK12 protein.

3.4. Expression Analysis of MdCDPKs During C. gloeosporioides Infection

To elucidate the transcriptional dynamics of MdCDPKs following C. gloeosporioides infection, the expression patterns of MdCDPK genes in ‘Hanfu’ leaves treated with C. gloeosporioides were examined by RT-qPCR. Following RT-qPCR analysis of 38 MdCDPK genes, we selected 9 genes exhibiting altered relative expression during 0–48 h (Figure 4). The relative expression levels of MdCDPK3, MdCDPK8, MdCDPK25, and MdCDPK31 showed a slight downward trend within 48 h post-inoculation with C. gloeosporioides. Only MdCDPK24 exhibited significantly increased expression, progressively rising from 0 to 48 h. These results suggest that MdCDPK24 is closely associated with the C. gloeosporioides infection process, warranting further investigation into its potential role in pathogen response. To understand the potential functions of MdCDPK24, subcellular localization of MdCDPK24-GFP was investigated in Nicotiana benthamiana leaves. In N. benthamiana cells transfected with pRI101-GFP, GFP fluorescence was uniformly distributed throughout the cells. N. benthamiana leaves expressing MdCDPK24-GFP showed cytoplasm-specific fluorescence, demonstrating cytoplasmic localization of the MdCDPK24-GFP fusion protein (Figure 5).

3.5. MdCDPK24 Positively Regulates Apple Bitter Rot Resistance

To further investigate whether MdCDPK24 is involved in resistance to C. gloeosporioides, we conducted stable overexpression of MdCDPK24 (MdCDPK24-OE) in the ‘Orin’ calli. The relative expression of MdCDPK24 significantly increased in MdCDPK24-OE calli compared with the control (Figure 6a). We inoculated MdCDPK24-OE calli with C. gloeosporioides for 10 days and found that the overexpression of MdCDPK24 significantly reduced the mycelium growth (Figure 6b). We also performed transient MdCDPK24 overexpression tests in the fruits of ‘Hanfu’ apple. Following the transient overexpression of MdCDPK24, apple fruit was inoculated with C. gloeosporioides (Figure 6c). We found that the lesion diameters on MdCDPK24-overexpressing (pRI101-MdCDPK24) fruits were significantly smaller than those on empty vector (pRI101-AN) control fruits at 5 d (Figure 6d). Longitudinal section analysis revealed significantly shallower lesion depths in fruits expressing pRI101-MdCDPK24 compared to the control group at 5 d (Figure 6e,f). These results demonstrate that MdCDPK24 overexpression reduces C. gloeosporioides lesion expansion and suppresses pathogen development.

4. Discussion

CDPKs serve as primary calcium receptors and central regulators of plant development and stress responses. Utilizing genomic data from ‘Hanfu’ apple, we identified the CDPK gene family members. Phylogenetic analysis and conserved domain characterization revealed evolutionary relationships within this family. Pathogen inoculation with C. gloeosporioides induced MdCDPK24 expression. Subsequently, subcellular localization of MdCDPK24 was performed. We characterized the role of MdCDPK24 in conferring resistance to apple bitter rot caused by C. gloeosporioides.

Since the whole genome sequencing of multiple plant species has been completed, the CDPK gene family has been systematically identified, cloned, and analyzed in several plants. Genome-wide analyses have identified 34, 29, and 30 CDPKs in A. thaliana [12], rice [13], and pear [14]. In our study, we found 38 CDPKs in ‘Hanfu’ apple (Table S1). The apple, pear, rice, and A. thaliana genomes harbor comparable numbers of CDPK genes. Plants may require approximately 30 CDPK genes to transduce Ca²⁺ signaling cascades. The conserved domain is closely related to gene function. The conserved CDPK consists of three domains: the variable N-terminal domain, the central kinase catalytic domain, and the C-terminal CDPK activation domain [38]. In apple, CDPK proteins exhibit highly conserved amino acid sequences, particularly in their catalytic domain (Figure 3b). When CDPK is activated by Ca²⁺, the catalytic domain phosphorylates multiple substrates, propagating calcium signals downstream to initiate environmental adaptation responses. AtCPK12 phosphorylates two abscisic acid-responsive transcription factors (ABF1 and ABF4) to regulate seed germination and post-germination growth [39]. OsCPK17 regulates rice cold stress tolerance through the phosphorylation of sucrose–phosphate synthase OsSPS4 and aquaporins OsPIP2;1/OsPIP2;6, thereby modulating sugar metabolism and membrane channel activity [40]. StCDPK7 is transcriptionally activated during the Phytophthora infestans infection of potato and phosphorylates phenylalanine ammonia lyase proteins [41]. These studies suggest that CDPKs in apple potentially regulate both developmental processes and stress responses through substrate phosphorylation mediated by their conserved kinase domains.

CDPK genes are ubiquitously distributed in diverse plant species and play critical roles in mediating abiotic stress responses. ABA signaling orchestrates S-type anion channel SLAH3 activation via spatial regulation within plasma membrane nanodomains, where CPK21 phosphorylates SLAH3 but is antagonized by ABI1 phosphatase. The regulatory components of ABA receptor 1/pyrabactin resistance-like protein 9 (RCAR1/PYL9) modulate this CPK21-SLAH3-ABI1 complex assembly, demonstrating that nanodomain partitioning controls phosphorylation-dependent anion channel gating during drought stress responses [42]. CPK4 and CPK11 act as positive regulators in calcium-mediated abscisic acid (ABA) signaling, where ABA-induced kinase activity phosphorylates transcription factors ABF1/ABF4 to modulate ABA-responsive gene expression. Genetic evidence demonstrates that cpk4/cpk11 mutants exhibit ABA- and salt-insensitive phenotypes during germination and seedling development, establishing their essential role in whole-plant salt stress adaptation [43]. These findings establish that CDPKs are central regulators of drought and salt stress responses. Notably, ABA orchestrates key CDPK-mediated signaling cascades during plant stress adaptation. Analysis of the MdCDPK24 promoter identified multiple ABRE elements, indicating that this kinase may mediate ABA-responsive signaling during abiotic stress responses (Table S3). Moreover, we identified multiple methyl jasmonate (MeJA) response elements, critical for mediating plant responses to both biotic and abiotic stresses. Exogenous ABA and MeJA application may transcriptionally induce MdCDPK24 expression to enhance multi-stress resistance in apple. Optimal dosage for field-scale deployment requires systematic optimization.

A phylogenetic tree enables the inference of gene evolutionary histories and the prediction of gene functions [44]. Consequently, phylogenetic analysis constitutes a fundamental component of gene family research. A phylogenetic tree analysis of 38 MdCDPKs and 34 AtCDPKs showed that the MdCDPK proteins were classified into four subfamilies (Figure 2). Proteins grouped within the same subfamily typically share conserved motifs or domains, which indicates close evolutionary relationships. Therefore, MdCDPKs and AtCDPK homologs within the same phylogenetic clade likely exhibit conserved functions in environmental stress responses. MdCDPK24 clusters with AtCDPK3 in the same clade position. AtCPK3 phosphorylates actin-depolymerization factor 4 (ADF4) to remodel the actin cytoskeleton, thereby enabling stomatal immunity and resistance to pathogenic bacteria [45]. Phylogenetic tree analysis implicated MdCDPK24 in biotic stress responses, though functional validation remains necessary. Determining subcellular localization is fundamental to characterizing gene function [46]. In A. thaliana, CDPK isoforms display distinct subcellular localization, including cytoplasm, nucleus, cell membrane, and peroxisome membrane compartments [47]. In tomato, CDPKs are predominantly localized to the cytoplasm based on predictive analyses, with a minority exhibiting plasma membrane association [48]. Subcellular localization results showed that the protein of MdCDPK24 was a cytoplasmic localization protein (Figure 5). Multiple enzymes regulating plant growth and stress responses—including superoxide dismutase [49], catalase [50], POD, and CAD [51]—localize in the cytoplasm, thus creating spatial proximity conducive to functional interactions with CDPK.

CDPKs also mediate biotic stress signaling pathways in plants. CPK5 acts as a master regulator of plant immunity through phosphorylation of the NADPH oxidase and respiratory burst oxidase homolog D (RBOHD) upon pathogen-associated molecular pattern (PAMP) perception, initiating a self-propagating ROS-CPK5 signaling circuit. This mutual activation cascade enables rapid systemic defense, potentiating salicylic acid-dependent resistance against Pseudomonas syringae pv. tomato DC3000 and coordinating defense-related transcriptional reprogramming in distal tissues [52]. The E3 ubiquitin ligases ARABIDOPSIS TÓXICOS EN LEVADUR (ATL31 and ATL6) enhance plant immunity by targeting CPK28 for proteasomal degradation upon flg22 elicitation, thereby stabilizing the BOTRYTIS-INDUCED KINASE1 (BIK1). This ubiquitin-proteasome pathway attenuates CPK28-mediated BIK1 turnover, revealing a dual-layer regulatory mechanism for immune signal amplification [53]. Mutational analyses reveal that phosphorylation of CPK28 at Ser318 is essential for ATL31 binding and subsequent ubiquitination. Further analyses confirm that CPK28 dimerization drives intermolecular autophosphorylation, creating a self-regulating loop that controls kinase stability through the ATL31/6 degradation machinery [54]. VpCDPK9 and VpCDPK13 activate salicylic acid and ethylene signaling pathways to confer powdery mildew resistance in grapevine [55]. In apple, multiple CDPK genes were significantly up-regulated after Alternaria alternata inoculation [56]. The potential biological function of apple CDPK genes in plant biotic stress responses remains unknown. Our study demonstrates that MdCDPK24 transcription is induced by C. gloeosporioides infection in apple and also positively regulates plant disease resistance (Figure 4 and Figure 6). However, the potential role of MdCDPK24 in regulating apple defense against C. gloeosporioides via protein–protein interaction and phosphorylation requires further experimental validation. Taken together, although an apple bitter rot resistance gene has been identified, substantial efforts remain to bridge the translational gap between theoretical discovery and breeding applications. Further investigation is required to determine whether this gene confers broad-spectrum resistance against major apple pathogens.

5. Conclusions

In this study, genome-wide identification in apple revealed 38 CDPK genes that were unevenly distributed across 14 chromosomes. Phylogenetic analysis with Arabidopsis orthologs classified these into four subgroups (I-IV). Following C. gloeosporioides infection, the expression level of MdCDPK24 was induced, which indicates that it might participate in apple resistance to the bitter rot pathogen. Subcellular localization analysis confirmed cytoplasmic-specific localization of MdCDPK24. Functional analysis demonstrated that overexpression of MdCDPK24 enhances resistance to bitter rot in apple calli and fruit. These findings provide valuable knowledge of the MdCDPK gene family and establish MdCDPK24 as a candidate gene for the molecular breeding of bitter-rot-resistant apple cultivars.