GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.


Introduction
GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at the N-terminus. Over 1100 members of the GDSL family of esterases/lipases have been found in 12 fully sequenced plant genomes [1], and they have become highly attractive topics because of their recently discovered properties and functions. Several plant GELPs have been isolated, cloned, and characterized. Members of the GELP family are primarily involved in the regulation of plant development, morphogenesis, secondary metabolite synthesis, and defense response [2,3]. In Arabidopsis, GELP77 functions in pollen dissociation and fecundity [4], secondary metabolism [5,6], and immunity [7]. The overexpression of AtGDSL1 promotes resistance to Sclerotinia sclerotiorum in oilseed rape by modulating salicylic acid (SA)-dependent and jasmonic acid (JA)-dependent pathways, thereby increasing phosphatidic acid accumulation and activating downstream stress response pathways following Sclerotinia infection [8]. OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, providing novel insights into lipid function in plant immunity [9]. In addition, GDSL lipases play roles in plant development, seed germination, morphogenesis, and pathogen defense responses [10,11]. However, the enzymatic or biological functions of most GELPs remain unclear.
Host plants recognize pathogens and produce high levels of defense genes, ROS generation, and corpus callosum deposition to inhibit pathogen infections [43,44]. We detected H2O2 accumulation at the infection site using 3′-diaminobenzidine (DAB) staining. Histological examination revealed that H2O2 accumulation was dramatically higher at the infection site in Fuji leaves than in Gala leaves ( Figure 1C). Callose synthase 2-like was also upregulated in Fuji leaves during infection ( Figure 1D). The amounts of callose deposits were higher in Fuji leaves infected with the deletion mutant of effector Sntf2 than in those infected with W16 ( Figure 1E). Sntf2 has previously been shown to play an important role in inhibiting plant defense responses to C. gloeosporioides infection [45]. These results indicated that C. gloeosporioides induces the plant immune defense response during the infestation of Fuji apple leaves.   Host plants recognize pathogens and produce high levels of defense genes, ROS generation, and corpus callosum deposition to inhibit pathogen infections [43,44]. We detected H 2 O 2 accumulation at the infection site using 3 -diaminobenzidine (DAB) staining. Histological examination revealed that H 2 O 2 accumulation was dramatically higher at the infection site in Fuji leaves than in Gala leaves ( Figure 1C). Callose synthase 2-like was also upregulated in Fuji leaves during infection ( Figure 1D). The amounts of callose deposits were higher in Fuji leaves infected with the deletion mutant of effector Sntf2 than in those infected with W16 ( Figure 1E). Sntf2 has previously been shown to play an important role in inhibiting plant defense responses to C. gloeosporioides infection [45]. These results indicated that C. gloeosporioides induces the plant immune defense response during the infestation of Fuji apple leaves.

GELP1 Expression Is Upregulated in Fuji during Infection
GDSL lipases/esterases play important roles in plant development and disease defense [9,46]. The screening of the Fuji Y2H library revealed a potential interacting protein of the virulence effector Sntf2 that encodes a GDSL lipase/esterase-like protein (Gene ID: 103451100), which we named GELP1 (Figure 2A). The transient co-expression showed that the signal of GELP1-eGFP was co-localized in the chloroplasts with the signal of Sntf2 ∆sp -TagRFP ( Figure S2A). However, the qRT-PCR results showed that GELP1 expression was significantly upregulated in Fuji at 24 hpi ( Figure 2B). To determine the role of GELP1 in the infection, we analyzed its expression in Gala at different stages of infection. The results showed that GELP1 was downregulated during the infection ( Figure 2C). Semiquantitative RT-PCR analysis showed that the transcription levels of GELP1 were higher in Fuji leaves than in Gala leaves ( Figure 2D). GELP1 was induced by C. gloeosporioides infection in Fuji ( Figure 2D). These results indicated that GELP1 participated in the immune defense response of apple. compared with those from samples at 0 hpi. Lowercase letters indicate significant differences (p < 0.01). (E) Aniline blue staining to observe callose deposition on apple leaves at 24 hpi.

GELP1 Expression Is Upregulated in Fuji during Infection
GDSL lipases/esterases play important roles in plant development and disease defense [9,46]. The screening of the Fuji Y2H library revealed a potential interacting protein of the virulence effector Sntf2 that encodes a GDSL lipase/esterase-like protein (Gene ID: 103451100), which we named GELP1 (Figure 2A). The transient co-expression showed that the signal of GELP1-eGFP was co-localized in the chloroplasts with the signal of Sntf2 Δsp -TagRFP ( Figure S2A). However, the qRT-PCR results showed that GELP1 expression was significantly upregulated in Fuji at 24 hpi ( Figure 2B). To determine the role of GELP1 in the infection, we analyzed its expression in Gala at different stages of infection. The results showed that GELP1 was downregulated during the infection ( Figure 2C). Semi-quantitative RT-PCR analysis showed that the transcription levels of GELP1 were higher in Fuji leaves than in Gala leaves ( Figure 2D). GELP1 was induced by C. gloeosporioides infection in Fuji ( Figure 2D). These results indicated that GELP1 participated in the immune defense response of apple.

GELP1 Is a GDSL Lipase/Esterase-Like Protein
NCBI BlastP analysis revealed that another GDSL lipase/esterase-like gene (GELP2) in the Golden Delicious genome (Gene ID: 103434228) is 93.37% similar to GELP1 ( Figure  S3A). Golden Delicious was also susceptible to GLSA ( Figure S3B). GELP1 and GELP2 are encoded as 362-aa proteins containing a GDSL domain, and hydrolase activity acts on

GELP1 Is a GDSL Lipase/Esterase-Like Protein
NCBI BlastP analysis revealed that another GDSL lipase/esterase-like gene (GELP2) in the Golden Delicious genome (Gene ID: 103434228) is 93.37% similar to GELP1 ( Figure  S3A). Golden Delicious was also susceptible to GLSA ( Figure S3B). GELP1 and GELP2 are encoded as 362-aa proteins containing a GDSL domain, and hydrolase activity acts on ester bonds, as predicted by InterPro 85.0 ( Figure 3A). RT-PCR analysis showed that the expression of GELP2 in Gala and Fuji leaves was similar to that of GELP1 ( Figure 3B). However, GELP2 expression was upregulated in Gala leaves at 12 hpi and downregulated at 24 hpi. Furthermore, cis-element analysis revealed that the promoter region of GELP1 contained defensive and stress-responsive cis-elements and SA-responsive cis-elements (Table S1). GELP2 is mainly involved in light-responsive cis-elements but also contains abscisic acid-and methyl jasmonate-responsive cis-elements (Table S2). Thus, GELP2 and GELP1 may respond to different stresses.
contained defensive and stress-responsive cis-elements and SA-responsive cis-elements (Table S1). GELP2 is mainly involved in light-responsive cis-elements but also contains abscisic acid-and methyl jasmonate-responsive cis-elements (Table S2). Thus, GELP2 and GELP1 may respond to different stresses.
SignalP analysis revealed that GELP1 contains an N-terminal signal peptide (SP) (1-24 aa). To determine GELP1 localization, we constructed GELP1 (with or without SP) and GFP-fused expression vector and expressed the GELP1-eGFP protein using Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. The GELP1-eGFP fluorescent signal accumulated in the endoplasmic reticulum (ER) and chloroplasts ( Figure  3C). However, the GELP1 signaling peptide was not associated with its localization in N. benthamiana ( Figure 3C). Therefore, exploring the localization of GELP1 in the leaves of Fuji apple and the function of the protein is essential.

GELP1 Suppresses the Formation of Infection Structures
To determine the antimicrobial activity of GELP1 against C. gloeosporioides, we expressed recombinant His-GELP1 fusion proteins in Escherichia coli M15 cells ( Figure 4A). The purified His-GELP1 was determined through Western blotting ( Figure 4A). No significant difference in appressorium formation was observed between the groups treated with His-GELP1 and His (the blank control) at 12 h of co-incubation ( Figure 4B). However, the formation of infection hyphae was suppressed by His-GELP1 at 24 h of co-incubation ( Figure 4B). We also inoculated the incubated conidia on GL-3 leaves to analyze whether His-GELP1 induces local and systemic plant resistance. The co-incubation of apple leaves for 24 h inhibited the germination and formation of C. gloeosporioides and SignalP analysis revealed that GELP1 contains an N-terminal signal peptide (SP) (1-24 aa).
To determine GELP1 localization, we constructed GELP1 (with or without SP) and GFP-fused expression vector and expressed the GELP1-eGFP protein using Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. The GELP1-eGFP fluorescent signal accumulated in the endoplasmic reticulum (ER) and chloroplasts ( Figure 3C). However, the GELP1 signaling peptide was not associated with its localization in N. benthamiana ( Figure 3C). Therefore, exploring the localization of GELP1 in the leaves of Fuji apple and the function of the protein is essential.

GELP1 Suppresses the Formation of Infection Structures
To determine the antimicrobial activity of GELP1 against C. gloeosporioides, we expressed recombinant His-GELP1 fusion proteins in Escherichia coli M15 cells ( Figure 4A). The purified His-GELP1 was determined through Western blotting ( Figure 4A). No significant difference in appressorium formation was observed between the groups treated with His-GELP1 and His (the blank control) at 12 h of co-incubation ( Figure 4B). However, the formation of infection hyphae was suppressed by His-GELP1 at 24 h of co-incubation ( Figure 4B). We also inoculated the incubated conidia on GL-3 leaves to analyze whether His-GELP1 induces local and systemic plant resistance. The co-incubation of apple leaves for 24 h inhibited the germination and formation of C. gloeosporioides and reduced the formation of infection hyphae compared with the control ( Figure 4C). These results suggested that GELP1 inhibited the formation of infection hyphae. reduced the formation of infection hyphae compared with the control ( Figure 4C). These results suggested that GELP1 inhibited the formation of infection hyphae.

GELP1 Enhances Plant Resistance to C. gloeosporioides
To further analyze the function of GELP1, we constructed an overexpression transgenic plant. All transgenic lines were screened using kanamycin. Transgenic lines overexpressing GELP1 (OE-GELP1) were confirmed using qRT-PCR and RT-PCR. GELP1 expression was significantly higher in the transgenic lines than in GL-3 ( Figure 5A) and Fuji ( Figure 5B). Compared with GL-3, the transgenic lines showed a similar phenotype on Murashige and Skoog medium ( Figure 5C) and showed good growth rate and redifferentiation. The pathogenicity of W16 in OE-GELP1 leaves was also investigated. The disease index of the overexpression strain was significantly reduced compared with that of GL-3 ( Figure 5D). In addition, after inoculation with spore suspension for 72 h, disease spots appeared on GL-3, whereas the transgenic plants had no disease spots, indicating that the disease resistance of OE-GELP1 was significantly improved ( Figure 5E).

GELP1 Enhances Plant Resistance to C. gloeosporioides
To further analyze the function of GELP1, we constructed an overexpression transgenic plant. All transgenic lines were screened using kanamycin. Transgenic lines overexpressing GELP1 (OE-GELP1) were confirmed using qRT-PCR and RT-PCR. GELP1 expression was significantly higher in the transgenic lines than in GL-3 ( Figure 5A) and Fuji ( Figure 5B). Compared with GL-3, the transgenic lines showed a similar phenotype on Murashige and Skoog medium ( Figure 5C) and showed good growth rate and redifferentiation. The pathogenicity of W16 in OE-GELP1 leaves was also investigated. The disease index of the overexpression strain was significantly reduced compared with that of GL-3 ( Figure 5D). In addition, after inoculation with spore suspension for 72 h, disease spots appeared on GL-3, whereas the transgenic plants had no disease spots, indicating that the disease resistance of OE-GELP1 was significantly improved ( Figure 5E).
x FOR PEER REVIEW 7 of 16 No significant difference in appressorium formation was found between W16 and GL-3 leaves at 24 hpi ( Figure 6A). However, the infectious phase of OE-GELP1 was significantly suppressed compared with that of GL-3 ( Figure 6A). In addition, aniline blue staining showed higher amounts of callose deposits in the infection sites of OE-GELP1-1 and OE-GELP1-2 leaves than in those of GL-3 leaves at 24 hpi ( Figure 6B). qRT-PCR analysis showed that the expression of Callose synthase 2-like was higher in OE-GELP1-1 leaves than in GL-3 leaves ( Figure 6B). In addition, MdLAPX6 and MAPKKK3 were upregulated in OE-GELP1-1 and Fuji leaves ( Figure 6C). MAPKKK3 encodes a component of the MAPK pathway, and MAPKKK3/MAPKKK5 is required for basal resistance to Pto-DC3000 [47]. These results suggested that GELP1 overexpression increased resistance to C. gloeosporioides in GL-3. No significant difference in appressorium formation was found between W16 and GL-3 leaves at 24 hpi ( Figure 6A). However, the infectious phase of OE-GELP1 was significantly suppressed compared with that of GL-3 ( Figure 6A). In addition, aniline blue staining showed higher amounts of callose deposits in the infection sites of OE-GELP1-1 and OE-GELP1-2 leaves than in those of GL-3 leaves at 24 hpi ( Figure 6B). qRT-PCR analysis showed that the expression of Callose synthase 2-like was higher in OE-GELP1-1 leaves than in GL-3 leaves ( Figure 6B). In addition, MdLAPX6 and MAPKKK3 were upregulated in OE-GELP1-1 and Fuji leaves ( Figure 6C). MAPKKK3 encodes a component of the MAPK pathway, and MAPKKK3/MAPKKK5 is required for basal resistance to Pto-DC3000 [47]. These results suggested that GELP1 overexpression increased resistance to C. gloeosporioides in GL-3. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 16

GELP1 Was Related with SA Accumulation
Exogenous SA application in apple leaves enhances resistance to GLSA [48]. In the present study, we sprayed SA onto Fuji and Gala leaves. Under SA stress, GELP1 expression was upregulated in Gala leaves ( Figure 7A) but showed no change at 0 and 24 h in Fuji leaves ( Figure 7A). However, the GELP1 expression in Gala leaves was similar to that in Fuji leaves at 24 h under SA stress ( Figure 7A). In the present study, MdWRKY15 expression was only slightly upregulated in OE-GELP1-1 leaves compared with GL-3 leaves at 24 hpi, and it was also upregulated in Fuji leaves at 24 hpi ( Figure 7B). The

GELP1 Was Related with SA Accumulation
Exogenous SA application in apple leaves enhances resistance to GLSA [48]. In the present study, we sprayed SA onto Fuji and Gala leaves. Under SA stress, GELP1 expression was upregulated in Gala leaves ( Figure 7A) but showed no change at 0 and 24 h in Fuji leaves ( Figure 7A). However, the GELP1 expression in Gala leaves was similar to that in Fuji leaves at 24 h under SA stress ( Figure 7A). In the present study, MdWRKY15 expression was only slightly upregulated in OE-GELP1-1 leaves compared with GL-3 leaves at 24 hpi, and it was also upregulated in Fuji leaves at 24 hpi ( Figure 7B). The transcription factor MdWRKY15 activates the SA synthetase MdICS [49]. Furthermore, WRKY17 expression was downregulated in OE-GELP1-1 leaves than in GL-3 leaves at 24 hpi and upregulated in GL-3 leaves at 24 hpi ( Figure 7C). Moreover, WRKY100 expression was only slightly upregulated in OE-GELP1-1 leaves ( Figure 7D). MdWRKY100 and MdWRKY17 have been linked to SA accumulation [31,32]. Reducing SA accumulation helps apples infected with Colletotrichum fructicola [31]. These results indicated that GELP1 promoted plant resistance to C. gloeosporioides by increasing SA accumulation. transcription factor MdWRKY15 activates the SA synthetase MdICS [49]. Furthermore, WRKY17 expression was downregulated in OE-GELP1-1 leaves than in GL-3 leaves at 24 hpi and upregulated in GL-3 leaves at 24 hpi ( Figure 7C). Moreover, WRKY100 expression was only slightly upregulated in OE-GELP1-1 leaves ( Figure 7D). MdWRKY100 and MdWRKY17 have been linked to SA accumulation [31,32]. Reducing SA accumulation helps apples infected with Colletotrichum fructicola [31]. These results indicated that GELP1 promoted plant resistance to C. gloeosporioides by increasing SA accumulation. qRT-PCR analysis of WRKY100 expression in OE-GELP1 and Fuji. The vertical axis represents the relative fold-changes of transcripts compared with GL-3 or 0 hpi samples. MdUBQ was used as the reference gene. Lowercase letters indicate significant differences (p < 0.01).

C. gloeosporioides causes semi-in vitro nutrient infection on the susceptible cultivar,
Gala, and mismatch or latent infection in vitro on the resistant cultivar, Fuji [50]. In the present study, in infected Fuji leaves, the formation of infection hyphae was inhibited, and the H2O2 accumulation and callose deposition increased. In addition, we identified a GDSL esterase/lipase At5g33370-like protein, GELP1, and found that its expression was significantly upregulated during Fuji infection. GDSL plays a role in plant growth and development, disease resistance, and lipid synthesis. Arabidopsis At5g33370 encodes the protein AtGELP95 (CUS2), which is involved in cutin polymer formation and cuticle permeability [51]. Tomato lipase GDSL1 is embedded in the cuticle matrix outside the epithelial cells and is associated with epidermal thickness, cuticle density, and resistance [52,53]. The GDSL esterase/lipase OsGELP78 modulates lipid metabolism to regulate rice resistance to disease [54]. In the present study, we found that GELP1 was highly similar to the LTL1-like GDSL esterase/lipase protein, whereas Arabidopsis thaliana Li-tolerant lipase 1 (AtLTL1) expression was activated by SA, suggesting that this lipase may also be involved in pathogen defense [55]. GELP1 overexpression increased apple resistance to C. gloeosporioides in GL-3. Callose deposits increased, and the expression of Callose synthase

Discussion
C. gloeosporioides causes semi-in vitro nutrient infection on the susceptible cultivar, Gala, and mismatch or latent infection in vitro on the resistant cultivar, Fuji [50]. In the present study, in infected Fuji leaves, the formation of infection hyphae was inhibited, and the H 2 O 2 accumulation and callose deposition increased. In addition, we identified a GDSL esterase/lipase At5g33370-like protein, GELP1, and found that its expression was significantly upregulated during Fuji infection. GDSL plays a role in plant growth and development, disease resistance, and lipid synthesis. Arabidopsis At5g33370 encodes the protein AtGELP95 (CUS2), which is involved in cutin polymer formation and cuticle permeability [51]. Tomato lipase GDSL1 is embedded in the cuticle matrix outside the epithelial cells and is associated with epidermal thickness, cuticle density, and resistance [52,53]. The GDSL esterase/lipase OsGELP78 modulates lipid metabolism to regulate rice resistance to disease [54]. In the present study, we found that GELP1 was highly similar to the LTL1-like GDSL esterase/lipase protein, whereas Arabidopsis thaliana Li-tolerant lipase 1 (AtLTL1) expression was activated by SA, suggesting that this lipase may also be involved in pathogen defense [55]. GELP1 overexpression increased apple resistance to C. gloeosporioides in GL-3. Callose deposits increased, and the expression of Callose synthase 2-like and MdLAPX6 was also upregulated. Apxs plays a role in scavenging ROS and protecting cells. In the present study, GELP1 is also involved in biotic stress responses.
The enzyme responsible for TAP in E. coli is a multifunctional GDSL esterase/lipase that functions as a thioesterase, protease, arylesterase, lysophospholipase, and esterase [56,57]. GDSL hydrolases have a flexible active site and switch conformation when different substrates are present, and some GDSL enzymes have a wide range of enzymatic activities, including esterase and protease activities in the same enzyme [58,59]. AtLTL1 and SlGDSL1 are orthologous extracellular acyltransferases in Arabidopsis and tomato, respectively, which catalyze the formation of line cuttings in oligomers using 2-MHG in vitro [52,60]. The recombinant GLIP1 protein possesses lipase and antimicrobial activities, which directly disrupt the integrity of fungal spores [9,61]. GLIP2 also possesses lipase and antimicrobial activities that inhibit the germination of fungal spores [62]. In the present study, recombinant GELP1 inhibited the formation of C. gloeosporioides infected hyphae. Plant GDSL lipase/esterase GLL25 is required for normal ER morphology and regular organelle distribution, and lipase/esterase activity is lost without a conserved active site of the GDSL motif [63].
Chloroplasts are important energy-converting cells in plants. In addition to photosynthesis, it plays a role in the synthesis of amino acids, fatty acids, plant growth substances, nucleotides, vitamins, and secondary metabolites [64]. Chloroplasts can be targeted as effectors, such as bacterial effectors HopI1, HopK1, and AvrRps4, which inhibit chloroplastderived ROS or SA [65,66]. In the present study, a small fraction of GELP1-eGFP fluorescence was observed in the chloroplasts. Lipids are widely distributed in plants and are thought to be involved in regulating plant immunity [9]. DGDG are abundant galactolipids present in the thylakoid membranes and photosynthesis systems I and II [67]. Interestingly, DGDG contributes to the biosyntheses of NO and SA, which are involved in defense responses [6]. In the present study, WRKY genes were upregulated in the GELP1 overexpression transgenic lines, which was related to SA accumulation. We speculate that the chloroplast location is linked to SA biosynthesis.
The defense responses of plants to pathogens are regulated by networks of SA, JA, and ET signaling pathways [68]. In Arabidopsis, PRLIP1 induction depends on a functioning SA and ET signal transduction pathway [69]. GLIP1 responds to SA and functions in resistance to Alternaria brassicicola [61]. It directly targets MPK3/MPK6 and its downstream transcription factor WRKY33. Arabidopsis GDSL lipase 1 (GLIP1) requires ET signaling components for its expression and regulates systemic immunity via the positive (i.e., ERF1 activation) and negative (i.e., EIN3 degradation) feedback regulation of ET signaling [70]. In the present study, GELP1 overexpression indirectly upregulated the expression of WRKY genes, which are associated with SA generation. GELP1 expression was also upregulated by the external application of SA. In another study, the overexpression of the GDSL motif lipase/hydrolase-like protein GLIP1 or treatment with exudates from 35S: GLIP1 plants induce systemic pathogen resistance, and this effect has been associated with JA/ET and SA [61]. In the present study, GELP1 overexpression increased resistance to C. gloeosporioides. In our next work, we will investigate whether GELP1 can elicit a broad spectrum of resistance to multiple pathogens.
WRKY transcription factors play important roles in resistance to various fungal diseases in apples [71,72]. MdWRKY100 positively regulates resistance to C. gloeosporioides infection [32]. RNAi-mediated silencing of MdWRKY17 in Gala plants promotes GLS tolerance, and MdWRKY17 negatively regulates SA levels, leading to GLS susceptibility [31]. Other studies have shown that SA is critical for GLS tolerance [48]. In plants, SA levels are primarily determined by the balance between SA biosynthesis (mainly controlled by ICS1) and degradation [73]. The transcription factor MdWRKY15 activates the SA synthetase MdICS1 [49]. SA confers enhanced resistance to GLSA [48]. In the present study, GELP1 overexpression increased resistance to C. gloeosporioides. The expression of MdWRKY15 and MdWRKY100 was upregulated, whereas that of MdWRKY17 was downregulated in the GELP1 overexpression transgenic lines. These findings highlight that SA plays a role in regulating resistance to C. gloeosporioides infection and that GELP1 contributes to this resistance by enhancing SA accumulation.

Materials and Growth Conditions
W16 was isolated and purified by our group [74] GL-3 (Royal Gala) plants were supplied by Professor Zhihong Zhang [75]. Table S3 lists the primers used in the present study. W16 was cultured on PDA agar plates at 25 • C in the dark. Transgenic lines overexpressing GELP1 (OE-GELP1) and GL-3 plants were grown on Murashige and Skoog medium in a climate-controlled culture room at 25 ± 1 • C with a light/dark photoperiod of 16/8 h [75,76]. Seedlings of N. benthamiana were grown in a greenhouse at 22-25 • C and 75% humidity. Healthy leaves of Fuji and Gala were obtained from seedlings in our research orchard (Institute of Pomology of Chinese Academy of Agricultural Sciences, CAAS, Xingcheng, Liaoning Province, China).

Phenotypic Analysis
Fresh conidial suspensions were harvested from the surface of PDA cultures (9 cmdiameter plates) with 10 mL of sterile distilled water. The conidial suspensions were centrifuged at 5000 rpm, resuspended in normal saline, and then washed three times. For plant inoculation, fresh conidial suspensions (1 × 10 6 conidia/mL) were sprayed onto the leaves. The inoculated apple leaves were grown at 28 • C with a humidity of 75%. The formation of fungal appressoria and infection hyphae in the leaves was assessed via light microscopy after discoloration by boiling with 95% anhydrous ethanol and transparency using chloral hydrate. Pathogenicity assays were performed by examining disease lesions at 3 dpi. The proportion of diseased lesions (lesion area/leaf area) was graded in accordance with the following scale: 1: ≤20%; 3: >20%, ≤40%; 5: >40%, ≤60%; 7: >60%, ≤80%; 9: >80%. The disease index was expressed as ∑ (number of lesions × associated lesion grade)/(total number of lesions × largest lesion grade).

Histochemical Assays
DAB staining was performed as previously described to evaluate H 2 O 2 production in plants [77]. Samples of inoculated leaves were collected at different stages of infection. DAB oxidation leads to the formation of a yellow polymer at the site of H 2 O 2 accumulation. Callose deposits were detected via aniline blue staining. Samples were obtained from WT-inoculated leaves, which were decolorized by boiling in 95% ethanol for 5 min, and then dipped in chloral hydrate to make them transparent. The processed leaf segments were stained with 0.05% aniline blue in 0.067 M K 2 HPO 4 (pH 9.6) [77]. For microscopy, the samples were preserved in 40% glycerol. The experiments were conducted at 0, 24, 36, and 48 hpi.

RNA Extraction and RNA Analyses
Total RNA was isolated from apple leaves using an RNA Prep Pure Plant Kit (TianGen Biotech, Beijing, China) in accordance with the manufacturer's protocol. First-strand complementary DNA (cDNA) was synthesized using a Two-Step RT-PCR SuperMix kit (TransGen Biotech). RT-PCR analysis of 100 ng total RNA was performed using RT-PCR SuperMix (TransGen Biotech) for the detection of GELP1 and MdUBQ. 5 The assay was conducted in triplicate. qRT-PCR was performed using the Tip Green qPCR SuperMix (TransGen Biotech). All of the qRT-PCR products were analyzed using Bio-Rad CFX Manager 3.0 software. Gene expression was measured by normalizing the expression values to those obtained from conidia or non-inoculated apple leaves using the β-tubulin gene as an endogenous reference. Each sample and the entire experiment were performed in three separate triplicates.

Yeast Two-Hybrid Assay
The coding sequences of SNTF2 were cloned into pGBKT7-BD as bait, and the Matchmaker GAL4 system (Shanghai OE Biotech, Shanghai, China) was used to screen a library of cDNA constructed from RNA isolated from infected Fuji leaves. The screening was performed in accordance with the manufacturer's instructions (Shanghai OE Biotech). The prey vector pGBKT7-GELP1 was constructed using the GELP1 cDNA from Fuji leaves to confirm the interaction between Sntf2 and GELP1. The corresponding prey and bait vectors were co-transformed into the Y2HGold strain. The transformed yeast strains were grown on a medium (SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade) at 30 • C for 3 days to select the positive transformants.

Transient Expression Analysis in N. benthamiana
For the subcellular localization assay, GELP1 was cloned into pGR35s-eGFP and transformed into Agrobacterium tumefaciens LBA4404. The transformed strains were resuspended in infiltration buffer (10 mM MgCl 2 , 10 µM AS, 10 mM 2-(N-morpholino) ethanesulfonic acid, pH 5.6) at OD 600 = 0.3 and injected into 4-week-old N. benthamiana leaves. The empty vector pGR35s-eGFP served as a positive control. After 2 days of infiltration, the injected leaves were observed with an epifluorescence microscope (Leica TCS SP8). For GFP fluorescence detection, the excitation and emission wavelengths were 488 and 495-545 nm, respectively.

Protein Extraction and Western Blot Analysis
GELP1 was cloned into the pQE30 vector and transformed into E. coli M15 cells. Transformed cell suspension (OD600 = 0.80) was cultured, and protein expression was induced by 100 µM isopyl β-D-thiogalactopyranoside at 180 rpm for 3 h at 37 • C. Induced samples were centrifuged at 10,000 rpm for 5 min, suspended with 1 × PBS (sodium phosphate buffer) (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3) with lysozyme and proteinase inhibitor, and then disrupted via sonication. After centrifugation at 12,000 rpm for 30 min at 4 • C, the supernatant was purified using Ni-NTA beads (CWBIO, CW0894S) in accordance with the manufacturer's protocol. The induced proteins were detected through SDS-PAGE and Coomassie Blue staining. For immune detection, the purified proteins were transferred onto polyvinylidene fluoride membranes and detected using the corresponding mouse anti-His antibody (1:1000; cat. no. HT501; TRANS, China) with secondary horseradish peroxidase-labelled goat anti-mouse IgG (1:2000; cat. no. A0216, Beyotime, Shanghai, China). Images were acquired using a Bio-Rad ChemiDoc imaging system.

Chemical Treatment
SA (Fluka) was dissolved in water (1 mM SA, 0.01% Tween 20) and sprayed onto the leaves (1 mL/plant). GELP1 transcript levels were measured through RT-PCR at 0, 12, 24, 48, and 72 h after treatment with 1 mM SA. Leaves of uniform size to the control were sprayed with distilled water. The antimicrobial activity of GELP1 was determined by adding recombinant proteins (0.5, 1, and 3 µg) to 10 µL of conidia suspension in (1 × 10 6 ), and the onion epidermis and leaf surface were inoculated at 25 • C. The formation rates of appressoria and infection hyphae were detected using light microscopy.

Generation of Transgenic GL-3 Plants with GELP1 Over-Expression
The GELP1 coding sequence was amplified and inserted into the pRPHA vector, which was then used to transform LBA4404 cells. The transformed strains were used to generate the overexpression transgenic line OE-GELP1. The Agrobacterium-mediated transformation of GL-3 was conducted as previously described [75].

Conclusions
We identified the GDSL esterase/lipase protein GELP1 and found that its expression was significantly upregulated in Fuji during C. gloeosporioides infection. GELP1 accumulated in the ER and chloroplasts and inhibited the formation of infection hyphae. GELP1 overexpression increased resistance to C. gloeosporioides. In addition, GELP1 was involved in SA biosynthesis. These results indicate that GELP1 contributes to C. gloeosporioides resistance by enhancing SA accumulation. This study provides insights into the mechanisms underlying GLSA resistance in apple and may serve as a basis for developing novel strategies to control the disease.