Targeting the Effector AwCES to Attenuate Virulence in the Postharvest Pathogen Aspergillus westerdijkiae

Abstract

Aspergillus westerdijkiae is a common pathogenic fungus responsible for postharvest fruit rot in pears, causing substantial economic losses. This fungus also produces ochratoxin A (OTA), which poses serious health risks to humans. During host colonization, fungal pathogens secrete effectors to facilitate invasion. Under host-mimicking culture conditions, transcriptomic analysis of A. westerdijkiae at 24 and 72 h post-inoculation (hpi), combined with signal peptide prediction, identified 272 and 214 up-regulated secreted protein-encoding genes, respectively. Among these, a carboxylesterase gene, AwCES, was found to be significantly up-regulated. Compared to the wild-type strain, deletion of AwCES resulted in reduced conidial production and germination rate. Further studies revealed that the deletion mutant showed significantly attenuated virulence on pear fruit. Moreover, the loss of AwCES impaired fungal adaptation to stress environments. Collectively, these findings demonstrate that AwCES plays a critical role in the growth, development, and pathogenicity of A. westerdijkiae.

1. Introduction

Pear (Pyrus bretschneideri) was one of the earliest domesticated fruits in the history of human agriculture and is the third most important temperate fruit in the world, after citrus and apple [1]. The fruit is characterized by its distinct aroma, agreeable taste, crisp and juicy texture, tender flesh, and relatively low pectin content. It is also nutritionally valuable, containing organic acids, vitamins, phenolic compounds, carotenoids, potassium, flavonoids, and dietary fiber, which contribute to both its culinary and potential health-beneficial properties [2]. However, after harvest, pears are highly susceptible to microbial infections under natural conditions, resulting in flesh softening, quality degradation, and compromised storability, thereby greatly restricting the economic benefits of the pear industry [3]. Control of pear fruit diseases is critically contingent upon elucidating the pathogenicity mechanisms of fungal pathogens and developing effective management strategies [4]. Consequently, understanding these pathogenic mechanisms constitutes an essential measure to safeguard the sustainable development of the pear industry. Aspergillus westerdijkiae, beyond its known infectivity in cereals and nuts, serves as a predominant postharvest pathogen affecting fruits including grapes, pears, and oranges [5,6]. Notably, this species was historically misclassified as Aspergillus ochraceus for an extended period. Postharvest diseases instigated by pathogenic fungi represent prevalent phytopathological conditions, with certain species concurrently producing mycotoxins during host colonization, thereby posing dual threats to agricultural commodities and human health [7]. Therefore, further exploring the virulence determinants and understanding the pathogenic molecular mechanisms of A. westerdijkiae are imperative for developing novel strategies to control plant diseases caused by this fungal pathogen [8].

Effector proteins, secreted by phytopathogens, are small molecular entities that modulate host cellular architecture and molecular functions to facilitate infection while concurrently eliciting plant defense responses [9]. Notably, fungal effector proteins typically lack conserved amino acid sequences [10]. Synthesized in the endoplasmic reticulum, these effectors traverse the ribosome/Golgi pathway and undergo vesicular transport for extracellular secretion, involving SNARE-mediated membrane fusion and exocytosis [11]. Upon secretion, effectors predominantly target the plant cell wall, which serves as a crucial physical barrier against microbial invasion. The majority of phytopathogenic fungi release cell wall-degrading enzymes (CWDEs) to penetrate this barrier during the process of host colonization [12]. Paradoxically, while phytopathogenic fungi employ CWDEs to disrupt plant cell wall integrity and promote invasion, the resulting degradation products serve not only as nutrients for the pathogen but also as pathogen-associated molecular patterns (PAMPs) that trigger PAMP-triggered immunity (PTI) in the host [13]. Chen et al. [14] demonstrated that effector PfAvr4-2 binds low-methyl-esterified pectin in the plant middle lamella, disrupting Ca²⁺-mediated cross-linking of homogalacturonan chains to weaken wall cohesion. Similarly, Verticillium dahliae deploys pectinase VdPEL1 and cutinase VdCUT11 as PAMPs to activate host defenses during infection [15]. Immune evasion represents a critical strategy for phytopathogenic fungi to circumvent host defenses. Fungal-secreted effectors achieve this by targeting core plant defense components and modifying PAMPs to avoid immune recognition [16]. Notably, LysM domain-containing effectors from phytopathogenic fungi bind chitin and peptidoglycan in plant cell walls, thereby suppressing chitin-triggered PTI. Exemplifying this mechanism, the LysM effector Slp1 from Aspergillus oryzae sequesters chitin oligomers, inhibiting chitin-induced immunity in rice [17]. Similarly, Takahara et al. [18] testified that effectors ChELP1 and ChELP2 bind chitin/chitin oligosaccharides to suppress MAPK activation during chitin-triggered immune responses in plants. Fusarium graminearum effector CgEP1 functions as a DNA-binding protein that non-specifically associates with maize genomic DNA during infection, thereby disrupting expression of immunity-related genes [19]. Han et al. [20] identified effector CcCAP1 as a dual-localized protein (cytoplasmic/nuclear) whose nuclear targeting and CAP domain primarily mediate suppression of host immunity. Furthermore, certain effectors subvert defense-related gene expression by interacting with transcription factors. As confirmed by Qi et al. [21], effector PpEC23 physically binds soybean transcription factor GmSPL12l to inhibit immune responses.

Carboxylesterases (CESs) are archetypal α/β-hydrolases featuring a conserved Ser-His-Glu/Asp catalytic triad, hydrolyze ester and amide bonds. Disruption of the Clt1 gene in Curvularia lunata, which regulates the synthesis of acetylxylan esterase and xylanase, thereby compromised xylan utilization. This led to a deficit in acetyl-CoA (essential for melanin precursors), with a consequent severe reduction in conidial production under xylan-based culture conditions [22]. Pamil et al. [23] demonstrated that knockout of kinesin-encoding KLP-7 in Botrytis cinerea attenuated pectin methylesterase activity, inducing hyperbranching, hyphal surface invagination, delayed conidial germination, and reduced germination rates versus wild-type. Cumulatively, esterase family proteins mediate critical regulatory roles in phytopathogenic fungi, governing secondary metabolite biosynthesis and developmental processes including sporulation and germination.

While effector biology in phytopathogenic fungi has garnered substantial research attention as a focal area in plant pathology, current investigations predominantly center on preharvest pathogens. Research on effectors from postharvest fungal pathogens remains markedly underserved, with studies addressing A. westerdijkiae effectors constituting a near terra incognita. This study employs transcriptomic profiling of A. westerdijkiae under host-mimicked conditions to systematically identify candidate effectors and conduct functional interrogation. Specifically, we elucidate the role of carboxylesterases (CESs) during host colonization, providing mechanistic insights for developing novel control strategies against postharvest pear pathology.

2. Materials and Methods

2.1. Experimental Strains

The strain A. westerdijkiae fc-1 was kindly provided by the Chinese Academy of Agricultural Sciences and maintained on a potato dextrose agar (PDA) plate in the dark at 28 °C. For conidia preparation, 10 mL of sterile distilled water was added to the one-week-old PDA plate, and conidia were collected by scraping the plates followed by filtration to remove the mycelium. The collected conidia were then resuspended in sterile distilled water to a final concentration of 10⁸ conidia/mL.

2.2. RNA-Seq and Effector Genes Prediction

Pear Juice Medium Preparation: Uniform pears at equivalent ripeness without surface damage were juiced. The crude juice was filtered through a quadruple-layer gauze, supplemented with pectinase (0.1% w/v), and incubated at 45 °C for 90 min. Enzymatic activity was terminated by boiling for 10 min. The solution was centrifuged at 10,000 rpm for 10 min to remove precipitates, and the pH adjusted to match Czapek–Dox medium. Under aseptic conditions, fresh spores of A. westerdijkiae were harvested from PDA plates by gentle swabbing with sterile cotton tips followed by aqueous suspension. The suspension was filtered through a triple-layer sterile gauze, and the filtrate centrifuged at 3000 rpm for 3 min to obtain spore pellets. Pellets were resuspended in 300 μL distilled water, quantified via hemocytometer, and adjusted to 1 × 10⁸ spores/mL. Aliquots were inoculated into Czapek–Dox medium and pear juice medium, then incubated at 28 °C with 180 rpm agitation. Spores from pear juice medium at 24 h (LP1) and 72 h (LP3), alongside Czapek–Dox controls (CS1/CS3), were collected, washed twice with 0.2 M PBS, and centrifuged at 4000 rpm for 5 min to eliminate residual liquid. For each condition, total RNA from four independent biological replicates was pooled with equal proportion, and a single sequencing library was constructed. Consequently, the design does not include independent biological replicate libraries for variance-based statistical testing.

Total RNA was extracted with a Trizol RNA Extraction Kit (SK1321). The integrity of the isolated RNA was verified by agarose gel electrophoresis, while its concentration was determined using a Qubit RNA Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). RNA specimens passing quality control were subjected to RNA-Seq analysis (Sangon Biotech, Shanghai, China). The libraries were subjected to sequencing on the HiSeq 2500 platform (Illumina, San Diego, CA, USA). After removing low-quality reads, adaptor sequences, and reads with N (uncertain bases) exceeding 10%, the remaining reads were assembled using the Trinity v2.15.0 platform to reconstruct a Unigene library.

Differentially expressed genes (DEGs) were screened using Cuffdiff v2.2.1 with thresholds of |log₂ (Fold Change)| > 2 and a false-discovery rate (FDR) of p < 0.05. Functional annotation of these DEGs was subsequently performed through GO enrichment analysis using Blast2GO and KEGG pathway mapping via the KEGG Automatic Annotation Server (KAAS v2.1; http://www.genome.jp/kegg/kaas/, accessed on 10 June 2023). In silico prediction of secreted protein genes was performed according to Ökmen et al. [24]. In brief, a sequential bioinformatic pipeline was employed to predict secreted proteins of A. westerdijkiae. Putative secreted proteins were first identified using SignalP-5.0 (https://services.healthtech.dtu.dk/service.php/SignalP-5.0, accessed on 10 June 2023) based on the presence of N-terminal signal peptides. These candidates were subsequently filtered using TargetP-2.0 (https://services.healthtech.dtu.dk/service.php/TargetP-2.0, accessed on 10 June 2023) and DeepTMHMM-1.0.19 (https://dtu.biolib.com/DeepTMHMM, accessed on 10 June 2023) to exclude proteins predicted for mitochondrial localization or containing transmembrane helices.

2.3. Quantitative Real-Time PCR

Total RNA was reverse-transcribed into cDNA using a AMV First Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China), according to the manufacturer’s instructions. qRT-PCR was performed on a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA) utilizing SYBR Green Fast qPCR Master Mix (Sangon Biotech, Shanghai, China). The amplification procedure was set as follows: 3 min at 95 °C followed by 45 cycles at 95 °C for 5 s, 60 °C for 30 s, and 75 °C for 30 s, respectively. The gene expression levels were determined by the comparative Ct (2^−ΔΔCt) method with 18s-rDNA as the reference gene, and each sample was examined in triplicate [25].

2.4. Construction of Deletion Mutant

The AwCES deletion mutant (ΔAwCES) was generated by homologous recombination according to Zhang et al. [26]. Approximately 900 bp upstream and 1100 bp downstream fragments of the AwCES gene were amplified from the wild-type strain A. westerdijkiae genomic DNA using CES-UF/CES-UR and CES-DF/CES-DR primer pairs, respectively. The hygromycin B-resistant gene (hyg) fragment gene was amplified from the pCAMBIA-1300 plasmid using the hyg-F/hyg-R primer pair. The three fragments were joined to generate the gene deletion cassette by overlap extension PCR using the primer pair CES-UP/CES-DOWN. The amplified products were transformed into the A. westerdijkiae protoplast using a polyethylene glycol (PEG)-mediated transformation. The resultant deletion transformants were screened on PDA plates containing 120 μg/mL hygromycin, and PCR was performed using specific primers to confirm the transformants. All primers used in this experiment are listed in Table S1.

2.5. Fungi Morphology and Growth Assays

To evaluate the radial growth of the wild-type and ΔAwCES strains, 20 μL of conidial suspension (1 × 10⁸ conidia/mL) was inoculated onto the center of PDA plates and incubated at 28 °C in the dark, and colony diameters were measured at 7 d after inoculation. Conidia on each plate were collected by washing with distilled water and counting with a hemacytometer. Conidial morphology was observed under a light microscope (Eclipse E200, Nikon, Tokyo, Japan). A sterilized cover glass was inserted into the PDA plate at an angle of 45°, and 10 μL of conidial suspension (10⁸ conidia/mL) was inoculated at the point where the coverslip met the medium mycelial morphology was observed under a light microscope after 5 d incubation.

For the conidiation assay, spore suspensions of A. westerdijkiae WT and ΔAwCES were prepared as described previously and adjusted to a concentration of 1 × 10⁷ spores/mL. Aliquots (100 μL) were spread evenly on PDA plates and incubated at 28 °C for 7 days. Plates were washed with 40 mL sterile water, filtered through three layers of lens paper, and the filtrate was diluted 10-fold. A 10 μL aliquot of the diluted spore suspension was loaded onto a hemocytometer, and spores were counted under an optical microscope. Each experiment included three biological replicates. Sporulation per unit area was calculated as total spore count divided by colony area.

For the conidia germination assay, 0.5 mL of conidial suspension (1 × 10⁸ conidia/mL) was inoculated into 50 mL of PDB medium containing 0.01% Tween-20 and incubated at 28 °C for 10 h. The number of germinated conidia was counted under a microscope to calculate the germination rate. Conidia were considered to be grown when the germ tube length exceeded the conidia diameter.

2.6. Stress Assay

To determine the role of the AwCES gene in response to various stresses, the wild-type and ΔAwCES strains of A. westerdijkiae were cultured on PDA plates containing different chemicals including osmotic stress reagents (0.5 M NaCl and 0.5 M KCl), oxidative stress reagent (10 mM H₂O₂), and cell wall stress reagents SDS and Congo red at concentrations of 400 μg/mL [27]. To assess growth inhibition, colony diameters were quantified after 7 d of incubation at 28 °C, and the percentage of radial growth inhibition was calculated.

2.7. Virulence Assay

Pear fruits were surface disinfected with 3% NaClO for 3 min and rinsed with sterile distilled water three times. Uniform wounds (3 × 3 mm) were made on the equator of pear fruits. Then, 20 μL of conidial suspension (1 × 10⁷ conidia/mL) of the wild-type and ΔAwCES strains was inoculated into each wound and then kept in plastic baskets sealed with a sheet of transparent plastic film to maintain a relative humidity of 90% at a storage temperature of 28 °C. The severity of the disease was determined by measuring lesion diameters during the storage for 7 d. CK (Control) refers to the blank control group, which was inoculated with an equal volume of sterile water. For each strain, pathogenicity tests were performed using 3 pear fruits per experiment, and the entire experiment was repeated three times independently.

2.8. Scanning Electron Microscopy (SEM)

Spore suspensions of A. westerdijkiae wild-type and ΔAwCES mutant prepared by method 2.1 were inoculated into mechanically wounded pears. Pear tissues were sampled at 0, 3, 6, 9, and 12 h post-inoculation. Samples were fixed overnight at 4 °C in 2.5% glutaraldehyde within 48-well plates under light-protected conditions. Post-fixation, glutaraldehyde was discarded and samples were washed twice with 0.2 M phosphate-buffered saline (PBS), with each incubation lasting 15 min. Ethanol gradient dehydration was performed sequentially (30, 50, 70, 80, 90, and 100%), with 15 min immersions per concentration [28]. Residual ethanol was aspirated, plates sealed with parafilm punctured by needle vents, and evaporated completely. Samples were pre-frozen at −20 °C for 4–6 h followed by −80 °C overnight, then lyophilized for 36 h until complete desiccation. Freeze-dried specimens were mounted on conductive stubs, sputter-coated with gold, and examined by SEM.

2.9. Ochratoxin A (OTA) Production Assay

HPLC analysis of OTA was performed according to our previously described method with minor modifications [29,30]. Spore suspensions of A. westerdijkiae wild-type and ΔAwCES were adjusted to 1 × 10⁸ spores/mL. Aliquots (1 mL) were inoculated into 15 mL pear juice medium and cultured at 28 °C with 180 rpm agitation for 7 d. Post-incubation, cultures were filtered and subjected to liquid–liquid extraction with equal volumes of chloroform. After phase separation, the lower organic phase was collected. This extraction procedure was repeated twice. The combined organic phases were evaporated to dryness under a gentle stream of nitrogen at 60 °C. The residue was redissolved in 1 mL of HPLC-grade methanol. After complete dissolution, the solution was filtered through a 0.22 μm membrane to remove impurities, transferred into an amber HPLC vial, and stored protected from light at −20 °C until analysis. Ochratoxin A (OTA) production was quantified by high-performance liquid chromatography (HPLC) using a Shimadzu AD20 system (Shimadzu, Kyoto, Japan) equipped with a Shim-pack VP-ODS C18 column (250 × 4.6 mm, 5 μm) and a fluorescence detector. The excitation and emission wavelengths were set at 333 nm and 460 nm, respectively. The injection volume was 10 μL. The mobile phase, consisting of 0.08% acetic acid in water and acetonitrile (52:48, v/v), was delivered at a flow rate of 1 mL/min under isocratic elution. The column temperature was maintained at 30 °C. The limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.2 ng/mL and 0.7 ng/mL, respectively, as established in our previous method validation [29,30].

2.10. Statistical Analysis

Data analysis was conducted with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Results are presented as mean ± standard deviation (SD), and statistical significance among groups was determined by one-way ANOVA followed by Duncan’s multiple range test, with a significance threshold of p < 0.05.

3. Results

3.1. Transcriptome Analysis A. westerdijkiae

Employing RNA-seq technology, four cDNA libraries (CS1, LP1, CS3, LP3) were constructed, each yielding over 5.46 Gb of sequencing data with Q30 scores exceeding 95.36% (Table S2). Given the absence of replicate libraries, the following differential expression analysis is exploratory and aims to generate a set of candidate genes for further validation. Differential expression analysis identified 4618 differentially expressed genes (DEGs) at 24 h, including 1627 up-regulated and 2991 down-regulated genes (Table S3). At 72 h, 2842 DEGs were detected, comprising 1458 up-regulated and 1384 down-regulated genes (Table S4). KEGG enrichment analysis revealed that these DEGs were predominantly enriched in pathways related to Cellular Processes, Environmental Information Processing, Genetic Information Processing, and Metabolism (Figure S1). qRT-PCR validation of five randomly selected DEGs demonstrated expression trends consistent with the RNA-seq data (Figure S2).

Fungal effector genes are typically stage-specifically expressed during infection and often encode secreted proteins that are highly up-regulated during host colonization. Therefore, we focused on up-regulated genes in A. westerdijkiae. Using SignalP-5.0, TargetP-2.0, and TMHMM-1.0.19 tools, we predicted secreted proteins among the up-regulated DEGs, excluding mitochondrial-targeted proteins and those containing transmembrane domains. Signal peptide screening identified 137 and 75 putative secreted effector candidates from the 24 and 72 h up-regulated DEG sets, respectively. After removing proteins of unknown function, those predicted at 24 h included glycoside hydrolases, peptidases, oxidoreductases, and lipases(Table S5), while the 72 h set contained peptidases, glycoside hydrolases, oxidoreductases, and lipases(Table S6). Notably, a carboxylesterase gene (TRINITY_DN4244_c1_g1, designated AwCES) exhibited significant up-regulation during host–pathogen interaction. Consequently, we selected AwCES for further functional characterization to elucidate its potential role in A. westerdijkiae.

3.2. AwCES Is Associate with Fungal Growth and Development

AwCES encodes a 519-aa protein with a 25-aa predicted N-terminal signal peptide. To investigate its potential roles in fungal growth and virulence, we generated an AwCES knockout mutant (ΔAwCES) using the illustrated homologous recombination strategies (Figure 1). To characterize the function of AwCES in fungal growth and development, the radial growth, conidial production, and germination of the ΔAwCES mutant and WT were also analyzed. Notably, the WT and ΔAwCES exhibit distinct colonial morphologies. ΔAwCES accumulated purplish-red secondary metabolites within the agar matrix (Figure 2a,b). As presented in Figure 2c, WT and ΔAwCES strains exhibited comparable growth rates on PDA medium. Hyphal architectures examined under differential magnification light microscopy after 5 d slide culture revealed uniform, turgid, and septate hyphae in WT. In contrast, ΔAwCES displayed pronounced hyperbranching with reduced branch elongation rates, accompanied by hyphal wrinkling and absence of discernible septa (Figure 3a). The ∆AwCES mutant produced 44% fewer conidia (0.84 × 10⁸ conidia/cm²) than the wild-type (1.21 × 10⁸ conidia/cm²) on PDA (Figure 3b). Wild-type spores initiated germination at 9 h, whereas mutant spores exhibited a delayed onset, commencing at 12 h. Following 12 h of incubation, the ∆AwCES mutant exhibited markedly impaired germination, with only 29.6% of conidia germinated compared to 88.3% in the wild-type strain (Figure 3c). In contrast, both strains produced comparable levels of ochratoxin A, showing no statistically significant difference (Figure 2d).

3.3. Involvement of AwCES in the Responses of A. westerdijkiae to Environment Stresses

To examine the link between AwCES and the chemical stress response in A. westerdijkiae, we examined the growth of WT and ΔAwCES mutant on PDA amended with an array of chemical reagents (Figure 4a).

Under hyperosmotic challenge, both WT and ΔAwCES strains developed altered colonial morphologies featuring sparse biomass distribution and enhanced peripheral aerial hyphae proliferation. Notably, colonies exhibited radial concavities extending centrifugally on NaCl-supplemented PDA. Quantitative assessment revealed significant osmotic hypersensitivity in ΔAwCES, with 5 d colony diameters measuring 6.38 cm (NaCl) and 7.26 cm (KCl) versus WT diameters of 7.80 and 8.14 cm, respectively, representing growth reductions of 18.2% and 10.8%. This phenotype demonstrates impaired osmoregulatory capacity in the mutant strain. Under H₂O₂ stress, ΔAwCES exhibited a 2.5% reduction in growth rate compared to WT. When exposed to cell wall-perturbing agents, growth rates declined by 1.3% (SDS-supplemented PDA) and 2.6% (Congo Red PDA) in ΔAwCES versus WT. In the ΔAwCES mutant, we observed compromised cell wall integrity and modulated stress responses, most notably a heightened sensitivity to hyperosmotic environments. These findings collectively suggest a role for AwCES in these processes.

3.4. AwCES Is Required for the Virulence of A. westerdijkiae in Pear Fruit

To evaluate the effect of AwCES deletion on the pathogenicity of A. westerdijkiae, pear fruit was inoculated with WT and ΔAwCES strains (Figure 5a). Lesion diameter progressively increased over time; however, at 3 and 5 d, ΔAwCES exhibited 22.4% and 26.1% reductions in virulence relative to WT, declining to 12.4% by 7 d (Figure 5b). These results demonstrate that AwCES deficiency attenuates pathogenicity throughout infection, with notably pronounced impairment during early-to-mid infection phases.

SEM analysis of A. westerdijkiae colonization on pear fruit is presented (Figure 6). At inoculation (0 h), WT and ΔAwCES conidia exhibited smooth, spherical morphologies with comparable dimensions, showing no discernible ultrastructural differences. By 12 h, WT conidia displayed pronounced surface involution with elongated germ tubes penetrating pear epicarp, whereas ΔAwCES mutants initiated germination with emerging germ tubes and minor surface wrinkling. The ΔAwCES mutant showed a significant reduction in infection efficacy in planta, suggesting that AwCES contributes to the pathogenic capacity of A. westerdijkiae.

4. Discussion

During host infection, fungal pathogens secrete a diverse repertoire of effector proteins to facilitate colonization. Consequently, identifying virulence-associated effector genes and elucidating their biological functions are essential for deciphering the molecular basis of plant–fungal interactions. This study employed transcriptomic profiling of A. westerdijkiae grown in Pear Juice Medium to identify 137 and 75 candidate effector genes at 24 and 72 h, respectively. Among these, AwCES was selected for further investigation due to its significant upregulation during host–pathogen interaction, aiming to elucidate its potential functions in pear infection and disease development.

Effector proteins serve as critical virulence weapons in fungal–plant interactions, playing pivotal roles during host colonization. Apart from conserved N-terminal signal peptides, effectors typically exhibit compact amino acid sequences with elevated cysteine residues. This study employed transcriptomic profiling to obtain amino acid sequences encoded by upregulated genes, leveraging these data to identify and functionally annotate putative effectors. Among 52 candidate effectors screened, 48% were functionally uncharacterized proteins, while the remainder participated in energy metabolism, biosynthesis of secondary metabolites, and carbohydrate synthesis/hydrolysis processes. The plant cell wall not only provides mechanical support but also functions as a physical barrier against pathogen invasion. During infection, pathogens secrete cell wall-degrading enzymes (CWDEs) to dismantle this barrier, facilitating host colonization and intracellular spread [31]. In Sclerotinia sclerotiorum and the white rot stramenopile (Coniothyrium diplodiella), genes encoding xylanases were highly upregulated during early infection stages, and their deletion attenuated virulence to varying degrees [32,33]. Among the 52 predicted effector proteins, various cell wall-degrading enzymes (CWDEs) were identified, including pectinases, cellulases, hemicellulases, and proteases. Additionally, glycoside hydrolases have been demonstrated to contribute to fungal pathogenesis. For instance, deletion of Bcara1 in Botrytis cinerea reduced arabinosidase activity and impaired virulence toward Arabidopsis thaliana [34]. Song et al. revealed that loss of endo-α-1,6-mannanase (GH76 family) compromised vegetative growth and pathogenicity in Aspergillus oryzae [35]. Notably, the AwCES gene, annotated as a carboxylesterase-encoding effector candidate, exhibited a 4.6-fold upregulation at 12 h. Crucially, no prior studies have definitively established the role of this carboxylesterase in fungal infection biology.

Following AwCES deletion, A. westerdijkiae exhibited altered secondary metabolism, particularly evidenced by increased pigmentation. The knockout mutant demonstrated reduced conidiation, delayed germination, decreased germination rates, hyphal hyperbranching, and surface invagination. Notably, hyphal growth remained unaffected. In Curvularia lunata, disruption of Clt1, a regulator of acetylxylan esterase and xylanase synthesis, compromised xylan utilization. This led to diminished acetyl-CoA production, which is essential for melanin precursors, and impaired conidiation under xylan-based culture [22]. Similarly, klp-7 deletion in Botrytis cinerea reduced pectin methylesterase activity, induced hyperbranching with hyphal constrictions, and delayed spore germination with reduced rates [23]. Prior studies have established that esterase family proteins are implicated in regulating secondary metabolite biosynthesis and developmental processes in pathogenic fungi. In alignment with these findings, our results suggest that deletion of AwCES in A. westerdijkiae likely impairs its putative carboxylesterase function, which may be linked to perturbed carbohydrate utilization. These disruptions could, in turn, underlie the observed hyphal morphological alterations, delayed germination, and reduced germination rates.

Upon activation of defense responses, plants produce resistance (R) proteins that recognize pathogen invasion to halt infection [36]. Conversely, pathogens secrete effectors to modulate the pH, osmotic, and oxidative environments of host tissues to facilitate colonization. In Valsa mali, deletion of VmPma1 significantly downregulated acid phosphatase expression, thereby impairing tissue acidification and virulence [37]. Furthermore, the integrity of the fungal cell wall and the ability to adapt to stressful environments are crucial for successful host colonization by pathogenic fungi. Our findings demonstrate that deletion of AwCES may impacts the susceptibility of the cell wall in A. westerdijkiae, accompanied by reduced growth rates under oxidative and hyperosmotic stress conditions compared to the WT. In Fusarium oxysporum, disruption of the protein phosphatase gene Ptc6 enhanced cell wall susceptibility and impaired CFW-induced Mpk1 phosphorylation, thereby attenuating virulence [38].

Following the targeted disruption of AwCES, in planta inoculation assays revealed a significant decline in the pathogenicity of A. westerdijkiae. Ultrastructural observation via SEM revealed markedly impaired host penetration capability in the mutant compared to the wild-type at 12 h, ultimately leading to attenuated virulence. Consistently, Feng et al. [39] identified a pectin methylesterase-encoding gene (Pcpme6) in Phytophthora capsici, and its knockout substantially reduced both PME activity and virulence. Similarly, in Peronophythora litchii, deletion of the pectin acetylesterase gene PAE5 did not affect hyphal growth in vitro but compromised host penetration capacity during infection, resulting in significantly diminished pathogenicity [40]. Consistent with these findings, deletion of a feruloyl esterase-encoding gene in Magnaporthe oryzae did not affect hyphal growth in vitro. However, during infection, most appressoria failed to propagate to adjacent plant cells post-penetration, ultimately leading to virulence attenuation [41]. Furthermore, in Aspergillus nidulans, a knockout of MoCreC, which encodes a key regulator in the CCR pathway, altered feruloyl esterase expression. This change resulted in reduced conidiation, compromised integrity of the cell wall and infection structures, and a consequent loss of pathogenicity [42]. Collectively, existing studies highlight the multifactorial nature of fungal pathogenicity attenuation. In this study, the deletion of AwCES impaired conidiation in A. westerdijkiae, a critical early infection step. Although the ΔAwCES mutant exhibited hyperbranching hyphae, its reduced germination capacity likely diminished tissue penetration. Furthermore, the loss of AwCES impaired fungal stress adaptation, potentially compromising its ability to overcome host defenses. All these changes might be responsible for the attenuated virulence of A. westerdijkiae on pear fruits. To further exploration the biological function of AwCES in A. westerdijkiae, more experiments, including the generation of complementary strains and identification of the targets of AwCES in host plants, are required in future research.

5. Conclusions

In this study, transcriptomic analysis of A. westerdijkiae cultured in pear juice medium and Czapek–Dox medium revealed numerous differentially expressed genes. Signal peptide prediction screening of the up-regulated genes identified various secreted proteins, primarily including glycoside hydrolases, peptidases, oxidoreductases, and esterases. A novel effector candidate gene, AwCES, was selected for functional characterization. Deletion of AwCES altered colonial morphology, reduced conidial production and germination rate, and influenced cellular responses to osmotic stress. Consistent with this, the ΔAwCES mutant exhibited attenuated virulence on pear fruit. These collective findings indicate that AwCES is required for normal growth, development, and pathogenicity in A. westerdijkiae.