The wild-type strain B05.10 of B. cinerea was preserved in the Key Laboratory of Plant Physiology and Molecular Pathology of Hebei Province. Mycelia were inoculated in a PD medium (PDA medium without agar) and cultured at 25°C for 5 days for RNA and DNA extraction. For protoplast preparation, the wild-type strain was inoculated on PDA medium for 7–10 days, and the mycelium was scraped using Fries medium and cultured at 25°C to obtain a conidial suspension.

The whole genome sequence of B05.10 was searched in the Ensembl fungi database using the amino acid sequence of human and mouse kynureninease. Multiple sequence alignment was performed to analyze the results, and the protein’s three-dimensional structure was predicted using the Swiss Model.¹ Expression profile data of the B. cinerea infection process was obtained from the GEO² database, and the expression data of the KMO gene at different times points were compared to the control (PRJNA628162). The log2 value were taken, and the expression level differences in each pathogen infection period were analyzed.

Based on the restriction enzyme digestion site of the BcKMOL gene and the multiple cloning site of the pBS-pUC vector, Snapgene was used to find a suitable restriction enzyme digestion site. Gene-specific primers containing the restriction enzyme digestion site were designed (Table 1), and the target fragment was amplified. After digestion and ligation into the pMD19 cloning vector, positive clones with correct sequencing were ligated into pUC, and the positive clones were verified. The knockout mutant vector was used to obtain the knockout mutant △BcKMOL by protoplast transformation. DNA level analysis, Southern Blot, and qRT-PCR were performed to confirm the knockout.

The genomic DNA of B. cinerea B05.10 wild-type and knockout transformants △BcKMOL was extracted, and single enzyme digestion was performed using the restriction enzyme Xba I. The enzyme-digested genomic products were detected using 0.8% agarose gel to ensure that the enzyme digestion was sufficient. After purification, the purified product was separated by overnight electrophoresis with 0.8% agarose gel, transferred to the membrane, and the hygromycin resistance gene was labeled with digoxin (Zhang et al., 2025) to make a probe for overnight hybridization (Supplementary Table S1). The specific steps were performed according to the (Roche) kit.

Snapgene was used to analyze the pEarleyGate104 expression vector and the CDS sequence of the BcKMOL gene. Specific primers (BcKMOL-pEarleyGate104-F/BcKMOL-pEarleyGate104-R; Table 1) were designed to amplify the full-length gene, and the correct positive clone was named pEarleyGate104-BcKMOL. BcKMOL-C transformants were obtained by ATMT transformation. PCR using the glufosinate gene on the vector and gene-specific primers was used to identify the transformants. Real-time PCR was performed to confirm the transformants, using the Tubulin gene as the internal reference.

The wild-type and mutant △BcKMOL were inoculated in a liquid PD medium, respectively, and grew for 10 days at 25°C in the dark. After treatment with a freeze-dryer, 80% methanol was mixed and placed in a 2 mL adapter. The liquid nitrogen was frozen for 5 min, repeated freezing and thawing twice, and 900 μL 10% methanol was added. The liquid nitrogen was repeatedly frozen and thawed and installed in a freeze grinder. Centrifuge at 4°C for 5 min at 12,000 rpm/min. 100 μL supernatant was added to 100 μL 20 ppb Trp-d5 solution and vortexed for 10 s. 150 μL supernatant was added to the detection bottle, and the contents of tryptophan, kynurenine, 3-hydroxy kynurenine and quinolinic acid, the key metabolites of kynurenine pathway, were determined by tryptophan targeted metabolic analysis.

The BcKMOL gene mutants were inoculated on the non-resistant PDA medium to observe the colony growth rate, recorded every 24 h. Conidia growth of the BcKMOL gene mutants were observed. Five-day-old mutant colonies were rinsed with 5 mL sterile ddH₂O, and 10 μL was placed on a slide for microscopic observation. Mycelium morphology was observed under a fluorescence microscope after CFW staining, and cell length and width were counted.

Tobacco leaves were surface-disinfected with 75% alcohol for 3 min and washed with sterile water 2–3 times. Uniform fungal plates were made using an 8 mm puncher from 7-day-old mutant and wild-type strains. Colonies were inoculated on tobacco leaves using 50% Tween drops and moisturized under dark conditions at 25°C. Disease incidence was observed, and lesion areas were statistically analyzed using a multi-spectral scanner. Three replicates were set for each strain, and one-way ANOVA was performed using GraphPad software (significant: *: 0.01 < p ≤ 0.05; highly significant: **: p ≤ 0.01).

The BcKMOL gene mutants were inoculated on the non-resistant PDA medium, and infection pads were observed under the microscope. The number and size of the infection pads were statistically analyzed. The BcKMOL gene mutants and wild-type strains were inoculated on a PDA medium containing 0.05% bromothymol blue and incubated in complete darkness at 22°C for 7 days to assess acid secretion. Cell wall-degrading enzymes activity was determined using an enzyme activity detection kit after filtering the culture medium of 14-day-old liquid PD cultures. qRT-PCR analyzed the expression levels of cell wall-degrading enzyme-related genes in the BcKMOL gene mutants.

The three-dimensional structure of BcKMOL was constructed, and docking software was used to analyze the target protein sequence. MEGADOCK was used to predict eigenvalues, and the protein–protein interaction data with antibacterial peptides were obtained. The top 200 protein groups were selected based on the MEGADOCK score and docked in batches using HDOCK docking software. The top 10 combinations were selected for AlphaFold3 interaction analysis.

The protein three-dimensional structure of BcKMOL, CAMPQ3966, and CAMPQ4589 was established using Swiss-Model,³ and molecular simulation docking was performed using HDOCK SERVER.⁴ The results were imported into Pymol for in-depth analysis (Wang et al., 2024).

The two antimicrobial peptides with the highest docking scores were selected as CAMPQ3966 and CAMPQ4589. After codon optimization, the prokaryotic expression vectors were constructed respectively, and the plasmids were extracted and transformed into E. coliBL21. The positive clones pET28a-CAMPQ3966 and pET28a-CAMPQ4589 were inoculated into 20 mL LB medium containing kanamycin sulfate and cultured overnight. The overnight culture broth was taken at a ratio of 1:20 and inoculated into LB medium preheated to 37°C and containing kanamycin sulfate. Conventional culture at 37°C for about 30–60 min or longer, until the OD600 of the bacterial solution reached 0.5–0.7; the pET28a-CAMPQ3966 strain was added with IPTG to a final concentration of 0.1 mM and induced at 16°C for 24 h. The pET28a-CAMPQ4589 strain was added with IPTG to a final concentration of 1 mM and induced at 28°C for 12 h. The bacterial liquid was collected into a centrifuge tube, centrifuged at 4°C, 15,000 g for 1 min, discarded the supernatant, and collected the precipitate; the supernatant was resuspended with Tris–HCL, added 5 × louding buffer, boiled for 10 min, centrifuged at 12,000 g for 3 min, and 20 μL of the supernatant was placed on 10% SDS-PAGE gel electrophoresis, 80 V electrophoresis for 1 h, 100 V electrophoresis for 1 h, Coomassie brilliant blue staining for 15 min, washed with water, and observed protein induction.

The antimicrobial peptide CAMPQ3966 was induced to express at 16°C for 24 h, and CAMPQ4589 was induced to express at 28°C for 12 h. Antibacterial peptides CAMPQ3966 and CAMPQ4589 were induced in large quantities and purified using an AKTA protein purification instrument after ultrasonic lysis and centrifugation.

The wild-type strain of B. cinerea and the BcKMOL mutants at the same growth period were inoculated on PDA solid plates. After 5 days of dark culture at 25°C, a bacterial plate with a diameter of 5 mm was punched with a sterile puncher for subsequent inoculation experiments. The same concentration of CAMPSQ3966 and CAMPSQ4589 proteins were coated on the right side of the culture dish, and the same volume of protein eluent was coated on the left side of the culture dish as the control group. The wild-type strain of B. cinerea and the BcKMOL mutants were inoculated to observe the growth of the strain.

The wild-type strain B05.10 of B. cinerea was inoculated on a PDA solid plate and cultured for 5 days. A 5 mm diameter plate was punched for subsequent inoculation experiments. N. benthamiana plants at 4–5 weeks were selected. CAMPQ3966 and CAMPQ4589 proteins were applied to the right side of tobacco leaves, with soluble Elution Buffer applied to the left side as a control. Elution Buffer was prepared from 5.844 g NaCl, 4 mL 20 mM Tris–HCl, 100 mL 1 M Imidazole, and diluted to 200 mL. After 24 h, leaves were inoculated with B05.10, and plants were placed in a humid environment at 25°C for dark culture. Tobacco leaf disease incidence was observed and photographed after 2 days of inoculation. The same number of diseased leaves were taken and RNA was extracted. The Tublin gene was used as the reference gene of Botrytis cinerea, and the fungal biomass during the disease period was detected by qRT-PCR. The protein CAMPSQ3966 and CAMPSQ4589 were mixed with Tween in equal proportions, and an appropriate amount was dropped on the surface of apples or pears. In the control group, the protein eluent was mixed with Tween in equal proportions, and an appropriate amount was added to the other side of the same apple or pear. The wild-type strain of B. cinerea at the same growth period was inoculated and placed in dark to observe the incidence.

RNA was extracted from the knockout transformants and wild-type strains, and reverse transcribed into cDNA. The expression of the target gene in the transformants was detected by Real-time PCR using Tubulin gene as an internal reference (Supplementary Table S1). The reaction system was: template (cDNA) 2.0 μL, Mix (5 U/μL) 10.0 μL, forward primer (10 μM) 0.4 μL, reverse primer (10 μM) 0.4 μL, ROX 1.0 μL, ddH₂O 6.2 μL, the total system was 20 μL. The reaction procedure was carried out in strict accordance with the Takara fluorescence quantitative RT-PCR kit.

The appropriate amount of mycelium or leaf lesion site was taken, grinded and crushed, diluted with PBS buffer solution with pH = 7.2, after repeated freezing and thawing, the ultrasonic crushing instrument was used for crushing. Centrifuging at 4°C, 2,000 rpm/min for 20 min, and the supernatant was carefully collected. The kynurenine monooxygenase activity of BcKMO protein was determined by the Human KMO ELISA kit (DLDEVELOP, Canada). The specific steps were carried out according to the instructions of the kit. The linear regression equation of the standard curve was calculated by using the concentration and OD value of the standard substance, and the OD value of the sample was substituted into the equation to calculate the enzyme activity of the sample.

Utilizing the amino acid sequence of kynurenine monooxygenase (KMO) from humans and yeast, a search was conducted in the Ensembl Fungi database for the corresponding sequence in B. cinerea B05.10 strain. The search revealed that Bcin01g01500, henceforth referred to as BcKMO-like or BcKMOL, exhibited high similarity to KMO in other species. The Swiss-Model website was used to analyze the advanced structure of BcKMOL and found that it was similar to the structure of other KMOs (Figure 1a). Multiple sequence alignment was performed using MEGA7.0 software, which highlighted several highly conserved sequences between BcKMOL, HsKMO (human KMO) and ScKMO (yeast KMO) (Figure 1b). By analyzing the expression profile data of the wild-type B. cinerea strain from the GEO database during its growth, development, and infection phases, it was observed that the expression level of the BcKMOL gene increased within the first 1–15 h post-inoculation (hpi) during pathogen growth and development. Notably, the expression level peaked at 16 hpi during pathogen infection but gradually declined thereafter (Figure 1c). These findings suggest that BcKMOL plays a role in the growth and development of B. cinerea. By detecting the activity of BcKMOL at 16 hpi, the enzyme activity of BcKMOL was significantly higher than that of uninfected (Supplementary Figure S3a).

To elucidate the impact of BcKMOL on growth and development of B. cinerea, we successfully generated knockout mutants and revertant strains of the BcKMOL gene (Supplementary Figures S1, S2). It was found that the deletion of BcKMOL affected the content of key metabolites of KP, in which the upstream product tryptophan was significantly accumulated, and the downstream metabolites 3-HK and QA were decreased. The content of other key metabolites such as KYNU, KYNA, and Nam also changed (Figure 1d). The contents of metabolites related to the other two pathways of tryptophan metabolism: the bacterial degradation pathway and the 5-hydroxytryptamine pathway also changed (Supplementary Figure S3b). It is speculated that BcKMOL encodes kynurenine monooxygenase and regulates the growth, development, and infection of B. cinerea.

We then observed the colony morphology, mycelial morphology, and growth rate of these BcKMOL gene mutants (Figure 2). Our findings revealed that the knockout mutants of the BcKMOL gene failed to produce sclerotia, and their conidia exhibited an elongated morphology compared to the wild type. Furthermore, the mycelial cells of the mutants were significantly longer and considerably narrower than those of the wild type. Notably, the BcKMOL-C mutant exhibited a substantial restoration in sclerotium production, spore morphology, mycelial cell length, and growth rate when compared to the wild type. These results collectively suggest that mutations in the BcKMOL gene exert a specific influence on the growth and development of B. cinerea, thereby implicating the BcKMOL gene in the regulation of the growth and development processes in B. cinerea.

To investigate the role of the BcKMOL gene in the pathogenesis of B. cinerea, we inoculated tobacco leaves with both the wild type and the BcKMOL gene mutants at the same growth stage. The results showed that while the △BcKMOL mutant was capable of producing visible lesions on tobacco leaves, the lesion area was significantly smaller compared to that caused by the wild type (Figure 3). Moreover, the pathogenicity of the BcKMOL-C revertant strain was restored, confirming that the BcKMOL gene exerts a positive regulatory effect on the pathogenesis of B. cinerea.

As a necrotrophic plant pathogenic fungus, B. cinerea develops multicellular structures known as infection cushions specifically for plant penetration. To elucidate the role of the BcKMOL gene in the pathogenic process of this fungus, we analyzed the infection cushions and acid production capabilities of B. cinerea wild-type, along with BcKMOL gene mutants (Figure 4). The results showed that the number and size of the infection cushions of the △BcKMOL mutants were significantly different from those of the wild type and the revertant mutant (Figure 4). Although the colony color of the △BcKMOL mutants was largely similar to that of the wild type, we further assessed the colony’s pH value and found no significant difference between the BcKMOL mutant and the wild type. These results suggest that the BcKMOL gene negatively regulates the formation of infection cushion in B. cinerea.

The host infection by fungi primarily depends on cell wall permeability and integrity, which are governed by the activity of cell wall degrading enzymes (Zhang et al., 2009). Consequently, we assessed the extracellular pectinase and cellulase activities in the △BcKMOL mutants. Our results indicated that the deletion of BcKMOL led to a marked reduction in the extracellular pectinase and cellulase activities of the pathogen, whereas these activities were restored in the BcKMOL-C strain (Figure 5a). Additionally, we examined the expression levels of genes associated with cell wall degradation enzyme activity (Table 1). We observed significant down-regulation in the expression of BccutB, Bcpgx1, Bcpg3, Bcpme2, BccutA, Bcpme1, Bcams, Bcpg5, and Bcpg4. Conversely, only the expression levels of Bcpg1, Bcmns, and Bcmnl were up-regulated (Figure 5b).

We utilized BcKMOL as the bait protein and employed a multi-step screening process involving MEGADOCK for rough screening, HDOCK for fine screening, and AlphaFold3 for one-to-one nuanced analysis. This approach allowed us to identify 20 potential candidates. Notably, the iptm + ptm values for the bait protein and the library proteins with IDs of CAMPSQ3966 and CAMPSQ4589 exceeded 0.75. Remarkably, the iptm + ptm value for the CAMPQ3966 protein reached 1.49, suggesting a very high probability of interaction with the bait protein (Table 2).

The binding sites of CAMPQ3966 and CAMPQ4589 to BcKMOL were subjected to further analysis using HDOCK and Pymol (Figure 6a). Specifically, the binding sites of CAMPQ3966 to BcKMOL encompassed THR (82)–THR (469), THR (85)–SER (466), SER (148)–ILE (472), and SER (184)–TRP (483). For CAMPQ4589 binding to BcKMOL2, the sites included ASN (8)–VAL (467), ASN (27)–LEU (419), SER (31)–GLY (463), GLY (56)–ARG (429), and GLN (143)–SER (470). Expression studies revealed that CAMPQ3966 could be produced in abundance at 16°C, while CAMPQ4589 was expressed in large quantities at 28°C and subsequently purified using an AKTA protein purification instrument (Supplementary Figures S4a,b). The wild type and BcKMOL mutants were treated with antimicrobial peptides, and it was found that the wild type and BcKMOL-C were sensitive to the antimicrobial peptides CAMPSQ3966 and CAMPSQ4589, and the growth was significantly inhibited, while the growth of △BcKMOL did not change significantly (Figures 6b,c), which indicated that the antimicrobial peptides CAMPSQ3966 and CAMPSQ4589 acted on BcKMOL.

The purified antimicrobial peptides CAMPSQ396 and CAMPSQ4589 were applied to the right side of the tobacco leaves, and the blank control was set on the left side. The results showed that the leaves smeared with CAMPSQ3966 or CAMPSQ4589 had a significant inhibitory effect on the infection of B. cinerea. After treatment with antimicrobial peptides, the lesion area of the same apple or pear was significantly lower than that of the control group (Figures 6d,e), and the biomass of B. cinerea at the lesion of the leaves was detected. It was found that the biomass of B. cinerea was significantly reduced after treatment with CAMPSQ3966 or CAMPSQ4589 (Supplementary Figure S4c). It is indicated that the antibacterial peptides CAMPSQ396 and CAMPSQ4589 can inhibit the growth of B. cinerea and inhibit the pathogenicity of the pathogen.