The Benzoylation of the Splicing Factor Skip Is Critical for Development, Oxidative Stress Response and Pathogenicity in Aspergillus flavus

TL;DR

This paper shows that the Skip protein and its benzoylation are essential for the growth, stress response, and pathogenicity of Aspergillus flavus, a fungus that infects crops.

Contribution

The study identifies lysine 325 as a benzoylated site on Skip and reveals its role in fungal development and pathogenicity.

Findings

Single-copy deletion of Skip in A. flavus caused slowed growth and reduced spore formation.

Benzoylation of Skip at lysine 325 is catalyzed by EsaA and is critical for morphogenesis and stress adaptation.

Mutation of K325 impaired pathogenicity and aflatoxin biosynthesis in A. flavus.

Abstract

Alternative splicing of pre-mRNA is a crucial mechanism in gene expression regulation. As a core component of the spliceosome, the biological function of the Skip protein in Aspergillus flavus remains unknown. Quantitative real-time PCR (qPCR) analysis revealed the presence of two skip gene copies in A. flavus. Single-copy deletion of Skip resulted in slowed growth, reduced conidiation, abolished sclerotial formation, increased aflatoxin biosynthesis, and diminished crop colonization. Meanwhile, Skip was found to regulate the oxidative stress response by modulating the alternative splicing of yapA. Subsequently, immunoprecipitation and Western blot analyses identified lysine 325 (K325) as the benzoylated site on the Skip protein, which catalyzed by the acyltransferase EsaA. Mutation of benzoylated site K325 directly impaired fungal morphogenesis, pathogenicity, and stress adaptation. These findings established the crucial role of Skip and its benzoylation in A. flavus and suggested a potential target for controlling its infection in important crops.

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1. Introduction

Aspergillus flavus, a widespread pathogenic fungus, poses a serious threat to global agriculture and food security [1]. It attacks important economic crops including maize and peanuts, causing extensive yield and quality losses. Meanwhile, A. flavus produces aflatoxin B1, a Group 1 carcinogen known for its hepatotoxicity, mutagenicity, and immunosuppressive effects [2]. According to a Food and Agriculture Organization estimate, approximately 25% of global grain harvests are contaminated by mycotoxins, with aflatoxins being an important contributor [3]. However, recent monitoring data reveal that the true contamination rate is far higher than this estimation [4]. Chronic low-dose exposure to aflatoxins is significantly associated with an increased risk of hepatocellular carcinoma [5], while acute high-dose exposure could lead to death. Given the high thermostability of aflatoxins, aflatoxins can accumulate in animals and humans and consequently pose a serious global health threat [2]. Thus, it is imperative to investigate the mechanisms of development and aflatoxin biosynthesis in A. flavus, which is essential for protecting the global food supply and public health.

In eukaryotes, gene expression is regulated at both the transcriptional and post-transcriptional levels. Post-transcriptional regulation includes precursor messenger RNA (pre-mRNA) splicing, 5′ capping, and 3′ polyadenylation. Among these processes, spliceosome-mediated pre-mRNA splicing plays a critical role in the regulation of gene expression [6]. The Ski-interacting protein (Skip) is an evolutionarily conserved protein that acts as a splicing factor as part of the spliceosome [7]. In yeast, Prp45 (Skip homology) is required for the early stages of cotranscriptional spliceosome assembly [7]. In addition, Skip can function as a transcriptional regulator in plants [8]. However, the role of Skip in fungal virulence and pathogenicity remains unknown.

Mounting evidence confirms that post-translational modifications (PTMs) play a pivotal role in epigenetic regulation, serving as a core pathway for modulating protein function [9]. Lysine acylation represents the most diverse class of PTMs, often occurring on histones and non-histone proteins [10]. Histone PTMs act as key dynamic regulators in chromatin structure and gene expression [11,12]. Meanwhile, acylation of non-histone proteins can directly modulate its function by altering stability, protein interaction and subcellular localization [13]. Lysine benzoylation (Kbz) is a recently discovered PTM [14] which was originally identified in histones as a transcriptional activation mark [15]. A subsequent study identified a broad spectrum of non-histone proteins as benzoylation targets in A. flavus [16], with the Skip protein being among them. However, the role of benzoylation on the Skip protein remains unclear.

The purpose of this study was to explore the biological functions of Skip and its benzoylation in A. flavus. We identified an indispensable role of Skip in morphogenesis, aflatoxin biosynthesis, stress resistance and crop colonization. We further determined that Skip was benzoylated at K325 by acyltransferase EsaA. Additionally, we found that his leads to mutation of the benzoylated site which disrupts fungal development, stress adaptation and pathogenicity, underscoring the essential role of benzoylation in A. flavus.

2. Results

2.1. Skip Exists as a Two-Copy Gene in Aspergillus flavus

Previous research revealed that Skip is essential for fungal growth [17,18]; however, some essential genes exhibited two copies of skip in A. flavus [19]. Therefore, we performed quantitative real-time PCR to determine the copy number of the skip gene in A. flavus. The qPCR analysis confirmed the presence of two copies of the skip gene in the A. flavus genome (Figure 1A). To explore the potential biological function of Skip in A. flavus, we examined its expression profile across different developmental states. Our results revealed that the skip gene exhibited high expression during the sclerotium formation state (SCR) and conidiation state (CON) following its vegetative growth state (VG) and aflatoxin synthesis state (AFS) (Figure 1B). This suggests that Skip plays a significant role in the development of A. flavus.

2.2. Skip Plays Important Roles in Sclerotial Formation and Conidiation

After identifying the copy number, we successfully constructed the single-copy knockout strain (skip^−/+^) and complementation strain (skip-com) (Figure S1). Subsequent phenotypic experiments demonstrated that the single-copy deletion of the skip gene abolished sclerotial formation in A. flavus (Figure 1C,D), while genetic complementation successfully rescued this developmental defect, confirming that Skip was essential for sclerotia formation. Consistently, the expression of nsdC, nsdD and sclR, which regulate sclerotial formation, was downregulated in the skip^−/+^ strain (Figure 1E). In addition to the defects observed in sclerotial formation, the skip^−/+^ mutant exhibited significantly reduced colony diameter and increased conidia production (Figure 1F–H). Consistent with the observed increase in conidial production in skip^−/+^ mutant, the expression levels of the conidiation-related genes abaA and brlA were also higher in the skip^−/+^ mutant than that in the WT and skip-com strains (Figure 1I), indicating that Skip plays a crucial role in conidiation.

2.3. Skip Regulates Aflatoxin Production

To investigate the role of Skip in aflatoxin biosynthesis, WT and skip mutants were inoculated into YES liquid medium, and the culture supernatants were collected for aflatoxin quantification. We performed a semi-quantitative analysis using thin-layer chromatography (TLC) and Fiji (ImageJ), which showed significantly increased AFB_1_ production in the skip^−/+^ mutant relative to the WT and *skip-*com strains (Figure 2A). Meanwhile, the transcriptional levels of key transcription factors (aflR and aflS) and structural genes (aflC, aflD, aflM, aflN, aflP and aflQ) within the aflatoxin biosynthetic cluster were significantly upregulated (Figure 2B,C). These findings suggested that Skip influences aflatoxin biosynthesis by modulating the expression of the aflatoxin biosynthetic cluster genes.

2.4. Skip Is Involved in Pathogenicity

Aspergillus flavus, a critical plant-pathogenic fungus, causes huge agricultural losses by contaminating key economic crops such as peanuts and maize [20]. To investigate the role of Skip in interaction between A. flavus and living seeds, we examined the ability of the WT, skip^−/+^ and skip-com mutants to produce conidia and aflatoxin in maize and peanut seeds. The result showed that the skip^−/+^ appeared to exhibit fewer conidia and more AFB_1_ than WT and skip-com strains in the maize kernels (Figure 3A–C). A similar assay in peanut seeds exhibited almost the same results (Figure S2). To further assess the pathogenicity of the WT and skip mutants, we quantified fungal biomass in maize seeds using qPCR, which revealed that skip^−/+^ exhibited a significantly reduced fungal DNA burden (Figure 3D). Taken together, these results indicated that reduced skip gene expression impairs colonization of A. flavus, highlighting an important role for Skip in crop infection.

2.5. Skip Regulates Oxidative Stress Response Through Alternative Splicing of yapA

Reactive oxygen species damage cellular components and disrupt homeostasis, while the oxidative defense system protects organisms from such damage [21]. Quantitative real-time PCR analysis revealed that the expression of the skip gene was significantly upregulated under oxidative stress conditions (Figure 4A), suggesting a potential role in the stress response. To explore the role of Skip in oxidative stress response, we performed a phenotypic assay by inoculating WT and skip mutants onto YGT agar plates containing 3.5 mM H_2_O_2_ (the optimal concentration, Figure S3). The skip^−/+^ mutant exhibited significantly greater growth inhibition compared to the WT and skip-com strains (Figure 4B,C).

YapA served as a master transcription factor controlling the oxidative stress response in fungi [22]. To elucidate the molecular mechanism underlying this, we analyzed the expression of key antioxidant defense genes. The qPCR results showed that transcript levels of the upstream transcription factor yapA and its downstream target catalase catA were both significantly downregulated in the skip^−/+^ mutant (Figure 4D). To further determine if Skip regulates oxidative stress through splicing, we analyzed the transcripts of yapA and catA. RT-PCR revealed that single-copy deletion of Skip caused specific and aberrant retention of the first intron in mRNA of yapA (Figure 4E). Since this intron harbors a stop codon (TGA), this mis-splicing resulted in a non-functional, truncated YapA protein. These results demonstrated that Skip controls the transcriptional activation of its downstream target by ensuring the correct alternative splicing of the transcription factor yapA mRNA, thereby mediating the oxidative stress response.

2.6. K325 Is the Benzoylated Site in Skip Protein

Our previous benzoylomic raw data, which are available in the China National GeneBank database (CNP0003875), identified a benzoylated site on Skip protein [16] located at K325 (Figure 5A and Figure S4). The sequence alignment result indicated that the benzoylated site K325 was evolutionary conserved among various Aspergillus spp. (Figure 5B). The benzoylomic data were validated by generating Skip-HA fusion mutants (K325-HA, K325R-HA, and K325A-HA) (Figure S5), with arginine (R) and alanine (A) substitutions mimicking the unacylated and null-mimetic forms of lysine (K), respectively. WB results showed that mutation of K325 had no impact on Skip protein expression (Figure 5C). Additionally, the immunoprecipitation and Western blot assays showed that K325-HA exhibited Kbz signal (Figure 5D), confirming that Skip is a benzoylated protein in A. flavus. Meanwhile, Kbz signal was decreased in K325R-HA and K325A-HA mutants (Figure 5D), suggesting that K325 is the benzoylated site on Skip in A. flavus.

2.7. Identification of EsaA as the Benzoyltransferase for Skip

Previous studies have indicated that EsaA possesses benzoyltransferase activity in yeast [15]. To investigate whether EsaA is responsible for the benzoylation of Skip, we generated an overexpression mutant (OE::esaA-skip-HA) and subsequently examined the Kbz levels of Skip in skip-HA and OE::esaA-skip-HA. The construction of OE::esaA-skip-HA mutant was systematically validated through genomic PCR and Western blot (Figure 6A–C). Comparative Western blot analysis revealed that overexpression of EsaA led to a marked and direct increase in the benzoylation levels of Skip (Figure 6D). These results indicated that EsaA overexpression mutant was successfully constructed and functions as the benzoyltransferase of Skip in A. flavus.

2.8. Mutation of Benzoylated Site in Skip Caused Increased Sclerotium but Decreased Conidiation

To investigate the effect of benzoylation on the Skip, we constructed benzoylated site mutants (skip^K325R^ and skip^K325A^). Phenotypic analysis revealed that benzoylated site mutation resulted in a marked increased sclerotial production (Figure 7A,B). Meanwhile, the expression of sclerotial formation-related genes (nsdC, nsdD and sclR) was significantly upregulated in skip^K325R^ and skip^K325A^ mutants (Figure 7C). The close correlation between the expression pattern and the sclerotial phenotype indicated that benzoylation of Skip was involved in regulating sclerotial formation in A. flavus.

Our previous results confirmed that Skip plays a crucial role in conidiation; following this, we hypothesized that its benzylation may potentially contribute to this process. To verify the hypothesis, we inoculated the WT and benzoylated site mutants onto YGT medium and observed their colony morphology and conidial morphology. Compared to the WT, skip^K325R^ and skip^K325A^ mutants exhibited a significantly smaller colony diameter (Figure 7D,E). Further quantitative analysis revealed that conidia production was significantly decreased in both the skip^K325R^ and skip^K325A^ mutants (Figure 7F). Meanwhile, the expression of brlA and abaA, which regulate conidial formation, was downregulated in the benzoylated site mutants (Figure 7G). These findings suggested that benzoylation of Skip was critical for conidiation.

2.9. Benzoylation of Skip Is Involved in Oxidative Stress Response

After confirming the important role of Skip in oxidative stress response, we further determined the specific function of its benzoylation. Phenotypic analysis revealed that the skip^K325R^ and skip^K325A^ mutants exhibited greater tolerance to oxidative stress, with a significantly lower inhibition of growth rate compared to the WT strain (Figure 8A,B). Consistent with this enhanced tolerance, qPCR analysis showed the expression of yapA and catA was significant increase in skip^K325R^ and skip^K325A^ mutants (Figure 8C). Meanwhile, RT-PCR analysis indicated no aberrant splicing of yapA and catA in skip^K325R^ and skip^K325A^ mutants, suggesting that the deficiency benzoylation of Skip does not impair its splicing function. These results indicated that benzoylation of Skip acts as a negative regulator, and its deficiency activates the fungal oxidative stress response.

2.10. Benzoylation Deficiency of Skip Results in Reduced Crop Colonization

To investigate the role of benzoylation in the pathogenicity of A. flavus, we inoculated live peanut and maize seeds with fresh conidia of WT, skip^K325R^ and skip^K325A^ strains. Crop colonization assays revealed that the benzoylated site mutants exhibited significantly reduced conidiation and AFB_1_ production (Figure 9A–C and Figure S6). Meanwhile, quantification of fungal biomass in infected maize kernels showed a significantly lower fungal burden in skip^K325R^ and skip^K325A^ mutants (Figure 9D). Since no significant difference in AFB_1_ production was observed between the WT, skip^K325R^ and skip^K325A^ mutants, the decreased AFB_1_ levels in infected maize were probably an indirect result of reduced fungal colonization by the mutants. Taken together, these data demonstrated that benzoylation of Skip was involved in regulating the pathogenicity of A. flavus.

3. Discussion

Skip is a conserved bifunctional eukaryotic protein that acts not only as a core factor associated with the spliceosome in pre-mRNA splicing, but also as a transcriptional coactivator [23,24]. Skip has been proven to play critical roles in the growth of fungi and plants. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the skip gene is essential for cell viability [17,18]. In rice, suppression of OsSKIPa leads to significantly reduced germination rates, cell viability and growth arrest [25]. Consistent with these results, we also found that a single-copy knockout of skip impaired vegetative growth in A. flavus. Meanwhile, our phenotypic analysis showed that the deletion of single copy skip resulted in increased conidiation and AFB_1_ production, and abolished sclerotia formation. This demonstrated that Skip is involved in regulating both fungal growth, development and secondary metabolism. In addition, our results showed that the fungal burden was significantly lower in kernels infected with the skip^−/+^ mutant than in those infected with the WT or skip-com strains, indicating that Skip is required for efficient host colonization. This finding was consistent with the demonstration of the splicing factor MoSrp1 directly affecting conidiation and pathogenicity by regulating the efficient splicing of the MoATF1 and MoMTP1 genes in Magnaporthe oryzae [26]. Altogether, these findings revealed that Skip was a key regulator coordinating growth, development, secondary metabolism, and crop colonization in A. flavus, thereby expanding the understanding of spliceosome factors in fungal pathogenesis.

Skip mediates stress responses by controlling the alternative splicing of stress-related pre-mRNAs and enhances cellular resistance through protein–protein interactions [27]. In this study, we found that the skip gene plays an important role in the oxidative stress response of A. flavus. The skip^−/+^ strain exhibited significantly increased sensitivity to oxidative stress, with a higher growth inhibition rate than the WT strain. This was consistent with findings in rice, while overexpression of Skip could enhance ROS scavenging capacity [25]. Meanwhile, our qPCR analysis revealed that the transcript levels of the transcription factor yapA and downstream antioxidant enzyme catA were significantly reduced in the skip^−/+^ strain. Given that Skip loss primarily causes aberrant splicing characterized by intron retention [28], we further performed RT-PCR analysis on yapA and catA mRNAs. The results indicated that pre-mRNA of yapA showed abnormal splicing in the skip^−/+^ mutant, while a portion of transcripts retained in the first intron. This first intron contains a stop codon, which would lead to premature translation termination and a non-functional protein. These results demonstrated that single-copy deletion of Skip could disrupts the oxidative defense by inducing aberrant splicing of yapA pre-mRNA, thereby reducing its transcription and translation, which in turn regulates the transcription of its downstream targets.

Skip’s function is regulated by diverse post-translational modifications. For example, H3K36 methylation promotes the interaction of Eaf6 with Prp45 (Skip homolog) and Prp19, facilitating spliceosome recruitment for effective and constitutive splicing [29]. In Arabidopsis, Skip undergoes 26S proteasome-mediated degradation [24]. Moreover, the N-terminal PINIT domain of the yeast SUMO E3 ligase Siz1 interacts with Prp45 and mediates its poly-SUMOylation [30]. Previous acylation proteomic studies confirmed the absence of acetylation, succinylation or 2-hydroxyisobutyrylation on the Skip protein in A. flavus [31,32,33]. Our earlier benzoylomic analysis identified a potential benzoylated site on the Skip protein [16]. In this study, our data confirmed that Skip is indeed a benzoylated protein, and benzoylation is mediated by the acyltransferase EsaA, whose overexpression significantly enhanced the benzoylation level of Skip. Although this demonstrated a key functional involvement of EsaA, it remains unclear whether its effect on Skip is mediated through a direct physical interaction or indirectly via global changes in the acyl-CoA pool or chromatin state, and the mechanism needs to be elucidated. In addition, EsaA also catalyzes histone crotonylation and acetylation [34,35], demonstrating its broad acyltransferase activity and suggesting that EsaA regulates fungal development and pathogenicity through a complex network. Taken together, these findings illustrated that Skip is a benzoylation protein in A. flavus, and may undergo multiple post-translational modifications in different organisms.

Lysine 325 (K325) is a benzoylated site on the Skip protein in A. flavus. The benzoylation level was significantly lower in site-directed mutant strains (K325R-HA and K325A-HA) compared to the K325-HA control strain. The residual benzoylation signal observed on the K325-mutated Skip protein could be attributed to two possibilities. One possibility was that Skip harbors other benzoylated sites which went unidentified in our benzoylomic sequencing. Previous research also showed that proteins often harbor multiple modification sites [36]. Alternatively, native Skip protein is known to form homodimers in fungi [17]. Due to the limitations of our current genetic model, we cannot delete both copies of the skip gene. Additionally, our mutation was only introduced into one genomic copy, so the resulting heterodimeric complex may contain one mutant and one wild-type Skip monomer. Consequently, immunoprecipitation targeting the mutant subunit would co-precipitate the benzoylated wild-type partner, thereby detecting the benzoylation signal.

Like other acylations [37], benzoylation is also capable of directly regulating the function of non-histone proteins [16]. Previous studies have shown that the acyltransferase GcnE affects aflatoxin biosynthesis and pathogenicity by regulating the benzoylation of the AdhB protein, while the benzoylation of AdhB is required for its functional execution in A. flavus [16]. The identified benzoylation site K325 is located in the SNW domain, which plays a critical role in the biological functions of Skip [24], suggesting that benzoylation directly regulates Skip’s function. As expected, the benzoylation-deficient point mutants exhibited distinct developmental phenotypes, including growth defects, reduced conidiation, increased sclerotia, impaired host colonization, and more tolerant to oxidative stress. Meanwhile, benzoylation deficiency in Skip did not induce aberrant alternative splicing of yapA, further indicating that benzoylation is not required for its core splicing function. Importantly, these development phenotypes contrast with those observed in the skip^−/+^ strain, suggesting that benzoylation likely acts as an inhibitory modification on Skip protein function. This was consistent with acylation suppressing protein activity through diverse mechanisms. For instance, acetylation can suppress the activities of ACSS1 and ACSS2 [38,39]. KAT9-mediated acetylation of glucose-6-phosphate dehydrogenase (G6PD) inhibits G6PD dimer formation, thereby reducing its enzymatic activity [40]. Given that Skip possesses a dimeric structure, we speculated that benzoylation might similarly inhibit its function by potentially interfering with dimer formation or stability; this hypothesis requires validation by further studies. Taken together, these data showed that the effect of benzoylation was not uniform across different modified proteins.

4. Conclusions

In conclusion, we identified the indispensable role of Skip in the regulation of development, aflatoxin biosynthesis, seed colonization and stress resistance in A. flavus. Moreover, our study demonstrated that benzoylation of the Skip protein regulates critical biological processes in this pathogenic fungus. These results advance our comprehension of Skip function and illuminate the broader role of benzoylation in controlling fungal development and pathogenicity.

5. Materials and Methods

5.1. Strains and Culture Conditions

All strains used in this study are listed in Table S1. All strains were cultured on YGT agar medium for conidiation and sclerotia assays, and the conidia were harvested and counted by hemocytometer and microscope [16]. Aflatoxins were extracted from YES liquid medium. Gene expression was analyzed across four developmental states induced under specific conditions: VG (vegetative growth; YES liquid, 37 °C, 24 h), CON (conidiation; YES solid, 37 °C, 48 h), AFS (aflatoxin synthesis; YES liquid, 29 °C, 48 h), and SCR (sclerotial formation; CM solid, 37 °C, 72 h) [16].

5.2. Construction of Mutant Strains

The mutant construction of A. flavus was carried out using the protocols described previously [41]. The single-copy deletion mutant (skip^−/+^) was constructed via overlap PCR fusion of the gene’s flanking sequences with the pyrG marker, followed by transformation into CA14 protoplasts. The complemented mutant (skip-com) was constructed using the described protocol [16], and the pyrG marker was subsequently excised by 5-FoA screening. Benzoylated site mutants and tagged fusion mutants were constructed through the fixed-point mutation primers. PCR and RT-PCR were used to confirm the positive transformants. All primers used for mutant construction and verification are listed in Table S2.

5.3. Detection of Skip Gene Copy Number

Quantitative real-time PCR is a well-established technique for determining gene copy numbers [19]. The SumO gene, confirmed as a single-copy gene in A. flavus [42], served as the endogenous control for normalization; associated qPCR primers are listed in Table S3. Each experiment was conducted with three biological replicates.

5.4. qPCR Assays

Total RNA was extracted from fungal mycelia using TRIzol reagent (BBI, B610409). cDNA was synthesized from 5 µg of total RNA using the HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China, R312-01). The qPCR was performed on a PikoReal 96 Real-Time PCR System with Hieff^®^ qPCR SYBR Green Master Mix (Yeasen, Shanghai, China, 11201ES08). Due to considerable differences in amplification efficiency among the qPCR primers for various target genes and the housekeeping gene, relative gene expression was calculated using the standard curve method [16]. The actin gene served as the housekeeping gene for normalization. All qPCR primers used in this study are listed in Table S3. Each experiment was conducted with three biological replicates.

5.5. Phenotypic Assays

A suspension of 10^3^ conidia was inoculated onto YGT plates. Colony morphology and diameter were recorded after 5 days of incubation at 37 °C, followed by conidial quantification. For sclerotia analysis, plates were incubated for 7 days at 37 °C, surface mycelia and conidia were removed with 75% ethanol, then the sclerotia were harvested and counted [43]. Each experiment was performed three times.

5.6. Aflatoxin Assays

A suspension of 10^4^ conidia was cultured on YES liquid medium at 29 °C for 6 days [16]. Then, aflatoxins were extracted by dichloromethane. AFB_1_ was separated by thin-layer chromatography and quantified by Fiji software [44]. This semi-quantitative approach primarily reveals relative differences in aflatoxin levels among different samples. Each experiment was performed three times.

5.7. Oxidative Stress Response Assays

A suspension of 10^3^ conidia was inoculated onto YGT plates which contained hydrogen peroxide (3.5 mM H_2_O_2_). This concentration was selected based on a preliminary dose–response experiment. After incubation, the colony diameter was measured, and the inhibition rate was calculated [45]. Each experiment was performed three times.

5.8. Infection Assays

The infection assay was performed according to previously reported methods [45]. Viable peanuts and maize seeds were sterilized, washed, and inoculated with fresh spores, then cultured at 29 °C for 7 days. Maize seeds and peanuts were inoculated with sterilized water as a control. After inoculation, peanuts and maize seeds were harvested for conidia counting, aflatoxin measurement and fungal burden quantification. Following spore counting, AFB_1_ was extracted from infected seeds into dichloromethane. After solvent evaporation and re-dissolution, total AFB_1_ was quantified by TLC and Fiji analysis. DNA was isolated from infected maize seeds by phenol–chloroform method. Fungal burden was quantified by qPCR with 28S-ITS2 specific primers, using equal amounts of seed DNA as template, and was calculated based on a standard curve method [45].

5.9. Western Blot

The protein was extracted by RIPA lysis buffer (Beyotime, Shanghai, China, P0013B), and separated by SDS-PAGE, then transferred to PVDF membranes (Millipore, Burlington, MA, USA, IPVH00010). The anti-Kbz primary antibody (PTM Biolabs, Zhejiang, China, PTM-762), anti-HA (CST, Danvers, MA, USA, 3724S) primary antibody, anti-Actin (Servicebio, Wuhan, China, GB113225) primary antibody, and anti-rabbit and anti-mouse secondary antibody (Thermo, Waltham, MA, USA, 31460 and 31430) were used for Western blotting assays. Signals were detected using an ultra-sensitive ECL substrate (Glpbio, Montclair, CA, USA, GK10008) on a GBox XT4 Chemiluminescence and Fluorescence Imaging System.

5.10. Validation of Benzoylation

Following immunoprecipitation with Anti-HA magnetic beads (MCE, HY-K0201), the bound proteins were eluted with 1× loading buffer and subjected to immunoblotting using anti-HA and pan anti-Kbz antibodies.

5.11. Statistical Analysis

Data were presented as the means ± standard deviation from at least three biological replicates; then, data were processed by GraphPad Prism 10. Statistical significance was evaluated by one-way ANOVA and LSD test. Meanwhile, the significance threshold was set at p < 0.05.