The Autophagy-Related Protein ATG8 Orchestrates Asexual Development and AFB1 Biosynthesis in Aspergillus flavus

Autophagy, a conserved cellular recycling process, plays a crucial role in maintaining homeostasis under stress conditions. It also regulates the development and virulence of numerous filamentous fungi. In this study, we investigated the specific function of ATG8, a reliable autophagic marker, in the opportunistic pathogen Aspergillus flavus. To investigate the role of atg8 in A. flavus, the deletion and complemented mutants of atg8 were generated according to the homologous recombination principle. Deletion of atg8 showed a significant decrease in conidiation, spore germination, and sclerotia formation compared to the WT and atg8C strains. Additionally, aflatoxin production was found severely impaired in the ∆atg8 mutant. The stress assays demonstrated that ATG8 was important for A. flavus response to oxidative stress. The fluorescence microscopy showed increased levels of reactive oxygen species in the ∆atg8 mutant cells, and the transcriptional result also indicated that genes related to the antioxidant system were significantly reduced in the ∆atg8 mutant. We further found that ATG8 participated in regulating the pathogenicity of A. flavus on crop seeds. These results revealed the biological role of ATG8 in A. flavus, which might provide a potential target for the control of A. flavus and AFB1 biosynthesis.


Introduction
Aspergillus flavus, a saprophytic soil fungus, infects insects and contaminates both preharvest and postharvest seed crops such as maize and peanuts, while also producing potent aflatoxins (AFs) [1,2].Among the AFs, aflatoxins B1, B2, G1, and G2 are the major four and aflatoxin B1 (AFB1), the most toxic and potent hepatocarcinogenic compound among them, primarily targets the liver [3,4].Chronic low-level exposure to AFB1 can lead to immunosuppression and hepatocellular carcinoma, while a single acute exposure could be fatal [5,6].Additionally, A. flavus, the second most common cause of aspergillosis after Aspergillus fumigatus, poses a significant threat to immunocompromised individuals [7][8][9].Sino-orbital, cerebral, and ophthalmic infections due to A. flavus are the major clinical types in aspergillosis, after pulmonary aspergillosis [10].It has emerged as a predominant pathogen in fungal sinusitis and keratitis globally [11].Aflatoxins and Aspergillus not only devalue contaminated crops and cause significant agricultural economic losses but also pose a substantial threat to human health [12,13].Therefore, tackling the problem of A. flavus and aflatoxin contamination is a pressing and crucial endeavor.
Numerous management strategies have been developed to address A. flavus contamination.One commonly employed approach is improving storage conditions [14].Moreover, the use of anti-mildew agents is crucial for preventing fungal contamination.
For instance, quercetin has been found to effectively inhibit the proliferation of A. flavus in a dose-dependent manner [15].Research has shown that the growth of A. flavus hyphae and the germination of its spores can be suppressed in the presence of α-Fe 2 O 3 nanorods under sunlight irradiation [16].Furthermore, p-anisaldehyde (AS) has been recognized as a natural antifungal agent against A. flavus, with a demonstrated ability to modulate AFB1 biosynthesis [17].In addition, biological control methods, including the use of Trichoderma strains, offer the potential for reducing A. flavus and its toxin levels through enzymatic degradation or complexation [18].Despite substantial efforts, A. flavus and AF prevention and control remain significant global challenges.Hence, investigating the growth and development processes of A. flavus in its host, along with the regulatory mechanisms of aflatoxin synthesis, will offer critical insights and a theoretical framework for effectively managing A. flavus contamination.
Autophagy is a cellular degradation process in which cytosolic components and organelles are enclosed within double-membrane vesicles known as autophagosomes.These autophagosomes subsequently merge with degradative organelles, such as the vacuole/lysosome, where the enclosed contents are broken down and the resulting macromolecules are recycled [19,20].ATG8, a ubiquitin-like protein, binds with the lipid phosphatidylethanolamine (PE) to form Atg8-PE, a crucial component for the formation of double-membrane autophagosomes [21].Consequently, ATG8 is considered a reliable autophagic marker for monitoring autophagy progression in cells at both microscopic and molecular levels [22].The molecular mechanisms of autophagy have been extensively investigated in yeast, where autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure depend on ATG8 [23].Furthermore, autophagy plays crucial roles in various aspects of filamentous fungi biology, including asexual and sexual development, nutrient deprivation responses, cellular stress responses, and pathogenicity.In Magnaporthe oryzae, mutant strains with impaired autophagy exhibit severely reduced or completely lost pathogenicity [24,25], along with decreased production of asexual spores [26].Autophagy also plays a critical role in the growth, development, and pathogenicity of Fusarium graminearum [27] and Fusarium oxysporum [28].Significantly, its potential role in mycotoxin biosynthesis has been reported in F. graminearum [29,30].
While the function of ATG8 has been reported in other fungi, its specific role in A. flavus remains uncharacterized.In this study, we identified ATG8 in A. flavus and examined its involvement in hyphal growth, sporulation, sclerotia development, stress tolerance, aflatoxin production, and pathogenicity.These findings will provide valuable insights for the development of potential novel control strategies.

Strains and Culture Conditions
The A. flavus strains utilized in this research are described in Table 1.All strains were inoculated onto potato dextrose agar (PDA) medium and incubated at 37 • C for 5 days.Spores were gathered employing a 0.001% Tween-20 solution, quantified with a hemocytometer, and the spore concentration was adjusted to 10 6 spores/mL for subsequent analysis.

Strain Construction
The deleted and complemented mutants of atg8 were constructed according to the homologous recombination principle.To generate the atg8 deleted strain (∆atg8), flanking regions (5 ′ and 3 ′ ) of the atg8 gene, along with argB sequences, were amplified from wild-type genomic DNA using the primers detailed in Table 2. Subsequently, these three fragments were fused by PCR, and the resulting PCR products were then introduced into the protoplasts of the parental strains TJES20.1 and TXZ21.3 following the previously described approach [32].The upstream homologous arm fragment (AP), downstream homologous arm fragment (BP), and open reading frame (ORF) were amplified from the genomic DNA of selected transformants using three primer pairs (atg8/p1 and argB/170R, argB/1205F and atg8/p6, atg8-orf/F and atg8-orf/R), respectively.This procedure yielded ∆atg8 and ∆atg8∆pyrG mutants.To generate the atg8 complemented strain (atg8 C ), the upstream region (comprising 5 ′ flanking regions and the coding sequence of agt8) and the downstream region (containing argB sequences) were amplified from wild-type genomic DNA.Subsequently, the purified PCR products were fused with the A. fumigatus pyrG gene.The resulting fused fragment was then introduced into protoplasts of the ∆atg8∆pyrG mutant to generate the atg8 complemented strain.Confirmation of all transformants was performed via PCR.The transformants were tested by amplifying AP, BP, and ORF fragments with three pairs of primers (c-atg8/p1 and pyrG/801R, pyrG/351F and c-atg8/p6, atg8-orf/F and atg8-orf/R), respectively.Those atg8 C transformants containing the AP, BP, and ORF fragments were successfully generated.

Aflatoxin B1 Extraction and Determination
For aflatoxin detection, 10 6 spores were inoculated into 8 mL of the YES liquid culture medium.After 5 days of incubation at 29 • C, 800 µL of the culture was mixed with an equal volume of dichloromethane, followed by centrifugation.Then, 700 µL of the lower liquid phase was collected and air-dried.Following drying, the tube walls were rinsed with 20 µL of dichloromethane, and the remaining 10 µL of the sample was analyzed using thin-layer chromatography (TLC) on a silica gel plate.AFB1 standard solution (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) was used as the control.The TLC results were recorded using a UV gel imaging system (BIO RAD ChemiDoc XRS, Hercules, CA, USA).Finally, Image J 1.8.0 was utilized to quantify the spots.

Stress Analysis
To investigate the role of ATG8 in A. flavus response to environmental stress, 2 µL of spore suspension was inoculated onto GMM solid medium supplemented with various stress-inducing agents: oxidative stress agents (6 mM and 8 mM H 2 O 2 ), cell membrane stress agents (0.005% and 0.01% Sodium dodecyl sulfate, SDS), cell wall stress agents (200 µg/mL and 300 µg/mL Calcofluor white (CFW); 200 µg/mL and 300 µg/mL Congo red, CR), and high osmotic stress media (1 M and 2 M KCl, 1 M and 2 M NaCl).The cultures were then incubated at 37 • C in darkness for 5 days.Afterward, colony diameters were measured, and inhibition rates were calculated.

ROS Measurement
To elucidate the impact of ATG8 on the levels of reactive oxygen species in A. flavus, 100 µL spore suspension (10 6 spores/mL) was inoculated into 900 µL of PDB medium and incubated at 37 • C for 12 h.Following this, A. flavus mycelia were harvested by centrifugation and washed 2-3 times with PBS.The mycelia were then treated with 10 µM 2 ′ -7 ′ -dichlorofluorescein diacetate (DCFH-DA) at 37 • C for 30 min in darkness and washed with PBS.Subsequently, fluorescence microscopy (Leica, Cellvizio system DualBand, Heidelberg, Germany) was employed to analyze the samples (Excitation = 488 nm; emission = 525 nm).

Seed Infections
In order to investigate the impact of ATG8 on the pathogenicity of A. flavus, peanut, and maize kernels were utilized in infection experiments.The peanut and maize kernels were first sterilized using 1% sodium hypochlorite and then rinsed three times with sterile water.Subsequently, they were sterilized with 75% ethanol and rinsed again three times with sterile water.Following this, five seeds were placed in a sterile culture dish, and each seed was inoculated with 10 µL of spore suspension (10 6 spores/mL).After a 5-day incubation period, infected seeds were assessed for conidia and aflatoxin production.

Real-Time Quantitative Reverse Transcription PCR
To evaluate the gene expression of the brlA pathway, A. flavus strains were grown in PDB at 37 • C under static liquid conditions for 48 h.To assess gene expression associated with aflatoxin production, strains were cultured in YES medium at 29 • C with shaking at 200 rpm for 48 h.For other RT-qPCR analyses, strains were cultured in PDB at 37 • C with shaking at 200 rpm for 48 h.The mycelium was collected and ground with liquid nitrogen.Subsequently, TRIzol (Invitrogen, Carlsbad, CA, USA) was employed to extract total RNA following the manufacturer's instructions.After RNA isolation, nanophotometer UV/Vis spectrophotometer (Implen, NanoPhotometer ® N50, Stuttgart, Germany) was employed to check the quality and quantity of RNA.cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA).The TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) was used in the Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA).Primers for gene expression detection are listed in Table 3, with the actin gene used as a reference in this experiment.

Statistical Analysis
In this study, all experiments were conducted independently at least three times.Error bars represent standard deviation.Statistical analyses employed two-tailed unpaired Student's t-test, two-way analysis of variance, followed by a multiple comparison test with Geisser-Greenhouse correction in GraphPad Prism 9, unless otherwise specified.Statistical significance was indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001.

Identification of ATG8 Protein in A. flavus and Prediction of the Tertiary Structure of ATG8 Protein
To elucidate the biofunction of ATG8 in A. flavus, we acquired the amino acid sequence of XP_002376260.1 from NCBI (http://www.ncbi.nlm.nih.gov/,accessed on 25 December 2021).Additionally, the amino acid sequences of ATG8 from various species (C.fimbriata, S. cerevisiae, A. oryzae, Homo sapiens, Mus musculus, A. fumigatus, P. oryzae, A. alternata, F. graminearum, N. crassa, Drosophila melanogaster) were retrieved through BLAST.Subsequently, a phylogenetic analysis of ATG8 proteins was conducted using MEGA 7.0.The analysis revealed that A. flavus ATG8 exhibited significant similarity to orthologs of A. fumigatus Af293, A. oryzae RIB40, and F. graminearum PH-1.Domain analysis revealed that the XP_002376260.1 protein contains the conserved domain Ubl_ATG8, showing structural similarity to known and validated ATG8 proteins (Figure 1A).Therefore, we named the XP_002376260.1 protein ATG8.Furthermore, the predicted tertiary structure of A. flavus ATG8 was successfully generated using SWISSMODEL (https://swissmodel.expasy.org/,accessed on 6 March 2022) (Figure 1B).

Deletion and Complementation of atg8 in A. flavus
The atg8 deletion and complementation mutants were created through homologous recombination (Figure 1C,E).The diagnostic PCR and RT-qPCR analysis were employed to confirm the successful construction of mutant strains.As illustrated in Figure 1D, AP and BP were obtained from the genomic DNA of the ∆atg8 strain, while the ORF could not be amplified.Conversely, AP, BP, and ORF fragments were successfully amplified from the genome of the atg8 C strain (Figure 1F).Subsequent RT-qPCR analysis further validated the deletion of the atg8 gene in ∆atg8 and its restored expression level in atg8 C (Figure 1G).These findings indicate the successful construction of the ∆atg8 and atg8 C strains.

ATG8 Is Crucial for Hyphal Growth and Conidiation in A. flavus
To investigate the impact of ATG8 on hyphal growth and conidiation in A. flavus, wildtype (WT) and mutant strains were cultured on various media (PDA, GMM, YES, and YGT) at 37 • C for 4 days.While colony growth of ∆atg8 was notably slower on GMM compared to WT and atg8 C , colony diameter remained unchanged for ∆atg8 mutants when cultured on PDA, YES, and YGT (Figure 2A,B).Additionally, Figure 2C illustrated a significant decrease in spore production in the ∆atg8 strain compared to WT and atg8 C across the four different culture media mentioned above.Microscopic examination revealed sparser conidiophores and smaller conidium heads in ∆atg8 compared to WT, with conidiophore morphology of atg8 C resembling that of WT (Figure 2D).Further assessment of conidia germination rates demonstrated significantly lower rates for ∆atg8 compared to WT and atg8 C at the 6th, 9th, and 12th hours (Figure 2F,G), indicating inhibition of spore germination due to atg8 deletion in A. flavus.Additionally, the measurement of expression levels of conidium-related genes (brlA, abaA and wetA) showed a sharp decrease in ∆atg8 mutants compared to WT and atg8 C (Figure 2E).Collectively, these findings confirm the significance of ATG8 in A. flavus development and sporulation formation.

ATG8 Contributes to Sclerotia Formation
A. flavus relies on the production of sclerotia to survive adverse environmental conditions [33].To investigate the importance of ATG8 in the sclerotium formation, WT and

ATG8 Contributes to Sclerotia Formation
A. flavus relies on the production of sclerotia to survive adverse environmental conditions [33].To investigate the importance of ATG8 in the sclerotium formation, WT and mutant strains were inoculated on GMMS media at 37 • C for 5 days.Figure 3A,B revealed a significant reduction in the number of sclerotia in ∆atg8 compared to WT and atg8 C strains.Further RT-qPCR demonstrated that the expression of sclerotium-related genes (sclR and nsdD) in the ∆atg8 mutants significantly decreased in comparison with those in WT and atg8 C (Figure 3C).These findings suggest that ATG8 is indispensable for sclerotium development in A. flavus.
J. Fungi 2024, 10, x FOR PEER REVIEW 10 of 18 mutant strains were inoculated on GMMS media at 37 °C for 5 days.Figure 3A,B revealed a significant reduction in the number of sclerotia in Δatg8 compared to WT and atg8 C strains.Further RT-qPCR demonstrated that the expression of sclerotium-related genes (sclR and nsdD) in the Δatg8 mutants significantly decreased in comparison with those in WT and atg8 C (Figure 3C).These findings suggest that ATG8 is indispensable for sclerotium development in A. flavus.

ATG8 Regulates Aflatoxin Biosynthesis in A. flavus
Aflatoxin B1 (AFB1), a notorious mycotoxin produced by A. flavus, contaminates different crops such as cotton and maize, and causes immense effects on the health of humans and animals [34,35].In order to reveal the effect of ATG8 on the AFB1 biosynthesis, we measured AFB1 production in WT, Δatg8, and atg8 C strains.TLC assays revealed that the Δatg8 mutant accumulated less AFB1 than the WT and atg8 C strains when cultured in YES media (Figure 4A,B).There are 29 genes associated with AF biosynthesis, stages: early, middle, and late stages (Figure 4C).Moreover, we evaluated the expression levels of AFrelated genes, which were significantly downregulated as depicted in Figure 4D.The results demonstrated that Δatg8 mutant strain disrupted the ability of A. flavus to produce AFB1.

ATG8 Regulates Aflatoxin Biosynthesis in A. flavus
Aflatoxin B1 (AFB1), a notorious mycotoxin produced by A. flavus, contaminates different crops such as cotton and maize, and causes immense effects on the health of humans and animals [34,35].In order to reveal the effect of ATG8 on the AFB1 biosynthesis, we measured AFB1 production in WT, ∆atg8, and atg8 C strains.TLC assays revealed that the ∆atg8 mutant accumulated less AFB1 than the WT and atg8 C strains when cultured in YES media (Figure 4A,B).There are 29 genes associated with AF biosynthesis, stages: early, middle, and late stages (Figure 4C).Moreover, we evaluated the expression levels of AF-related genes, which were significantly downregulated as depicted in Figure 4D.The results demonstrated that ∆atg8 mutant strain disrupted the ability of A. flavus to produce AFB1.

Response of ATG8 to Multiple Stresses in A. flavus
To characterize the role of ATG8 in fungal susceptibility to environmental stresses, we treated the Δatg8 with various inhibitors.Figure 5A,B show that the Δatg8 mutant exhibited heightened sensitivity to 200 µg/mL CFW, a cell wall stress agent.Furthermore, compared to the WT and atg8 C strains, the sensitivity of Δatg8 mutant to 300 µg/mL CFW, 200 and 300 µg/mL CR, also known as a cell wall stress agent, increased.Then, we determined the sensitivity of Δatg8 mutant to a cell membrane stress agent.Adding SDS to PDA media revealed a downregulation in sensitivity to 0.005% SDS for the Δatg8 mutant.In contrast, the Δatg8 mutant exhibited heightened sensitivity to 0.01% SDS compared to the WT and atg8 C strains (Figure 5C,D).Furthermore, we examined the response of A. flavus to hyperosmotic stress following ATG8 deletion by supplementing PDA media with

Response of ATG8 to Multiple Stresses in A. flavus
To characterize the role of ATG8 in fungal susceptibility to environmental stresses, we treated the ∆atg8 with various inhibitors.Figure 5A,B show that the ∆atg8 mutant exhibited heightened sensitivity to 200 µg/mL CFW, a cell wall stress agent.Furthermore, compared to the WT and atg8 C strains, the sensitivity of ∆atg8 mutant to 300 µg/mL CFW, 200 and 300 µg/mL CR, also known as a cell wall stress agent, increased.Then, we determined the sensitivity of ∆atg8 mutant to a cell membrane stress agent.Adding SDS to PDA media revealed a downregulation in sensitivity to 0.005% SDS for the ∆atg8 mutant.In contrast, the ∆atg8 mutant exhibited heightened sensitivity to 0.01% SDS compared to the WT and atg8 C strains (Figure 5C,D).Furthermore, we examined the response of A. flavus to hyperosmotic stress following ATG8 deletion by supplementing PDA media with KCl or NaCl.As shown in Figure 5E,F, the sensitivity of the ∆atg8 mutant decreased at all tested concentrations of KCl or NaCl.The abovementioned data indicate the involvement of ATG8 in responding to these stress stimuli in A. flavus.
KCl or NaCl.As shown in Figure 5E,F, the sensitivity of the Δatg8 mutant decreased at all tested concentrations of KCl or NaCl.The abovementioned data indicate the involvement of ATG8 in responding to these stress stimuli in A. flavus.In fungi, autophagy has been recognized as essential for oxidative stress resistance [36].Therefore, the resistance ability of Δatg8 mutant against oxidative stress was measured.Sensitivity assays on PDA revealed that Δatg8 was very sensitive to the oxidative agent H2O2 (Figure 6A,B).To further confirm that autophagy is involved in resistance to reactive oxygen species (ROS) in A. flavus., we cultured the strains in PDB at 37 °C for 12 h and observed them under fluorescence microscopy.The Δatg8 hyphae, stained with DCFH-DA, emitted significantly brighter green fluorescence compared to WT and atg8 C strains, indicating a higher accumulation of ROS in the Δatg8 strain (Figure 6C).RT-qPCR In fungi, autophagy has been recognized as essential for oxidative stress resistance [36].Therefore, the resistance ability of ∆atg8 mutant against oxidative stress was measured.Sensitivity assays on PDA revealed that ∆atg8 was very sensitive to the oxidative agent H 2 O 2 (Figure 6A,B).To further confirm that autophagy is involved in resistance to reactive oxygen species (ROS) in A. flavus, we cultured the strains in PDB at 37 • C for 12 h and observed them under fluorescence microscopy.The ∆atg8 hyphae, stained with DCFH-DA, emitted significantly brighter green fluorescence compared to WT and atg8 C strains, indicating a higher accumulation of ROS in the ∆atg8 strain (Figure 6C).RT-qPCR analyses also revealed significantly decreased expression levels of sod1 and yap1 in ∆atg8 (Figure 6D).These data indicate the involvement of autophagy in antioxidant stress systems.
analyses also revealed significantly decreased expression levels of sod1 and yap1 in Δatg8 (Figure 6D).These data indicate the involvement of autophagy in antioxidant stress systems.

Effect of ATG8 Mutants on Pathogenicity to Crop Seeds
A. flavus is known to produce aflatoxins on various crops, including corn, peanuts, cottonseed, and nuts, resulting in significant agricultural economic losses and posing health risks [37,38].The pathogenicity of ATG8 on crop seeds was detected.The result indicated that the Δatg8 mutant exhibited reduced mycelia vigor compared to the WT and atg8 C strains (Figure 7A).We further detected that in the conidia production from the infected crop seed, as shown in Figure 7B, compared to the WT and atg8 C strain, the conidia production of Δatg8 mutant is significantly reduced.All the above results suggested that the Δatg8 mutant is impaired in both infection and sporulation on crop seeds.Additionally, AFB1 production in infected crop seeds was quantified using TLC, and the results showed that the Δatg8 mutant produced little detectable AFB1 in the maize kernels and peanut seed compared to the WT and atg8 C strains (Figure 7C,D).These results underscore the crucial role of ATG8 in crop infection by A. flavus.

Effect of ATG8 Mutants on Pathogenicity to Crop Seeds
A. flavus is known to produce aflatoxins on various crops, including corn, peanuts, cottonseed, and nuts, resulting in significant agricultural economic losses and posing health risks [37,38].The pathogenicity of ATG8 on crop seeds was detected.The result indicated that the ∆atg8 mutant exhibited reduced mycelia vigor compared to the WT and atg8 C strains (Figure 7A).We further detected that in the conidia production from the infected crop seed, as shown in Figure 7B, compared to the WT and atg8 C strain, the conidia production of ∆atg8 mutant is significantly reduced.All the above results suggested that the ∆atg8 mutant is impaired in both infection and sporulation on crop seeds.Additionally, AFB1 production in infected crop seeds was quantified using TLC, and the results showed that the ∆atg8 mutant produced little detectable AFB1 in the maize kernels and peanut seed compared to the WT and atg8 C strains (Figure 7C,D).These results underscore the crucial role of ATG8 in crop infection by A. flavus.

Discussion
Autophagy is a highly conserved evolutionary process where proteins, membranes, and organelles are broken down and repurposed to sustain energy balance within eukaryotic cells [39,40].In this study, the ATG8, a marker to monitor autophagosome formation [36], was characterized in A. flavus.We identified the atg8 gene and found that the ATG8 protein plays a crucial role in various aspects of A. flavus biology, including vegetative growth, conidial development, pathogenicity, resistance to ROS, and AFB1 biosynthesis.
Autophagy is crucial for fungi, influencing their growth, morphology, and development.Studies in various fungi, such as S. cerevisiae [41], F. verticillioides [42], and A. alternata [36], have shown that mutations or deletions in the ATG8 gene lead to developmental defects or reduced vegetative growth.Our studies were consistent with recent research on ATG8 functions in fungi.Specifically, our study reveals that ∆atg8 mutants exhibit similar defects, with the deletion of atg8 resulting in reduced vegetative growth and developmental abnormalities in conidia.These findings collectively support the idea of the conserved roles of ATG8 in regulating cellular differentiation in both yeast and filamentous fungi.
Previous research has highlighted the crucial involvement of autophagy in pathogenicity [43,44].Histone acetyltransferase acetylates autophagy-related proteins, regulating both appressorium formation and pathogenicity in M. oryzae [39].Furthermore, inhibiting autophagy may decrease the pathogenicity of F. graminearum [30,45].In Colletotrichum fructicola, deletion of the cfatg8 and cfatg9 genes impaired appressorium function and caused defects in pathogenicity [46].Our study further demonstrates that ∆atg8 mutants in A. flavus exhibit pathogenicity defects.The reduced pathogenicity of ∆atg8 on maize kernels and peanut seeds may arise from various phenotypic abnormalities, including reduced hyphal growth and developmental defects of conidia.These findings highlight the significance of autophagy in pathogenicity.
The ability of fungi to infect hosts is closely linked to their capacity to withstand diverse environmental stresses.To investigate the involvement of ATG8 in A. flavus response to various environmental stresses, inhibitors such as KCl, NaCl, SDS, CFW, and CR were tested.The results indicate that ATG8 plays a significant role in A. flavus response to environmental stresses, including osmotic stress, cell membrane stress, and cell wall stress (Figure 5).The fluorescence microscopy showed that deletion of atg8 leads to the accumulation of high ROS levels.Furthermore, we observed downregulation of genes involved in ROS detoxification in the ∆atg8 mutant (Figure 6).Consistent with our results, in A. alternata, the ∆AaAtg8 failed to detoxify ROS effectively, resulting in ROS accumulation [25].Overall, our findings suggest that autophagy-mediated ROS detoxification plays a critical role in the oxidative stress response.
Aflatoxins have acutely toxic, immunosuppressive, carcinogenic, and teratogenic effects [47][48][49]; therefore, the prevention and control of aflatoxin are crucial.Our study revealed a striking decrease in AFB1 production in the ∆atg8 mutant compared to the WT and atg8 C strains, indicating that autophagy is necessary for AFB1 biosynthesis in A. flavus.Furthermore, RT-qPCR analysis revealed a downregulation of aflatoxin biosynthesis gene expression in the ∆atg8 mutant.It is speculated that ATG8 regulates AFB1 biosynthesis by controlling the gene cluster responsible for aflatoxin biosynthesis in A. flavus.
In summary, we identified the ATG8 protein and constructed atg8 deletion and complementation mutants using a homologous recombination strategy.We proposed that ATG8 is vital for the growth, conidial development, stress resistance, AFB1 biosynthesis, and pathogenicity of A. flavus.Given the crucial role of ATG8 in maintaining autophagy and pathogenicity, future investigations will explore ATG8-interacting proteins to enhance our comprehension of the autophagy regulatory network in A. flavus.

Figure 2 .
Figure 2. Effect of ATG8 on growth and conidia of A. flavus.(A) The growth of WT, Δatg8, and atg8 C strains on PDA, GMM, YES and YGT at 37 °C for 4 days in the dark; (B) Colonial diameter of WT, Δatg8, and atg8 C strains on the mentioned media; (C) Conidia count of WT, Δatg8, and atg8 C strains on the mentioned media; (D) Conidiophore morphology of WT, Δatg8, and atg8 C strains following 12 h of cultivation in darkness at 37 °C; (E) Relative expression levels of the conidium-related genes brlA, abaA, and wetA in WT, Δatg8, and atg8 C strains; (F) Conidium germination of WT, Δatg8, and atg8 C strains under dark conditions at 37 •C for 3, 6, 9, and 12 h; (G) Conidium germination rate of WT, Δatg8, and atg8 C strains at 3, 6, 9, and 12 h.**, *** and ns indicate p < 0.01, p < 0.001 and not significant, respectively.

Figure 4 .
Figure 4. ATG8 contributes to aflatoxin biosynthesis.(A) Thin-layer chromatography analysis of AFB1 production by WT, Δatg8, and atg8 C strains in YES liquid media after 5 days of culture at 29 °C; (B) Quantification analysis of AFB1 production in panel A; (C) AF gene cluster in A. flavus categorized into three stages; (D) Relative expression levels of genes related to AF biosynthesis.** and *** indicate p < 0.01 and p < 0.001, respectively.

Figure 4 .
Figure 4. ATG8 contributes to aflatoxin biosynthesis.(A) Thin-layer chromatography analysis of AFB1 production by WT, ∆atg8, and atg8 C strains in YES liquid media after 5 days of culture at 29 • C; (B) Quantification analysis of AFB1 production in panel A; (C) AF gene cluster in A. flavus categorized into three stages; (D) Relative expression levels of genes related to AF biosynthesis.** and *** indicate p < 0.01 and p < 0.001, respectively.

Figure 5 .
Figure 5. ATG8 involvement in A. flavus response to environmental stresses.(A) Fungal strains exposed to cell wall stress induced by CFW and CR on PDA for 4 days; (B) Growth inhibition rates under cell wall stress caused by CFW and CR; (C) Fungal strains under cell membrane stress induced by SDS on PDA for 4 days; (D) Growth inhibition rates under cell membrane stress caused by SDS; (E) Fungal strains under osmotic stress induced by KCl and NaCl on PDA for 4 days; (F) Growth inhibition rates under osmotic stress caused by KCl and NaCl.*, *** and ns indicate p < 0.05, p < 0.001 and not significant, respectively.

Figure 5 .
Figure 5. ATG8 involvement in A. flavus response to environmental stresses.(A) Fungal strains exposed to cell wall stress induced by CFW and CR on PDA for 4 days; (B) Growth inhibition rates under cell wall stress caused by CFW and CR; (C) Fungal strains under cell membrane stress induced by SDS on PDA for 4 days; (D) Growth inhibition rates under cell membrane stress caused by SDS; (E) Fungal strains under osmotic stress induced by KCl and NaCl on PDA for 4 days; (F) Growth inhibition rates under osmotic stress caused by KCl and NaCl.*, *** and ns indicate p < 0.05, p < 0.001 and not significant, respectively.

Figure 7 .
Figure 7. ATG8's role in A. flavus colonization on crops.(A) Colonization of peanut seeds and maize kernels by WT, Δatg8, and atg8 C strains at 29 °C in darkness for 5 days; (B) Conidia count statistics of the fungal strains on peanut seeds and maize kernels; (C) TLC analysis of AFB1 yield by the fungal strains on the kernels; (D) Quantification analysis of AFB1 production in panel C. ** and *** indicate p < 0.01 and p < 0.001, respectively.

Figure 7 .
Figure 7. ATG8's role in A. flavus colonization on crops.(A) Colonization of peanut seeds and maize kernels by WT, ∆atg8, and atg8 C strains at 29 • C in darkness for 5 days; (B) Conidia count statistics of the fungal strains on peanut seeds and maize kernels; (C) TLC analysis of AFB1 yield by the fungal strains on the kernels; (D) Quantification analysis of AFB1 production in panel C. ** and *** indicate p < 0.01 and p < 0.001, respectively.

Table 1 .
Aspergillus strains used in this study.

Table 2 .
Gene-specific primers used for deleted and complemented mutants.