A. flavus CA14Δku70ΔpyrG was used as the parental strain for transformation. CA14Δku70 was used as the wild type (WT) for phenotypic analysis. WT, mutant and revertant (RT) stains were cultured on YG or Minimal medium (MM) (Armitt et al., 1976). Five millimoles of uracil and uridine were added for the strains with pyrG auxotrophy. The spores were collected by using 0.2% Tween-20 (v/v) from plates with 48 h of incubation at 37°C. Mycelia was harvested from liquid medium cultivation at 37°C with shaking at 200 rpm, washed with distilled water, frozen in liquid nitrogen, and grounded using mortar and pestle. The mycelium powder was stored at -80°C for RNA extraction. S. cerevisiae EBY.VW4000 strain was cultured in YPM or S. cerevisiae EBY.VW4000 harboring plasmid pRS424-EGFP was grown in the synthetic medium (SD) supplemented with drop-out amino acids lacking tryptophan (SD-Trp^-), added 1% maltose and other carbon sources unless otherwise mentioned and incubated at 28°C.

The sugar transporter mutant Δ1982, Δ5374 and Δ9351 and RT strains were constructed by homologous recombination strategy. Each upstream and downstream flanking homologous arm (~1 kb) was generated by PCR. Likewise, the pyrG (~1.6 kb) fragment was PCR amplified from the pEXPYR plasmid. Three fused fragments were assembled in pCE-Zero vector. The deletion cassette was transferred to CA14Δku70ΔpyrG protoplasts. Transformants were screened on MM supplemented with 1 M sorbitol and then verified by PCR. For generation of RT constructs, RT constructing containing upstream flanking region, gene, pyrithiamine (PT) marker and pyrG were PCR amplified. PT fragment was PCR amplified from plasmid pPTRII. All the fragments were fuse-cloned to pCE zero vector and transferred to STP mutant protoplasts on MM screening plates supplemented with 0.1 µg/mL PT. The appeared colonies were screened and further PCR verified by primer pairs ( Supplementary Table S2 ).

The hexose transporter-deficient S. cerevisiae strain EBY.VW4000, which cannot grow on glucose but can grow on maltose, was used to validate the function of A. flavus STP genes. ORFs of G4B84_001982 and G4B84_005374 were amplified from A. flavus cDNA with BamH I sites and cloned into the BamHI-linearized yeast shuttle vector pRS424-EGFP (under the HXT7 promoter and terminator), generating plasmids pRS424-1982-EGFP and pRS424-5374-EGFP. Primer sequences are listed in Supplementary Table S2 . These plasmids were transferred to EBY.VW4000 strain by electroporation, and transformants were selected on SD-Trp^- medium. Integration was confirmed by PCR of genomic DNA using gene and GFP-specific primers. Single colonies were tested for growth on SD-Trp^- medium with glucose. Cultures were grown to OD₆₀₀ 0.8 - 1.0, washed with PBS, serially diluted (1:10), and spotted on SD-Trp^- plates containing different sugars. Plates were incubated for 3 days and photographed.

MM supplemented with 1% of various sugars, glucose, N-acetyl-D-glucosamine (GlcNAc), glucosamine (GlcN), fructose, xylose, galactose, sucrose, maltose, mannose, arabinose, glycerol, or ethanol as the sole carbon source was prepared to assess sugar utilization by mutant strains. Fresh conidia from WT, mutant, and RT strains were serially diluted (10⁵-10²), and 10 μL aliquots were spot-inoculated onto the media. Plates were incubated at 37°C and photographed after two days.

Conidia from WT, mutant, and RT strains were point-inoculated at the center of MM plates and incubated at 37°C. Colony diameters were measured along the same axis every 24 h for 10 days to assess growth rates, with colony morphology photographed on day 3 and 10. For conidiation analysis, conidia were harvested, washed and resuspended by using 0.2% Tween-20 and centrifuged at 5,000 rpm. Two-fold serial dilutions were prepared, and conidia were counted using a hemocytometer under a microscope.

Conidia (1×10⁵ CFU/mL) from WT, mutant, and RT strains were inoculated into 200 µL of liquid MM in 96-well plates and incubated at 37°C. Spore germination was observed under a microscope at 8, 10, and 15 hours. For each time point, ten random fields of view were examined, and the number of germinated versus total spores was counted to calculate germination rates.

WT, mutant, and RT strains were point-inoculated at the center of YG plates and incubated at 28°C in the dark for 7 days. Sclerotia were counted, and conidia and mycelia were washed with 75% ethanol prior to imaging. Each assay was performed in triplicate, and statistical analysis was conducted to assess differences in sclerotium formation between strains.

Serially diluted conidia (10⁵-10²) from each strain were point-inoculated onto MM plates containing various stress agents and incubated at 37°C. Stress conditions included 1.2 M sorbitol, 0.8 M NaCl, and 0.6 M KCl (osmotic stress); 5 mM H₂O₂ (oxidative stress); and 50 µg/mL Congo Red (CR) and 100 µg/mL Calcofluor White (CFW) for cell wall stress. Plates were photographed after 2 days.

Cell wall analysis was performed with slight modifications to a previously described method (François, 2006). A. flavus mycelia were cultured in MM liquid medium at 37°C, 200 rpm for 48 h, harvested by filtration, and ground in liquid nitrogen. The resulting powder was treated with SDS-BME buffer (50 mM Tris, 50 mM EDTA, 2% SDS, 1 mM TCEP) and boiled for 40 minutes. Cell wall fractions were washed with Milli-Q water until foam disappeared and freeze-dried. Ten milligrams of dried wall material were hydrolyzed with 75 μL of 75% H₂SO₄ at room temperature for 3 hours, diluted to 2 N H₂SO₄ with 0.95 mL Milli-Q water, and boiled at 100°C for 4 hours. The hydrolysate was neutralized using Ba(OH)₂, and BaSO₄ precipitates were removed after overnight incubation at 4°C. The supernatant was analyzed for monosaccharide content by HPAEC-PAD using a CarboPac PA10 column with an AminoTrap guard column, eluted at 1 mL/min with 18 mM NaOH at room temperature.

Virulence assays in Galleria mellonella infection model were conducted according to previously described method (Ahamefule et al., 2020; Champion et al., 2016). To assess virulence in G. mellonella, larvae were divided into control (0.02% Tween-20), WT, mutant, and RT groups, with 90 larvae per group. Each larva was injected with 10 µL of 1×10⁶ CFU/mL conidial suspension into the hind proleg using a Hamilton syringe. Larvae were incubated at 37°C, and survival rate was recorded at 24-, 48-, and 72-h post-infection. Larvae were considered dead if immobile with signs of melanization or darkening.

The pathogenicity of mutant strains on crop seeds was assessed as previously described (Yang et al., 2018). Uniform-sized fresh corn and peanut seeds were selected, and endosperm was removed using toothpicks to prevent germination and provide infection site. Seeds were surface-sterilized with 0.05% sodium hypochlorite for 3 min, followed by 75% ethanol for 1 min, and rinsed three times with sterile water. Sterile seeds were placed in 100 mL flasks and inoculated with 1×10⁶ CFU/mL conidia of each strain. After incubation at 28°C for 30 minutes, seeds were cultured in the dark for 6 days with constant humidity maintained by wet filter paper. Post-infection, seeds were transferred to 50 mL tubes containing 20 mL of 0.2% Tween-20 and shaken vigorously for 5 min to release conidia. Spore suspensions were serially diluted, and spores were counted using a hemocytometer. Each experiment was performed in triplicate and repeated three times.

Aflatoxin was extracted from 500 µL of culture filtrate using an equal volume of chloroform. The organic layer was collected, evaporated at 70°C, and analyzed by thin-layer chromatography (TLC). A solvent system consisting of acetone: chloroform (1:9, v/v) was used. Aflatoxins were visualized under UV light at 365 nm.

All statistical analyses were conducted using GraphPad Prism 8. Data are presented as mean ± standard deviation (SD). Two-group comparisons were analyzed using a Student’s t-test, while multiple comparisons were assessed using one-way ANOVA and pot hoc tests Dunnett’s was conducted.

A BLASTp search of the A. flavus genome using S. cerevisiae hexose transporters and sensors (Snf3 and Rgt2) identified approximately 100 candidate proteins, from which the top seven hits were selected for further analysis ( Supplementary Table S1 ). These candidates, ranging from 513 to 672 amino acids in length, all belong to the STP subfamily of the MFS family.

To assess their expression dynamics, RT-PCR was performed at 0, 8, 24, and 48 h in the presence of 1% glucose ( Figure 1A ). Among the seven candidate genes, G4B84_001982 showed the highest transcript levels across all stages, followed by G4B84_005374, which peaked during germination and hyphal growth. These patterns suggest that the corresponding MFS transporters may play important roles in fungal development and adaptation.

Phylogenetic analysis supported their functional classification, revealing that these transporters cluster with known fungal sugar transporters ( Supplementary Figure S1 ). Specifically, XP_041141740.1 (encoded by G4B84_001982) grouped with N. crassa Rco-3 (a high-affinity glucose transporter), T. reesei Str1 (xylose transporter), A. niger XltA, and A. nidulans MstC, while XP_041145042.1 (encoded by G4B84_005374) and XP_041148888.1 (encoded by G4B84_009351) clustered with C. graminicola Hxt2, another high-affinity glucose transporter.

Structural analysis using TMHMM and PROTTER predicted XP_041141740.1, XP_041145042.1 and XP_041148888.1 harbored a conserved structure of 12 transmembrane α-helices with cytoplasmic N- and C-termini, a hallmark of MFS transporters (Saier, 1999). Intriguingly, while most candidates had short C-termini, XP_041148888.1 possessed an unusually long 173-amino-acid C-terminal tail, a typical feature of sugar sensors ( Figures 1B, C ), suggesting a potential sugar-sensing function in A. flavus.

Although MFS sugar transporters share low amino acid sequence similarity (12 - 18%), they possess a highly conserved architecture comprising 400 – 600 amino acid residues and 12 transmembrane (TM) helices arranged in N- and C-terminal domains that ensure efficient transport activity (Madej et al., 2014). For example, TMs 1, 4, 7, and 10 are critical for carbohydrate transport, with many residues directly interacting with substrates; TMs 2, 5, 8, and 11 link the N- and C-terminal domains and contribute to substrate binding and translocation; and TMs 3, 6, 9, and 12 provide structural stability. MFS transporters typically share the same three-dimensional fold and functional characteristics (Law et al., 2008; Taveira et al., 2024 ( Supplementary Figures S2A, B ).

Consistent with other hexose transporters, these proteins contain the Sugar Porter signature motifs, including the conserved D(N)RXGRR sequences between TM2-TM3 and TM8-TM9, and the PESPR motif at TM6 (Law et al., 2008). Most residues within these motifs are charged or polar, forming an extensive hydrogen-bond network that mediates interactions between the TM helices and the intracellular domains. Additionally, an aromatic residue–rich sequence (YFFYY) and the signature motif GR- - -G-G-G- - - - - -P-Y-SE-AP- -RG- - - - - -QL-TT-G (indicated by black bars in Supplementary Figure S2C ) are conserved (Taveira et al., 2024). Notably, single point mutations of these conserved motif residues in bacterial homologs of glucose transporters (GLUT1 - 4) abolish transport activity entirely (Sun et al., 2012). Collectively, these structural and sequence analyses strongly suggest that G4B84_001982, G4B84_005374 and G4B84_009351 are functional sugar transporters or sensors involved in sugar uptake in A. flavus.

For function analysis, mutants of G4B84_001982, G4B84_005374 and G4B84_009351 were generated via gene replacement using pyrG as a selectable marker ( Supplementary Figure S3 ). Sugar substrate specificity assays ( Figure 2A ) showed that the Δ1982 mutant exhibited severe growth defects in the presence of all tested hexoses (glucose, fructose, sucrose, mannose, and maltose), pentoses (xylose and arabinose), and amino sugars (GlcNAc and GlcN). Additionally, Δ1982 failed to grow on non-fermentable carbon sources such as ethanol and glycerol, indicating that this transporter may be involved in the uptake of a broad range of sugars and carbon sources.

The Δ5374 mutant displayed markedly reduced colony growth and an albino phenotype on monosaccharides such as glucose, fructose, and mannose, as well as the disaccharide sucrose. However, its growth was not affected by alternative carbon sources, suggesting a selective, yet biologically significant role in sugar transport.

In contrast, the Δ9351 mutant exhibited only minor defects in colony expansion and conidiation, with no substantial growth impairment across all the tested sugars. Despite its long C-terminal region - a signature of sugar sensors – the deletion of G4B84_009351 did not result in a notable phenotype under these conditions. Consequently, subsequent analyses focused on the two mutants with pronounced phenotypic alternations: Δ1982 and Δ5374.

To evaluate the impact of these mutations on fungal growth, colony diameters were compared among the strains ( Figures 2B, C ). The Δ1982 displayed the most pronounced growth defect at 72 h and persisted throughout the observation period. Although Δ5374 grew better than Δ1982, both mutants displayed rough, irregular colonies lacking the radial symmetry and smooth margins observed in the WT strain. Furthermore, the condition was significantly impaired in both mutants ( Figure 2D ). Collectively, these findings suggest that the STP genes, particularly G4B84_001982 and G4B84_005374, play essential roles in sugar utilization, growth, and asexual development in A. flavus.

In liquid MM medium, STP mutant strains exhibited delayed germination compared to the WT strain. At 8 h, WT conidia had begun to swell and initiate germ tube formation, whereas the mutant strains had not yet reached the swelling stage. By 10 h, small hyphal extensions were observed in the WT, with a complete hyphal network established by 15 h, marking the transition to active mycelial growth. In contrast, the mutant strains showed significantly reduced germination rates, with no observable hyphal network formation. Specifically, Δ1982 exhibited less than 50% germination, while Δ5374 reached only 62%, compared to 100% germination in both WT and RT strains. These results demonstrate that deletion of STP genes significantly impairs conidial germination and early hyphal development in A. flavus ( Figures 3A, B ).

To investigate the functional roles of A. flavus STPs, the coding sequences of G4B84_001982 and G4B84_005374 were cloned into the yeast expression vector pRS424-EGFP and subsequently transformed into the S. cerevisiae hexose transporter-deficient strain EBY.VW4000. Confocal microscopy confirmed that both STPs were correctly localized to the plasma membrane in yeast ( Figure 4B ). Transformants were selected on maltose medium, which served as a positive control, while the strain carrying the empty vector served as a negative control.

Drop assay on SD-Trp^- plates revealed that expression of 1982 and 5374 restored the growth of EBY.VW4000 on glucose, confirming their function as glucose transporters ( Figure 4A ). Notably, strain 1982 also supported growth on fructose, GlcNAc, sucrose, mannose, xylose, and galactose, indicating broad substrate specificity. In contrast, strain 5374 facilitated growth on sucrose and mannose, suggesting a more limited but overlapping sugar transport capability ( Figure 4 and Supplementary Figure S5 ). These findings demonstrate that the two STPs mediate uptake of multiple sugar substrates when expressed in yeast.

Since sugars are key precursors for cell wall polysaccharide biosynthesis, we investigated whether disruption of STP would affect the cell wall integrity (CWI) of A. flavus. As shown in Figure 5A , the growth of the mutants was markedly inhibited in the presence of CR and CFW. To quantitatively assess CWI defects, we measured the cell wall components of each strain in glucose-grown cultures ( Figure 5B ). Compared to the WT strain, both Δ1982 and Δ5374 showed significant reductions in glucan (by 50% and 35%, respectively) and galactomannan (by 83% and 70%, respectively) content. Interestingly, Δ1982 exhibited a 42% increase in chitin content, while Δ5374 showed a 27% decrease. Mannan content was slightly reduced in Δ1982 (4%) but decreased substantially in Δ5374 (46%). The observed reduction in glucan content aligns with the CR sensitivity of both STP mutants ( Figure 5A ), further supporting our finding that two STPs are engaged in maintaining CWI in A. flavus.

Given the observed CWI defects, we further assessed the sensitivity of STP mutants under various environmental stress conditions. As shown in Figure 5A , both Δ1982 and Δ5374 mutants exhibited pronounced sensitivity and significantly impaired growth under ionic (0.8 M NaCl, 0.6 M KCl) and non-ionic (1.2 M sorbitol) osmotic stress, as well as oxidative stress induced by H₂O₂. In contrast, the WT strain maintained normal growth under these conditions. The higher inhibition rates observed in the mutant strains indicate that these STP genes are critical for mediating resistance to osmotic and oxidative stress. These findings suggest that STP-mediated sugar uptake plays an essential role in enabling A. flavus to adapt to hostile environments, potentially contributing to its survival and pathogenicity under adverse conditions.

The role of STPs in sclerotia formation was further examined. The Δ1982 showed a near-complete abolition of sclerotia formation, and Δ5374 produced very few sclerotia, both before and after ethanol wash, while abundant sclerotia were observed in the WT and RT strains ( Figure 6A ). Quantitatively, Δ1982 and Δ5374 exhibited 99% and 85% reductions in sclerotia production, respectively ( Figure 6B ). These findings suggest that STP deletion impairs A. flavus adaptation to adverse conditions, consistent with their heightened sensitivity to chemical and osmotic stresses.

The above findings highlight the critical role of STP genes in growth, conidiation, sclerotia formation, and stress responses-key factors for survival, host invasion, and pathogenicity. We therefore hypothesized that deletion of STPs may compromise the pathogenicity of A. flavus.

After 6 days of inoculating corn and peanut seeds with WT, mutants, and RT strains, seed colonization by Δ1982 and Δ5374 was visibly reduced compared to WT and RT strains ( Figure 7A ). Conidia recovered from infected seeds confirmed a significant reduction in spore production by the mutants ( Figure 7B ). We hypothesized that this colonization defect might result from the mutants inability to utilize seed-derived nutrients. To test this, growth assays were performed on corn or peanut powder agar plates. Δ1982 failed to grow on peanut powder, and Δ5374 showed limited growth on corn powder, supporting the notion that these mutants are nutritionally impaired ( Supplementary Figure S4 ).

A. flavus produces AFB1 which poses significant threat to humans, animals and plants. Furthermore, keeping the important physiological and cellular role of STP genes in A. flavus, we analyzed the amount of AFB1 accumulated in the infected seeds. TLC analysis revealed that AFB1 was only accumulated in the seeds infected by the WT and RT strains, whereas no AFB1 was detected in the seeds infected by Δ1982 and Δ5374 ( Figure 7C ). These results clearly demonstrate that the deletion of STPs resulted in its inability to colonize and grow on crop seeds, and AFB1 could not be accumulated in the mutant infected crops. Therefore, studying and targeting A. flavus STP genes more in depth might be a practical strategy to reduce aflatoxin contamination.

A. flavus is a major cause of invasive aspergillosis and superficial infections, relying on sugar acquisition from the host to establish infection. To assess the role of STPs in virulence, G. mellonella larvae were infected with WT, STP mutants, and RT strains, and survival rates were monitored over 72 h using Kaplan-Meier analysis ( Figure 8 ). The mutants showed significantly reduced virulence compared to WT and RT strains. At 48 h post-infection, over 50% mortality was observed in larvae infected with WT or RT strains, whereas mortality rate was only 44% for Δ5374 and only 10% for Δ1982. By 72 h, survival rates were 6 - 10% for WT, but remained high at 90% and 30% for Δ1982 and Δ5374, respectively. These results indicate that STP genes are important for A. flavus pathogenicity in this model.