The FlbC transcription factor contributes to the generation of strain heterogeneity in Fumagillin mycotoxin production in Aspergillus fumigatus

doi:10.21203/rs.3.rs-4435109/v1

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The FlbC transcription factor contributes to the generation of strain heterogeneity in Fumagillin mycotoxin production in Aspergillus fumigatus

https://doi.org/10.21203/rs.3.rs-4435109/v1

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Aspergillus fumigatus shows inter-strain heterogeneity in the repertoire of secondary metabolites such as mycotoxin fumagillin but the mechanism by which this heterogeneity arises in those production is still not understood. Here, we investigates the effect of the flbC gene on fumagillin production in A. fumigatus by introducing flbC deletions into laboratory strains, A1151, A1159, and A1280 from different backgrounds and examines the effect of the flbC gene on inter-strain heterogeneity. We found that, although all three laboratory strains were capable of producing fumagillin, there was heterogeneity in the effects of flbC gene deletion on fumagillin production. This heterogeneity may be dependent on differences in the expression levels of the fma gene family involved in fumagillin production and different levels of transcriptional activation by transcription factors FapR and LaeA. The flbC gene expression level peaks at the same time as peak mycelial growth but shows differential expression level, suggesting that the differences among strains in the range of expression levels are dependent on differences in the upstream expression of flbC. Thus, our findings show that the different interactions between flbC and factors regulating the expression of fumagillin gene cluster are the probable cause for heterogeneity in fumagillin production in the organism.

Aspergillus fumigatus

flbC

fumagillin

secondary metabolism

heterogeneity

Aspergillus fumigatus is an opportunistic pathogen, which infects human beings. Although A. fumigatus infection in healthy individuals has not established, persons with a history of chemotherapy, immunosuppressive drug use, or congenital immunodeficiency syndrome can have lethal lesions¹. The human-pathogenicity characteristic of A. fumigatus is determined by the interaction of several virulence factors in the organism. The mycelia produce secreted proteins², polysaccharides³, and a wide variety of secondary metabolites⁴. In recent years, inter-species, inter-strain, and even individual spore heterogeneity in these phenotypes and genotypes has been noted^5–10. Heterogeneity has also been observed between clinical and environmental isolates¹¹. In basic research, several strains with different backgrounds are currently used as reference strains; however, notable differences have been reported between these strains in terms of pathogenicity¹², genome structure^12,13, secondary-metabolite production capacity¹⁴, single nucleotide polymorphisms (SNPs) ¹⁵, chromatin modifications¹⁶, and transposable element distribution¹⁶. Vigorous research is now underway to understand how this heterogeneity arises between species or strains, mainly through large-scale data. Af293 is the most commonly used A. fumigatus strain for phenotyping in basic research because it was the first strain to have its entire genome sequenced and is currently the only strain that has been allocated to the chromosome¹⁷. In addition, CEA17¹⁸, D141¹⁹, and ATCC46645²⁰ strains have been established and used as laboratory strains. Heterogeneity among strains has made it more complicated to understand the physiology of the species by comparison between each study.

Although the secondary metabolites produced by Aspergillus spp. are not explicitly associated with pathogenicity in humans, a correlation between the repertoire of secondary metabolites that can be produced and pathogenicity has been reported²¹. Inter-species and inter-strain heterogeneity is also observed in the repertoire of secondary metabolites²¹. Many A. fumigatus strains that secrete fumagillin, pseurotin, fumitremorgin, and gliotoxin are pathogenic, and closely related species of A. fumigatus, A. oerlinghausenensis and A. fischeri can produce fumagillin and pseurotin as secondary metabolites; however, these organisms are not pathogenic to humans. Comparisons between strains with different repertoires of secondary metabolite secretion have shown that strains with a more severe clinical course tend to secrete a greater variety of metabolites. Although no single secondary metabolite is known to determine the human pathogenicity of A. fumigatus, the relationship between strain heterogeneity in secondary-metabolite production repertoire and pathogenicity remains unclear.

The fma gene family^22,23, required for fumagillin biosynthesis in A. fumigatus, forms a cluster in the subtelomeric region of chromosome 8²⁴. The gene cluster has eight Fma enzymes that catalyze sequential reactions to biosynthesize the end product and the transcription factor FapR/FumR^24,25, which positively regulates the expression of the fma gene family upstream. Because fumagillin precursors are secreted by strains lacking each gene of the fma gene family, each fma gene is essential for the biosynthesis of fumagillin²³. Loss of the gene encoding the histone methyltransferase LaeA affects the production of fumagillin and other secondary metabolites^26,27. Inside the fumagillin gene cluster, there is an intertwined group of genes involved in pseurotin biosynthesis, which is synchronously regulated by FapR and LaeA²⁴. In addition, a gene cluster for the biosynthesis of fumitremorgin is present in the neighborhood²⁴, suggesting that fumagillin-pseurotin-fumitremorgin is synchronously affected by the regulation of chromatin structure. Chromosome 8 has a lower frequency of trimethylation modifications of the ninth lysine residue of histone H3 than other genomic regions and has many LINE transposon sequences¹⁶, suggesting that the area around the fumagillin gene cluster may be a region of relatively high genomic instability. Consistent with this, approximately 60% of A. fumigatus isolates have lost a part of or almost all the fumagillin gene cluster in the subtelomeric region of chromosome 8¹⁵. Thus, the role of the fma gene cluster and its upstream transcription factors in the production of fumagillin, a typical secondary metabolite, is becoming clearer; however, the mechanism by which this heterogeneity arises in the fumagillin production is not yet known.

Secondary metabolite production is often regulated synchronously between several secondary-metabolite-producing systems rather than independently of each other²⁸. Lind et al. reported that the BrlA transcription factor, which is central to the central developmental pathway, affects the expression of almost all secondary metabolite products²⁹. The central developmental pathway is based on sequential transcriptional activation of a series of transcription factors, namely BrlA, AbaA, and WetA, which are important for the transcription of genes involved in morphogenesis³⁰. During the asexual life cycle of A. fumigatus, dormant spores first swell and germinate to form filamentous mycelia, followed by the asexual reproductive spores. These series of shifts are synchronized with the continuous activation of the central developmental pathway. The comparison of the mRNA expression profiles of laeA- and brlA-gene deficient strains and the secondary-metabolic production profiles have shown that despite the high correlation between the two profiles and the fumagillin-fumitremorgin-pseurotin gene cluster, there is negative correlation between the expression patterns of the these gene clusters²⁹. The central developmental pathway regulated by BrlA is further regulated upstream by several Flb transcription factors, which are together termed the upstream developmental activator (UDA) pathway. In Aspergilus nidulans, FlbB, FlbC, and FlbE are transcription factors that constitute the UDA pathway³¹. FlbB and FlbE cooperate, while FlbC alone activates the transcription of brlA³¹. In A. fumigatus strains deficient in flbB and flbE genes, reduced brlA gene expression, delayed mycelial growth, and reduced fumagillin and gliotoxin production have been observed³². However, the effects of the flbC gene on A. fumigatus growth, brlA gene expression, and secondary metabolite production have not yet been investigated in detail. In addition, in the aforementioned comparisons of brlA, laeA, and flbB/E gene-deficient strains, experiments were conducted using a single reference strain for each strain and inter-strain heterogeneity was not considered.

Therefore, this study aimed to investigate the effect of the flbC gene on fumagillin production in A. fumigatus by introducing flbC deletions into several laboratory strains from different backgrounds and to consider the effect of the flbC gene on inter-strain heterogeneity. In particular, changes in the expression pattern of the brlA gene over time, fumagillin production capacity, expression of the fma gene cluster, and expression of the upstream transcription factors laeA and fapR were compared.

Expression patterns of the flbC gene in the three strains

A1151, A1159, and A1280 as laboratory strains with suppressed non-homologous repair for genetic modification were deposited in FGSC. The strains CEA17, D141, and ATCC46645 were the parental strains of A1151, A1159, and A1280, respectively³³. The flbC gene in strain Af293 (Afu2g13770) was searched in the draft genomes of strains CEA17 and found AFUB029400-T. Using this sequence as a reference, primers were designed to amplify the entire CDS length of the flbC gene. Amplification was attempted using the genomic DNA of the three strains as a template, and amplification products were obtained in all groups. The sequences in the CDS of the amplified products were checked for point mutations and were found to be completely homologous in all three strains, suggesting that there is probably no difference in the amino acid sequence of the FlbC protein produced in the three strains. Next, to compare the expression patterns of the flbC gene in the three strains, RNA was extracted from spores and mycelia obtained at different incubation times, and the relative amounts of flbC gene transcripts were quantified over time. The flbC gene transcript was not amplified immediately after the start of incubation, when almost all spores formed buds, but was detected after approximately 8 h and reached peak expression in all three strains after 16 h (Fig. 1). Thereafter, a gradual downward trend was observed in A1151 and A1280, followed by a slight increase in A1159 at 72 h. These trends are consistent with previous research, in which the flbC transcript levels in A. nidulans during vegetative growth peaked at approximately 18–24 h and decreased thereafter³⁴. However, some heterogeneity appeared among the three strains, with expression ratios of approximately 3-fold in A1151, 4-fold in A1159, and 9-fold in A1280 at 16 h. In summary, the flbC gene appears to reach peak expression level approximately 16 h after the start of culture, that is, at the same time as peak mycelial growth. Comparison of three representative laboratory strains suggested that the CDS internal sequence of the flbC gene and the timing of the maximum expression level were generally the same, but the range of expression changes was slightly different. These results suggest that the differences among strains in the range of expression changes are dependent on differences in upstream expression of flbC.

Effect of flbC expression heterogeneity on strain phenotype

To investigate the effect of heterogeneity in the flbC expression pattern on the phenotype of the three laboratory strains, we produced flbC gene disruption mutant strains (ΔflbC) in each background. Because the internal sequence of flbC was exactly the same, we planned highly efficient gene disruption using the CRISPR-Cas9 method with the PAM sequence common to the three strains. The ΔflbCs were generated by inducing double-strand breaks via Cas9 (CRISPR#1, 2) binding, followed by insertion of pyrithiamine resistance gene markers through homologous recombination, using each of the three strains as parent (Fig. 2a, b). ΔflbCs were slightly reduced at radial growth circle than the parental strains (WT) on MM at 37°C (Fig. 2c). The three WT strains grew differentially under these conditions, with the A1280 strain growing slightly weaker on day 3 or earlier than the other two. The radial growth of the flbC gene-deficient strain was almost identical to this trend. Notably, when grown with the cell wall effector Congo red, A1280 showed weaker growth among the WT strains; however, growth was restored by flbC gene deletion (Fig. 2d). This was in contrast to A1151 and A1159, whose growth was suppressed by Congo red treatment. These findings suggest that the differences in the growth phenotypes observed in the three laboratory strains may be due to the function of the flbC gene.

Effect of flbC expression patterns on brlA expression

BrlA, which plays a central- and entry point- role in the central developmental pathway, is regulated by FlbC transcription factor^30,34. To investigate how different expression patterns of the flbC gene affect the brlA expression, we measured brlA transcript over time in WT and ΔflbC of the three laboratory strains. WT of the A1151 and A1159-background reached peak mRNA expression at 16–24 h, with an approximate 3- to 4-fold increase than the expression at 4 h, whereas this increase was suppressed in the ΔflbC (Fig. 3, upper and middle panel). In contrast, the WT of A1280-background showed little increase in brlA gene expression until 48 h after the start of incubation, and ΔflbC in the A1280-background also showed little change in brlA gene expression (Fig. 3, bottom panel). In summary, brlA gene expression level in WT of A1151 and A1159 peaked at 16–24 h, whereas this peak was removed by flbC gene deletion. In contrast, A1280 showed a flat expression up to 48 h in brlA gene expression in both WT and ΔflbC rather than a peak-like pattern. These results suggest that the brlA gene expression pattern is different among the three laboratory strains and that there is heterogeneity in the regulatory role of the flbC gene on brlA gene expression.

Effect of flbC gene on the heterogeneity of secondary metabolite production

To investigate the effect of the flbC gene on the heterogeneity of secondary metabolite production, we measured the fumagillin-production capacity of these strains. Remarkably, given that previous studies have shown the loss of fumagillin-producing capacity in several A. fumigatus strains¹⁵, all three WT strains produced fumagillin (Fig. 4). The effect of flbC gene deletion on fumagillin production was tripartite: in A1151, fumagillin production was below the detection limit due to flbC gene deletion; in A1159, fumagillin production was reduced but not eliminated due to flbC gene deletion; and A1280 produced sufficient amount of fumagillin in both WT and ΔflbC, with little difference in production level (Fig. 4). These results indicate that although all three laboratory strains are capable of producing fumagillin, there is heterogeneity in the effects of the flbC gene deletion. To investigate whether the heterogeneity in producing fumagillin is dependent on differences in the expression levels of the genes responsible for fumagillin biosynthesis, we measured the transcripts belonging to fma gene family involved in the fumagillin gene cluster (fma-TC / fmaA, fma-P450 / fmaG, fma-C6H / fmaF, fma-AT / fmaC, and fma-ABM / fmaE) and those not involved in fumagillin biosynthesis but located within the gene cluster (Afu8g00430, Afu8g00440 / psoF and Afu8g00500). In A1151-background, the expression levels of many fumagillin biosynthesis-related genes, including fma-TC / fmaA, fma-P450 / fmaG, fma-C6H / fmaF, and fma-AT / fmaC, were significantly reduced in ΔflbC than those in WT (Fig. 5a). Some genes not involved in fumagillin biosynthesis were also downregulated (Fig. 5b). A comparison of the A1159 background strains showed the same trend, except for fma-AT / fmaC (Fig. 5a). In contrast, comparisons of A1280 background strains showed significant differences in the reduced expression of fma-TC / fmaA and fma-C6H / fmaF, but the extent of the reduction was weaker than that in A1151 and A1159, and differences in the expression of other fma genes was not significant (Fig. 5a). In summary, in the ΔflbC of A1151 and A1159 backgrounds, the expression of the fma gene family was generally and coordinately suppressed, consistent with the reduced fumagillin production, while in A1280, where flbC gene deletion did not result in reduced fumagillin production, the degree of suppression of fma gene family expression was weaker than in the other two strain. Thus, the heterogeneity in the three laboratory strains between the WT and ΔflbC with regard to fumagillin production may be dependent on differences in the expression levels of the fma gene family and probably correlates with the degree of hetero-chromatinization of the fumagillin gene cluster.

Expression of the upstream transcription factors laeA and fapR

The production of secondary metabolites requires the coordinated action of global and cluster-specific transcription factors. In the case of fumagillin, the transcriptional activation of biosynthetic genes is achieved by the histone methyltransferase LaeA, which acts as a global transcription factor, and FapR, which acts as a cluster-specific transcription factor. The fapR gene is located within the fumagillin gene cluster, and production of the FapR transcription factor is initiated by the release of heterochromatin from the cluster by LaeA. To determine whether there are differences in the upstream gene of the fma gene family, we measured the expression levels of laeA and fapR genes in the A1151 and A1280 backgrounds between WTs and ΔflbCs, which had contrasting difference in the fumagillin production. In the A1151-background strains, laeA gene expression peaked after 16–24 h and then decreased in the WT, whereas the peak was delayed to 48–72 h in the ΔflbC (Fig. 6a, left panel). In contrast, in A1280, the expression of the laeA gene peaked at 16 h in the WT as same as that in A1151, while the ΔflbC showed a similar pattern to that of the WT (Fig. 6a, right panel). The fapR gene showed reduced gene expression at 16–24 h in the ΔflbC than that in the WT of the A1151 background (Fig. 6b, left panel), while there was no significant difference in expression levels at 24 h in the A1280 background (Fig. 6b, right panel). In summary, in the A1151 background strain, the flbC gene deletion resulted in delayed expression of the laeA gene and suppressed the expression of fapR, but not in the A1280 background. These results indicate that the heterogeneity in fumagillin production between strains A1151 and A1280 may be due to the different levels of transcriptional activation of FapR and LaeA. More specifically, this suggests a dependence on different direct or indirect interactions between flbC and genes regulating the expression of the fumagillin gene cluster, such as laeA and fapR.

Effect of heterogeneities on virulence

The analysis of the three laboratory strains shows that the deletion of the flbC gene may create new heterogeneity or reduce that which was present originally. To estimate the effects of these heterogeneities on virulence, a pathogenicity test was conducted on silkworms. We found that the deletion of the flbC gene probably does not contribute to silkworm-pathogenicity of the strains because there was no significant difference in the survival among WT and ΔflbC of any background (Fig. 7). Deletion of the flbC gene reduced the growth of the laboratory strains on MM and showed differences in the production of the mycotoxin fumagillin; however, these differences were not observed in the pathogenicity test.

This study shows that the flbC gene in A. fumigatus may increase or decrease the heterogeneity of hyphal growth, the expression patterns of brlA transcripts and secondary metabolites. FlbC is a C₂H₂ transcription factor that is widely conserved in Ascomycetes, but not in Basidiomycetes and Zygomycetes³⁴. The flbC gene has been found in A. nidulans³⁴, A. flavus³⁵, Trichoderma guizhouense³⁶, and Beauveria bassiana³⁷. In A. oryzae, it regulates the production of amylolytic enzymes, such as glucoamylase and α-amylase, during individual media growth³⁸. Fusarium graminearum deficient of flbC gene showed no phenotype³⁹. FlbC regulates brlA gene expression, along with the concerted action of FlbB, FlbD, and FlbE³⁰. Thus, the extent to which any flb family molecule contributes to the asexual life cycle varies among species. These Flbs also have differnt localizations in fungal cells, with FlbB and FlbE tending to accumulate at the tips of filamentous fungi, while FlbC is present in all nuclei, including metulae and phialides³⁴. This is not surprising, given that the plasticity and velocity of filamentous growth, as well as the cell wall and cell surface structures, differ between species⁴⁰. Genomic heterogeneity between clinical and environmental isolates is known, and transcriptome heterogeneity exists within the same strain, even within individual spores^5,9–11. The reason for the various levels of heterogeneity arising in A. fumigatus will become increasingly clear in future studies.

As we showed, in the A1151 and A1159 backgrounds, loss of the flbC gene was associated with reduced fumagillin production. These strains showed reduced expression of most members of the fma gene family and, in correlation, delayed the expression of laeA gene and suppressed the expression of fapR gene in A1151 cells. The effect of laeA and fapR gene expression on fumagillin production may be explained by the following facts presented by Dhingra et al. ²⁵. The fapR/fumR gene-deficient strain completely loses its ability to produce fumagillin, and the expression of the fma gene family is also almost completely suppressed. The expression levels of fapR/fumR and fmaB are suppressed by more than 90% in the laeA gene-deficient strain, and fmaB gene expression is also suppressed in the fapR gene-deficient strain. In strains deficient in the VeA transcription factor, which forms a heterotrimer with the LaeA protein, fapR gene expression is repressed, and fumagillin production is reduced by more than 80%. Thus the expression of the fma gene family; global transcription factors such as LaeA and VeA, which are upregulated; and FapR, a fumagillin cluster-specific transcription factor, play important roles in fumagillin production. However, the mechanism by which flbC gene deletion causes delayed expression of the laeA gene remains unclear. Several studies have suggested a link between central developmental pathways and secondary metabolic production. Lind et al. reported altered production patterns of many secondary metabolic products in brlA and laeA gene deletion strains²⁹. Kim et al. have also shown that deletion of the flbB and flbE genes results in repression of the laeA gene and reduced production of gliotoxin and fumagillin³². However, they did not discuss the mechanisms of laeA repression, but showed an altered expression pattern of the brlA gene due to flb gene deletion⁴¹. Moon et al. showed by multi-omics analysis that the flbC gene may be a direct target of LaeA and VeA transcription factors⁴². Rocha et al. revealed the regulation of flbC gene expression by the MAPK transcription factor RlmA⁴³. A feedback regulatory mechanism may be present between FlbC and global transcription factors such as LaeA and RlmA. As we have shown, the Congo red sensitivity of the A1280 WT strain was restored by the loss of flbC gene, which suggests a link between the cell wall integrity pathway and flbC.

Loss of the brlA gene, which is thought to be located downstream of FlbC, results in increased production of fumagillin²⁹. This seems to contradict our finding that flbC gene deletion reduced fumagillin production in A1151 and A1159. This may be due to perturbations in LaeA or FapR expression. However, we may have to consider the compensatory effects induced by gene loss (or overexpression), as is the case when certain important genes are involved in the basic properties and survival of mycelia. Considering that BrlA is an important transcription factor that plays a critical role in central developmental pathways, and that it may be important for brlA expression to be oscillatory, unidirectionally increasing or decreasing its expression may have drastic effects^44,45. This, in turn, is also true for flbC, and is a refutation and claim of the present study. The fact that fumagillin was reduced in flbC-deficient strains may not necessarily indicate that the FlbC transcription factor is “directly” involved in fumagillin production.

Notably, among the three standard laboratory strains, heterogeneity was observed not only in the gene-deficient strains but also in the WT strain. A1151, A1159, and A1280 were clinical isolates, but each isolate was isolated from a person with a different disease condition³³. Comparisons of the three strains have already been made at various levels, including whole genome, transcriptome, metabolome, leukemia model mouse infectivity, and macrophage phagocytosis capacity^12,13,17,46. The fumagillin gene cluster is located in the subtelomeric region of chromosome 8, where transposable elements and chromatin modifications are frequently found in Af293 and CEA17 backgrounds^16,29. Perrin et al. focused on the genomic instability of secondary-metabolite gene clusters and suggested that the expression profiles of secondary metabolites may differ among strains, showing that the structure of the subtelomeric region varies among strains, even within the same species²⁷. Thereafter, Lind et al. organized these mechanisms and divided them into different types²⁹. The present study just demonstrates this hypothetical model. However, the heterogeneity in fumagillin production identified in this study is probably not due to genomic instability in the subtelomeric region of chromosome 8 because WT of A1151, A1159, and A1280 expressed fma genes and produced fumagillin. According to Lind et al., the fumagillin gene cluster is relatively stable because it has fewer SNPs with non-synonymous substitutions than the other genomic regions²⁹. FlbC of the three strains behaves slightly differently with regard to the regulation of the fumagillin gene cluster; however, understanding this difference will probably require more than a comparison of genomic DNA between the strains. Nevertheless, with the advent of the CRISPR-Cas9, as we have used, recombinants of various strains from one species can be obtained efficiently and quickly. Although we obtained mutants by homologous recombination of drug resistance markers, “co-genome editing⁴⁷,” which does not use homologous recombination of markers, is now being developed. This enables the genome editing of a large number of clinical and environmental isolates and the use of these isolates as parental strains. Comparison of more strains than that in this study, which compared only three strains, is now possible.

Thus, our findings show that the different interactions between flbC and factors (LaeA and FapR) regulating the expression of fumagillin gene cluster are the probable cause for heterogeneity in fumagillin production among the strains. Moreover, our study serves as a basis for future studies on co-genome editing of large number of clinical and environmental isolates and the use of these isolates as parental strains.

Strains, media, and culture conditions

The A. fumigatus strains used in this study are listed in Supplementary Table S1. A. fumigatus strains A1151, A1159, and A1280 were obtained from the Fungal Genetics Stock Center (FGSC). The strains were grown on minimal medium [MM; 1% (w/v) glucose, 0.6% (w/v) NaNO₃, 0.052% (w/v) KCl, 0.052% (w/v) MgSO₄·7 H₂O, 0.152% (w/v) KH₂PO₄, and Hunter's trace elements, pH 6.5]. Plasmids were amplified in E. coli DH5α. Colony growth rates were measured as previously described⁴⁸. Briefly, conidia from each strain were point-inoculated onto the center of MM agar plates with or without 400 µg/mL CongoRed (Fujifilm, Japan). Colony diameters were measured after 3, 4, and 5 d of incubation at 37°C. The growth rates of all the individual strains were measured 5 times.

Generation of flbC mutants

Each sgRNA target sequence was explored using the web service CHOPCHOP (https://chopchop.cbu.uib.no/) and two synthesized DNA oligonucleotides, primer No. 3 and 4 (Supplementary Table S2). These sequences contained the T7 promoter, an sgRNA target sequence, and a sequence overlapping the Cas9 scaffold. The synthesis and purification of sgRNAs were performed using the Guide-it sgRNA In Vitro Transcription Kit (TAKARA, Japan). After the synthesized sgRNAs (sgRNA-1 and − 2) were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), they were used for ribonucleoprotein (RNP) formation with Cas9.

Repair templates were constructed based on the method described by Arai et al., with slight modifications⁴⁹. The pyrithiamine resistance cassette (ptrA marker) was obtained from the pPTRII plasmid (TAKARA). The KOD One PCR Master Mix (TOYOBO, Japan) and primer No. 1 and 2 (Supplementary Table S2) were used for PCR amplification to construct the repair template. The PCR products were purified using a FastGene Gel/PCR Extraction kit (NIPPON Genetics Co., Ltd., Japan) and quantified using a NanoDrop spectrophotometer.

A. fumigatus protoplasts were generated, and fungal transformation was performed as described by Kadooka et al., with slight modifications⁵⁰. Briefly, conidia were incubated in YG mediem (2% glucose, 0.5% yeast extract, and Hunter’s trace element) for 16 h at 37°C. Following incubation, the cell walls of germlings were digested with 32 mg/mL Vino-Taste Pro (Novozymes, Denmark) for 4 h 30 min at 30°C. Subsequently, 10 µg Guide-it Recombinant Cas9 (TAKARA) and 10 pmol each of sgRNA-1 and − 2 were mixed and incubated for 5 min, generating RNPs as described by Arai et al..⁴⁹ Protoplasts were transformed with 2–3 µg of repair templates and RNPs and plated onto MM agar supplemented with 0.6 M KCl. Following 24 h incubation at room temperature, agar plates were overlaid with MM top agar containing 0.1 µg/mL pyrithiamine hydrobromide (Sigma-Aldrich, USA). The flbC mutants were isolated and confirmed using colony PCR with primer No. 5 and 6 (Supplementary Table S2). At least three deletion strains were isolated in each case.

Quantitative real-time PCR analysis

Quantification of mRNA expression was performed using THUNDERBIRD SYBR qPCR mix (TOYOBO) and StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). The strains were grown at 37°C in liquid MM at each time point. Fungal RNA was isolated using the Sepasol-RNA I Super G (Nacalai Tesque, Japan) and reverse-transcribed into cDNA using 1 µg total RNA and ReverTra Ace qPCR RT Kit (TOYOBO). RT-PCR was performed using primers No. 7–32 (Supplementary Table S2), and gene expression levels were calculated using ΔΔCt method.

HPLC analysis

Each strain, maintained on a PDA plate, was inoculated into a 100-mL Erlenmeyer flask containing 50 mL of the production medium (3.0% sucrose, 3.0% soluble starch, 1.0% malt extract, 0.30% Ebios, 0.50% KH₂PO₄, and 0.050% MgSO₄·7H₂O; adjusted to pH 6.0 before sterilization). The production culture was carried out at 25°C for 7 days under agitation (150 rpm). Each culture broth was treated with 50 mL of acetone after 7 days and filtered. The filtrate, after the evaporation of acetone, was extracted with EtOAc (50 mL), and the extract was concentrated in vacuo.

The production of secondary metabolites was monitored using a Chromaster HPLC system (Hitachi High Technologies Co., Ltd., Tokyo, Japan) [column, CAPCELL PAK C18 MG-II (Osaka Soda. Co., Ltd., Osaka, Japan), i.d. 4.6 mm × 250 mm; flow rate, 0.8 mL/min; mobile phase, linear gradient from 20 to 100% CH₃CN containing 0.05% TFA for 30 min and 100% CH₃CN containing 0.05% TFA for 5 min; detection, UV 365 nm]. Each extract was prepared to 10 mg/mL CH₃OH solution, and 5.0 µL of each sample was injected for the HPLC analysis.

Silkworm larva infection model

Silkworm (Bombyx mori) larvae were maintained in an incubator at 27°C in the dark at a controlled temperature until conidial infection. A. fumigatus conidia were resuspended at a concentration of 1 × 10⁸ conidia/mL in phosphate buffered saline (PBS), and then silkworm 5th-instar larvae (2.0 g, n = 10) were infected with 1 × 10⁶ conidia (50 µL/larva) by injecting the hemolymph using a disposable 1 mL syringe with a 27G needle (TERUMO, Tokyo, Japan). The uninfected controls received PBS. Larvae were raised in the dark at 37°C for 4 d. Generalized Wilcoxon tests were performed using GraphPad Prism 8 software to compare survival curves, and differences were considered statistically significant at p < 0.05. Fertilized silkworm eggs of Bombyx mori (Hu·Yo × Tukuba·Ne) were purchased from Ehime Sansyu (Ehime, Japan) and fed an artificial diet (Silk Mate 2S; Nihon Nosan Kogyo, Kanagawa, Japan, and Silkmate; Katakura Industries, Tokyo, Japan).

Acknowledgments

This work was supported in part by Grant-in-Aid for Scientific Research (C) (22K06600 to Y.T., , 21K06631 and 24K09865 to H.Y.) from the Japan Society for the Promotion of Science (JSPS KAKENHI). We express our thanks to Ms. M. Iwamoto of Tohoku Medical and Pharmaceutical University for her technical assistance. We would like to thank Editage (www.editage.jp) for English language editing.

Author contributions

Y.T. conceived and designed the experiments. Y.T., I.A, R.K., H.Y. and A.Y. conducted the experiments. R.U. and M.S. helped with the design and coordination of the study. Y.T., H.Y. and A. Y. wrote the paper. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

Data availability statement

All data generated or analyzed during this study are included in Supplementary Table S3. A full-length version of the figure obtained by agarose gel electrophoresis is shown in Supplementary FigS1, and no contrast adjustment was made when used in the figures.

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No competing interests reported.

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The FlbC transcription factor contributes to the generation of strain heterogeneity in Fumagillin mycotoxin production in Aspergillus fumigatus

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