Abstract
Conidiation is an important reproductive process in Aspergillus. We previously reported, in A. nidulans, that the deletion of a putative glycosyltransferase gene, rseA/cpsA, causes an increase in the production of extracellular hydrolases and a severe reduction in conidiation. The aim of this study was to obtain novel genetic factors involved in the repression of conidiation in the rseA deletion mutant. We isolated mutants in which the rseA deletion mutant conidiation defect is suppressed and performed a comparative genomic analysis of these mutants. A gene encoding a putative transcription factor was identified as the associated candidate causative gene. The candidate gene was designated as srdA (suppressor gene for the conidiation defect of the rseA deletion mutant). The conidiation efficiency of the rseAsrdA double-deletion mutant was increased. Introduction of wild-type srdA into the suppressor mutants caused a conidiation defect similar to that of the rseA deletion mutant. Notably, the conidiation efficiencies of the rseAsrdA double-deletion and srdA single-deletion mutants were higher than that of the wild-type strain. These results indicate that srdA is a novel genetic factor that strongly represses conidiation of the rseA deletion mutant, and a putative transcriptional regulator, SrdA is a negative regulator of conidiation in A. nidulans.
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Introduction
Filamentous fungi are known for their ability to secrete a wide variety of extracellular enzymes1. Aspergillus species, common filamentous fungi found in diverse environments, are distributed worldwide. Some Aspergillus species have been used widely in industrial biotechnology. Koji-molds, including Aspergillus oryzae, Aspergillus sojae, and Aspergillus luchuensis, have been used in traditional fermented food production in East Asia2. Meanwhile, A. nidulans has been used as a model organism for the study of molecular genetics in filamentous fungi3, and the regulatory mechanisms for the production of extracellular hydrolases (amylolytic and cellulolytic enzymes)4,5. The regulation of conidiation has also been studied in A. nidulans, and a large number of genes are known to be involved in the formation and maturation of conidia6,7,8.
The production of extracellular enzymes, using Aspergillus, is performed under solid-state cultivation (SSC) and liquid cultivation (LC). Production of extracellular enzymes in Aspergillus species is highly promoted under SSC, but less stimulated under LC9,10. During the fermentation process, fungi cells undergo fermentation-specific environmental stresses. The stresses under the SSC could lead to promote extracellular enzyme production. Therefore, fungal stress response is important for enzyme production. Intracellular signaling pathways, such as the high osmolality glycerol (HOG) and the cell wall integrity (CWI) pathways, have been reported to play important roles in yeast (Saccharomyces cerevisiae) stress response11,12. The A. nidulans HOG and CWI pathways have also been investigated13,14.
Previously, we have reported that the deletion of a putative glycosyltransferase gene, rseA/cpsA, in A. nidulans under SSC, causes an increase in the production of extracellular hydrolases15. We showed that the HOG pathway was involved in the elevated production of hydrolases, and the rseA deletion mutant displayed a severe conidiation defect15. However, the mechanism of how rseA deletion causes substantial reduction of conidiation remains unclear.
In this study, we aimed to obtain novel genetic factors involved in the repression of conidiation in the rseA deletion mutant. We isolated mutants in which the conidiation defect in the rseA deletion mutant is suppressed, then we identified the suppressor mutant-associated causative gene. Additionally, we constructed and characterized the double deletion mutant of rseA and the causative gene.
Results
Isolation and characterization of suppressor mutants
Previously, we observed a severe conidiation defect in the rseA deletion mutant15. In the present study, we isolated and characterized suppressor mutants, that is, mutant strains in which the conidiation defect of the rseA deletion was suppressed. Spontaneous mutants with a green color occurred in the reddish-brown colonies of the rseA deletion mutant (DRA), when grown on MMGp agar plates (Fig. S1A). This suggests that the conidiation defect in DRA was largely restored in these spontaneous mutants. As shown in Fig. S1A, mutants B and C were observed in one DRA colony (left panel), and mutant D was observed in another DRA colony (right panel). We isolated the conidia from mutants B and D for further analysis. The strains isolated from mutants B and D were designated as SMGC-1 and SMGC-2, respectively.
SMGC-1 and SMGC-2 formed colonies with appearances similar to that of the wild-type strain (wtRA; Fig. 1A). In DRA, conidiophores were scarcely observed and the conidiophore’s morphologies were aberrant. In contrast, the morphologies of SMGC-1 and SMGC-2 conidiophores were similar to that of wtRA (Fig. 1B).
Characterization of the ΔrseA mutant (DRA), SMGC-1, and SMGC-2. (A) Colony growth appearances of DRA, SMGC-1, SMGC-2, and the wild type strain (wtRA) on MMGp agar plates (incubated at 37 °C for 5 days). (B) Scanning electron micrographs of the mutant (DRA, SMGC-1, SMGC-2) and wtRA conidiophores. (C) The mutant and wtRA conidiation efficiencies (Conidia/plate × 108). (D) Average diameter (mm) of the mutant and wtRA colonies. (E) Number of conidia per mm2 of the mutant and wtRA colonies (Conidia/mm2 colony × 105). Bars indicates standard deviations. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 (Welch’s t test, p-values were adjusted for multiple comparison using holm’s method).
The conidiation efficiency of DRA was drastically decreased. However, the conidiation efficiencies of SMGC-1 and -2 were approximately twofold higher than that of wtRA, indicating that conidiation was restored (Fig. 1C). Compared to wtRA, the colony size of DRA was reduced to 82.6% ± 1.5. Colony growth improvement was observed in SMGC-1 and -2. Relative to wtRA, SMGC-1 and -2 colony growth was 95.4% ± 1.0 and 96.4% ± 1.1, respectively (Fig. 1D). The number of conidia per mm2 in the DRA colony was 0.47% ± 0.27 of that of wtRA (Fig. 1E). Conversely, the number of conidia observed per mm2 in SMGC-1 and -2 was significantly greater than that of the wtRA (219% ± 38.8 and 232% ± 48.3, respectively) (Fig. 1E). Using Southern hybridization, we confirmed the deletion of rseA in the SMGC-1 and -2 (Fig. S1B). These results confirm that SMGC-1 and -2 are suppressor mutants of the DRA conidiation defect and are not revertants of the rseA deletion.
Comparative genomic analysis of the suppressor mutants and the ΔrseA mutant
To identify the causative gene(s) for the suppressor mutations in SMGC-1 and -2, we performed whole-genome sequencing. In the genome sequence of SMGC-1, two deletion mutations and two single-nucleotide substitutions were observed (Table 1A). In SMGC-2, one insertion and two single-nucleotide replacements were detected (Table 1B). Notably, mutations in AN5849 have been found in both SMGC-1 and -2 genome sequences. The gene structures of AN5849 in the wild-type and suppressor mutants are shown in Fig. 2A. In the wild-type strain, the open reading frame of AN5849 consists of 3571 bp with seven exons (determined by RNA-seq analyses) (FungiDB: https://fungidb.org/fungidb/app). One base deletion in exon 1 and one base insertion in exon 6 of AN5849 was detected in SMGC-1 and -2, respectively. The one-base alterations (deletion and insertion) result in frameshifts and cause pre-terminations. Therefore, AN5849 is one of the candidate genes responsible for suppressor mutations.
Positions of mutations in the srdA gene and gene product domain organization. (A) Gene structures of srdA (AN5849) in the wild-type strain, SMGC-1, and SMGC-2. Positions of exons, introns, and mutation points of srdA in SMGC-1, and SMGC-2 are indicated. (B) Domain organization of SrdA. Positions of a fungal Zn2Cys6 binuclear domain, nuclear localization signal motifs (NLSs), and a type 2 peroxisomal localization signal motif (PTS2).
AN5849 encodes a putative Zn2Cys6 transcription factor. Zn2Cys6 transcription factors are unique to fungi. Fifty-five Zn2Cys6 transcription factors, Gal4, Hap1, and Leu3, have been found in S. cerevisiae16. Some Zn2Cys6 transcription factors in A. nidulans have been characterized (e.g., AlcA, PrnA, AflR, NirA, SclB, and McrA)17,18,19,20,21,22. However, the function of AN5849 has not been investigated. The domain structure of AN5849, based on the results of Pfam and WoLF-PSORT searches is shown in Fig. 2B23,24. AN5849 consists of 1077 amino acids, and a fungal Zn2Cys6 binuclear cluster domain motif is located at the N-terminal region (amino acid residues 234 to 267). Three nuclear localization signal (NLS) motifs and one peroxisome targeting signal (PTS2) motif have also been found in AN584923,25. We designated AN5849 srdA as the suppressor gene for the conidiation defect of the rseA deletion mutant.
Conidiation of the ΔrseAΔsrdA and ΔsrdA mutants
To determine whether srdA was the causative gene for suppressor mutations of the ΔrseA mutant conidiation defect, we constructed and characterized a ΔrseAΔsrdA mutant (A1145DRDS) and the corresponding ΔrseA mutant (A1145DR). Colony appearances and SEM micrographs of these strains are shown in Fig. 3A–C. Similar to DRA, defects in conidiophore formation were observed in A1145DR. In contrast, the morphologies of the A1145DRDS conidiophores were similar to that of the wild-type (A1145WT). The conidiation efficiency of A1145DR was low (Fig. 3D). However, the conidiation efficiency of A1145DRDS was higher than that of A1145WT (Fig. 3D). Compared to the wild-type, the colony diameters of A1145DR and A1145DRDS significantly decreased, whereas the colony diameter of A1145DRDS was larger than that of A1145DR (Fig. 3E). Compared to the wild-type, the number of conidia per mm2 in the A1145DR colony decreased (0.37% ± 0.24). In contrast, that of A1145DRDS increased (227% ± 15.3) (Fig. 3F). These A1145DRDS phenotypes are similar to those of SMGC-1 and -2 (Fig. 1). We further examined srdA as the causative gene for suppressor mutations, by introducing wild-type srdA into SMGC-1 and -2 (Fig. 4). The srdA-introduced strains SMGC-1-PTSR and SMGC-2-PTSR displayed conidiation defects similar to that observed in DRA. Therefore, we concluded that the mutations on srdA observed in SMGC-1 and SMGC-2 are responsible for suppression of conidiation defects in the rseA deletion mutant.
Characterizations of the ΔrseΑ (A1145DR), the ΔrseAΔsrdA (A1145DRDS), and the ΔsrdA (A1145DS) mutants. (A) Colony growth appearances of A1145DR, A1145DRDS, A1145DS, and A1145WT on MMGp agar plates (incubated at 37 °C for 5 days). (B) Scanning electron micrographs of A1145DR, A1145DRDS, and A1145DS. (C) Scanning electron micrographs of A1145DR, A1145DRDS, and A1145DS at higher magnifications. (D) Conidiation efficiencies. (E) Average diameter of the colonies. (F) The number of conidia per mm2 colony of the samples. Bars indicate standard deviations. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 (Welch’s t test, p-values were adjusted for multiple comparison using Holm's method).
Introduction of wild type srdA into SMGC-1 and SMGC-2. (A) Colonies of the wild-type strain (wtRA), the ΔrseA mutant (DRA), the suppressor mutants (SMGC-1 and SMGC-2), the control strains in which only a ptrA marker gene was introduced (SMGC-1-PT and SMGC-2-PT), and the strains in which both ptrA and srdA were introduced (SMGC-1-PTSR and SMGC-2-PTSR). (B) Stereomicroscope observations of colony centers. 5 × 104 of conidia of these strains were inoculated on MMGp agar plates and incubated at 37 °C for 3 days.
We also constructed the ΔsrdA mutant, A1145DS. The colony appearance of A1145DS was similar to that of A1145WT and A1145DRDS. The conidiation efficiency, colony diameter, number of conidia per mm2 colony, and conidiophore morphology of A1145DS were all similar to that of A1145DRDS (Fig. 3). These results indicate that the srdA mutation is epistatic to the rseA mutation with regard to A. nidulans conidiation and growth.
Extracellular enzyme production of the ΔrseAΔsrdA mutant
We previously reported that extracellular hydrolase production increased in the ΔrseA mutant under a solid-state cultivation condition15. To determine whether the srdA deletion contributes to the increased extracellular hydrolase production of the ΔrseA mutant, we examined the extracellular endo-xylanase production by A1145DRDS (Fig. 5A). Endo-xylanase activity per g solid-state culture (SSC) was increased in A1145DR (5.26 ± 0.31 U/g wet SSC) compared to that of A1145WT (4.60 ± 0.24 U/g wet SSC) and decreased in A1145DRDS (4.50 ± 0.25 U/g wet SSC). To evaluate the growth of the fungal strains under SSC conditions, we estimated mycelial mass, by the chitin content in the dry SSC of the strains (A1145WT, 3,300 ± 703 μg/g; A1145DR, 1,706 ± 125 μg/g; A1145DRDS, 1,766 ± 124 μg/g) and those in the dry mycelia from liquid culture (A1145WT, 60.6 μg/mg; A1145DR, 81.1 μg/mg; A1145DRDS, 79.9 μg/mg). The masses of A1145WT, A1145DR, and A1145DRDS in the dry SSC were estimated at 54.4 ± 11.6 mg/g, 21.0 ± 1.5 mg/g, and 22.1 ± 1.6 mg/g, respectively. Therefore, the mycelial masses of A1145DR and A1145DRDS grown under SSC conditions were less than that of A1145WT. The extracellular endo-xylanase activities per mg of mycelia of A1145WT, A1145DR, and A1145DRDS were 0.087 ± 0.015 U/g wet SSC/mg/g dry SSC, 0.251 ± 0.012 U/g wet SSC/mg/g dry SSC, and 0.205 ± 0.020 U/g wet SSC/mg/g dry SSC, respectively (Fig. 5B). These results showed that the capacity of extracellular endo-xylanase production by A1145DRDS was significantly higher than that of A1145WT, with a capacity similar to that of A1145 DR. This finding suggests that the srdA deletion does not significantly affect the ΔrseA mutant ability to produce extracellular endo-xylanase.
Extracellular endo-xylanase production, sensitivities to cell wall perturbating agents, and the phosphorylation statuses of MAP kinases in the ΔrseA mutant (A1145DR), the ΔrseAΔsrdA mutant (A1145DRDS), and the wild type strain (A1145WT). (A) Endo-xylanase production under the solid-state cultivation condition. (B) Endo-xylanase production per mg mycelia. Experiments were conducted in triplicate. Bars indicate standard deviations. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 (student’s t test, p-values were adjusted for multiple comparison using holm’s method). (C) Growth sensitivities to calcofluor white (CFW). The concentrations in the medium are shown to the right of CFW (μg/mL). (D) Growth sensitivities to caspofungin (CAS). The concentrations in the medium are shown to the right of CAS (μg/mL). (E) Phosphorylation statuses of HogA and MpkA. Relative signal intensities of western blot bands are indicated. Signal intensity of A1145WT was defined as 100. Ratios of phosphorylated HogA per total HogA, and those of phosphorylated MpkA per total MpkA are also visible. CBB staining of the PAGE samples were used as loading control. The original data of the western bolts and SDS-PAGE are shown in Figs. S7 to S11.
Growth sensitivity of the ΔrseAΔsrdA mutant to cell wall perturbating agents
Since the ΔrseA mutant of A. nidulans was highly sensitive to calcofluor white (CFW) and resistant to caspofungin (CAS)15,26, we examined the growth sensitivity of A1145DRDS to these cell wall-perturbating agents (Fig. 5C,D). While A1145DR was more sensitive to CFW than A1145WT, the sensitivity of A1145DRDS was similar to that of A1145WT. In contrast, compared to A1145WT, A1145DRDS was resistant to CAS, which was similar to that of A1145 DR. These results suggest that the cell wall architecture of the ΔrseAΔsrdA mutant is partially different from that of the ΔrseA mutant.
Activation state of intracellular signaling pathways in the ΔrseAΔsrdA mutant
We previously reported that intracellular signaling pathways, such as the HOG and CWI pathways, were highly activated in the rseA deletion mutant15. To evaluate the effects of srdA deletion on the activation of these pathways, in the ΔrseA mutant, we determined the phosphorylation states of HogA and MpkA, the mitogen activated protein (MAP) kinases in the HOG and CWI pathways, in A1145DRDS (Fig. 5E). The signal intensity of phosphorylated HogA increased in A1145DR compared to that in A1145WT. However, phosphorylated HogA in A1145DRDS was similar to that of A1145WT. The signal intensity of total HogA decreased in A1145DR and A1145DRDS. Therefore, the ratio of phosphorylated HogA to total HogA increased in both A1145DR and A1145DRDS. Similarly, the ratio of phosphorylated MpkA to total MpkA was higher in A1145DR and A1145DRDS (Fig. 5E). However, these ratios in A1145DRDS were lower than that in A1145DR. These results indicate that HOG and CWI pathway are activated in the ΔrseAΔsrdA mutant. However, activation levels are attenuated by the srdA deletion.
Production of the extracellular endo-xylanase and the activation of the HOG pathway in the ΔsrdA mutant
The production of endo-xylanase in A1145DS was measured under SSC conditions (Fig. S2A,B). Endo-xylanase activity per g SSC of A1145DS was lower than that of A1145WT (4.79 ± 0.09 U/g wet SSC vs 6.15 ± 0.56 U/g wet SSC). To evaluate the growth of these strains under SSC conditions, we estimated mycelial mass, by the chitin content in the dry SSC of the strains (A1145DS, 2437 ± 113 μg/g; A1145WT, 4629 ± 321 μg/g) and those in the dry mycelia from liquid culture (A1145DS, 72.0 μg/mg; A1145WT, 60.6 μg/mg). The mycelial mass of A1145DS was significantly lower than that of A1145WT (33.8 ± 1.6 mg/g dry SSC vs 76.4 ± 5.3 mg/g dry SSC). Consequently, the activity per mg mycelia (U/g wet SSC/mg/g dry SSC) of A1145DS increased (0.142 ± 0.006) compared to that of A1145WT (0.80 ± 0.05) (Fig. S2B). These results indicate that the srdA deletion induced a modestly higher production of extracellular endo-xylanase in A. nidulans. The ratio of the phosphorylated HogA to total HogA was decreased in A1145 DS (Fig. S2C). The results indicate that, srdA deletion did not activate the HOG pathway in the wild-type strain.
Presence of SrdA orthologs in 23 species of filamentous fungi
We searched for SrdA orthologs in 23 filamentous fungi, belonging to Ascomycota, and constructed a phylogenetic tree of the SrdA orthologs using MEGA11 (Fig. 6)27. Amino acid sequences of the SrdA orthologs were aligned using the Clustal W algorithm28. The taxonomy of each fungal species is shown according to the NCBI Taxonomy browser29. The amino acid identity among the SrdA orthologs is indicated in Fig. S3. SrdA orthologs were conserved among Eurotiomycetes (> 38% amino acid identity) and in Dothideomycetes, Leotiomycetes, and Sordariomycetes (SrdA orthologs amino acid identity ranged between 25 and 32%). rseA/cpsA orthologs have been characterized in A. nidulans, A. fumigatus, Pyricularia oryzae, and Neurospora crassa15,26,30,31,32. SrdA orthologs were also identified in these fungal strains. In addition, the 23 filamentous fungi described above possessed RseA/CpsA orthologs (Fig. S4). RseA/CpsA orthologs are distributed in Basidiomycota (Agaricomycetes and Tremellomycetes) and Mucoromycota (Mucoromycetes). However, no SrdA orthologs have been identified in Basidiomycota and Mucoromycota. Furthermore, the budding yeast S. cerevisiae, fission yeast Schizosaccharomyces pombe; and dimorphic yeasts Candida albicans and Yarrowia lipolytica do not possess Rse/CpsA nor SrdA orthologs.
A phylogenetic tree of SrdA orthologs in the 23 filamentous fungi. NCBI RefSeq accession numbers or GenBank accession numbers of the SrdA orthologs are indicated in parentheses.
Discussion
Conidiation and noteworthy capacity for production of extracellular enzymes are important factors that drive the industrial utilization of filamentous fungi. The deletion of rseA causes hyper-production of extracellular enzymes in A. nidulans, but results in a conidiation defect15. In this study, we identified the causative gene that suppresses the conidiation defect in the ΔrseA mutant. Conidiation of the ΔrseA mutant was restored by the srdA deletion (Fig. 3). This suggests that SrdA plays a major role in the repression of the conidiation of the ΔrseA mutant.rseA encodes a putative GT family 2 glycosyltransferase, which is homologous to a hyaluronic acid synthetase (Cps1) of Cryptococcus neoformans33,34. Deletion of rseA/cpsA affects hyphal growth and morphogenesis of A. nidulans and its secondary metabolite and extracellular enzyme production15,26. Recently, it was reported that rseA/cpsA orthologs influence plant and human pathogens30,32,35. Since deletion of rseA or its ortholog in N. crassa affects cell wall composition and causes hyperactivation of the CWI pathway, it has been proposed that RseA and its ortholog play a role in cell wall biogenesis15,26,31. Although the growth sensitivity of the ΔrseAΔsrdA mutant to CFW was similar to that of the wild-type strain, the mutant was resistant to CAS (Fig. 5C, ΔrseAΔsrdA CAS sensitivity is similar to that of ΔrseA). These results indicate that restoration of cell wall integrity in the ΔrseAΔsrdA mutant is limited. In addition, hyperactivation of the CWI pathway was observed which supports the previous statement. The HOG pathway was activated in the ΔrseAΔsrdA mutant as well as in the ΔrseA mutant (Fig. 5E). Since this pathway is known to be involved in maintaining the cell wall integrity of A. nidulans36, the results suggest that the observed HOG pathway activation in the ΔrseAΔsrdA mutant is due to perturbation of fungal cell wall integrity.
An increase in conidiation efficiency was observed in both the ΔrseAΔsrdA and ΔsrdA mutants (Fig. 3). This suggests that SrdA is a negative regulator of A. nidulans conidiation. Moreover, we observed that the expression levels of brlA, abaA, and wetA were significantly increased in A1145DRDS compared to those in A1145DR and were higher in A1145DS than in A1145WT (our unpublished results). These data are consistent with our hypothesis that SrdA acts as a negative regulator of conidiation and functions upstream of BrlA. Many genes negatively affect conidiation in A. nidulans8. Among these genes, nsdD and rocA encode putative transcription factors, and their deletions cause hyper-conidiation on agar media37,38. NsdD and RocA are thought to function upstream of brlA, which is the first transcription factor to function in the central regulatory cascade of A. nidulans conidiation6,7,8. Furthermore, a putative Zn2Cys6 transcription factor, SfgA, acts as a negative regulator of conidiation37,39. In addition to these three negative regulators, SrdA may repress the conidiation of A. nidulans. Further investigation is required to clarify their functional relationships.
In the conidiation regulators of A. nidulans, CsgA, McrA, SclB, SfgA, and ZcfA are Zn2Cys6 transcription factors, as well as SrdA. SclB and McrA positively regulate conidiation, whereas CsgA and ZcfA are necessary for proper asexual and sexual development21,22,40,41. It has been reported that the heterodimer of the velvet proteins, VosA-VelB, directly binds to the promoter region of the target genes and regulates conidiation and spore viability42,43,44. The expression of sclB and mcrA are regulated by the VosA-VelB complex21,22. Although SrdA is not a positive regulator, VosA-VelB complex may be involved in regulating srdA expression. To date, the genes regulated by SrdA remain unknown. Transcriptome analysis of the ΔrseAΔsrdA mutant and ΔsrdA mutant will provide useful information for understanding the biological functions of SrdA.
Two hundred and six of 1077 amino acid residues in the C-terminal region of SrdA were deleted in the SMGC-2 mutant. However, the phenotype was similar to that of the ΔrseAΔsrdA mutant (Fig. 3). This result indicated that the C-terminal of SrdA contains a region required for functional conidiation. Since an NLS is present in this region, it is possible that the mutant protein could not localize and therefore not function in the nuclei. It has been reported that the C-terminal domains of AmyR, a Zn2Cys6 transcription factor for amylolytic genes, are necessary for the regulation of subcellular localization and sensing the stimulation of inducers45. Therefore, it is suggested that the C-terminal region of SrdA is indispensable for sensing intracellular signal(s) that regulate localization and/or function.
Extracellular endo-xylanase production per milligram of mycelia was significantly increased in the ΔrseAΔsrdA mutant (Fig. 5B), and we observed that the HogA pathway was activated in this mutant (Fig. 5E). These results are consistent with our previous proposal, which suggests that the HOG pathway is involved in the increased production of extracellular hydrolases in the rseA deletion mutant15. Endo-xylanase production per milligram of mycelia was partially increased in the ΔsrdA mutant, whereas the phosphorylation of HogA was remarkably attenuated, compared to the wild-type strain (Fig. S2B,C). Therefore, the mechanism of increased extracellular endo-xylanase production, caused by srdA deletion, is suggested to be independent of HOG pathway activation.
In this study, we indicated that loss of function of srdA caused suppression of conidiation defect in rseA deletion mutant. Moreover, SrdA function in conidiation regulation was analyzed. srdA orthologs are conserved in many filamentous fungi in Ascomycota. Therefore, it is expected that the reduced conidiation phenotypes of some koji-molds, caused by genetic manipulations for improvement in the production of enzymes and metabolites, could be suppressed by the deletion of srdA orthologs. Furthermore, we believe that the elucidation of SrdA function will contribute to understanding the developmental control of Aspergillus species.
Although rseA/cpsA is known to be involved in the regulation of secondary metabolite production, the effect of srdA mutations on secondary metabolite production in rseA deletion mutant has not been investigated in this study, because the fungal strains constructed in this study possess a mutation in veA (veA1). The gene, veA, plays crucial roles in regulating the secondary metabolite production46. Furthermore, it has also been reported that LaeA and velvet (VeA) family proteins coordinately regulate not only secondary metabolite production, but also the developments of A. nidulans47. Thus, under the veA+ background, the phenotype of ΔsrdA-single deletion mutant and ΔrseAΔsrdA-double deletion mutant might be different from the phenotype of those observed in this study.
Methods
Fungal strains and media
The fungal strains used in this study and their origins are listed in Table 2. The fungal strains were cultured in YG complete medium48 and MMG minimal medium49. Pyridoxine-auxotrophic mutants were cultured in pyridoxine-supplemented MMG medium (MMGp; 0.5 μg/mL pyridoxine). Fungal strains possessing pyrG89 and riboB2 mutations were cultured in MMG medium supplemented with uracil (10 mM), uridine (10 mM), and riboflavin (2.5 μg/mL). Agar plates, used for fungi culture, were prepared by adding 1.5% agar to the culture media.
General DNA techniques and fungal transformation
Oligonucleotide primers used in this study are listed in Table S1. High-fidelity enzymes, KOD plus Neo (Toyobo, Osaka, Japan) and PrimeSTAR MAX DNA polymerase (Takara Bio, Shiga, Japan), were used to amplify DNA fragments for plasmid construction and fungal transformation. The DNA polymerases were used according to the manufacturer’s instructions. The sequences of the DNA fragment inserts, contained in the plasmid DNA, were confirmed using standard Sanger sequencing (Eurofins Genomics K.K., Tokyo, Japan). Fungal transformations were performed using the protoplast-PEG method50. Southern hybridization was performed using a digoxigenin (DIG) labeling system (Roche Diagnostics, Basel, Switzerland) according to the instruction manual of Roche Diagnostics.
Construction of ΔrseA deletion mutants with riboflavin auxotrophy
In A. nidulans A114551, rseA was replaced with pyrG as previously described15. The obtained transformants were designated as ΔrseA-riboB2-1–3. The rseA deletions were confirmed by Southern hybridization (Fig. S5A).
Construction of ΔrseAΔsrdA and ΔsrdA mutants
The gpdA promoter, riboB, and gpdA terminator coding regions were amplified by PCR from A. nidulans A26 genomic DNA. The three amplified DNA fragments were ligated using fusion PCR52. The pUC-PTgpdA-riboB plasmid was generated by cloning the fusion PCR product into pUC118, using the SLiCE reaction53. A mutation, causing one amino acid exchange (E217K), was found in the riboB of pUC-PTgpdA-riboB. However, this mutation did not affect the intended function as a marker gene. Therefore, the plasmid was utilized in the present study.
A 5.9-kb DNA fragment containing srdA (AN5849) was amplified by PCR from the genomic DNA of A. nidulans A26. The PCR product was cloned into pUC118 to generate the pUC-AN5849 plasmid. The riboB marker cassette, amplified from pUC-PTgpdA-riboB, was introduced into pUC-AN5849 by the SLiCE reaction53 to generate the pUC-AN5849-DEL plasmid. A DNA fragment containing the srdA deletion was amplified by PCR from pUC-AN5849-DEL and introduced into A. nidulans ΔrseA-riboB2 and A. nidulans A1145 by the protoplast-PEG method50. The products, ΔrseAΔsrdA mutants (A1145DRDS-1, -2, and -3) and an ΔsrdA mutant with uracil-auxotrophic properties (ΔsrdA-pyrG89), were obtained. pyrG was introduced into ΔsrdA-pyrG89 by the protoplast-PEG method, as previously described15. The pyrG complemented ΔsrdA-pyrG89 mutants were designated as A1145DS-1, -2, and -3 (i.e., ΔsrdA mutants). Southern hybridization was used to confirm the srdA deletion and pyrG introduction (Figs. S5B and S6A). As each of A1145DRDS-1–3 and each of A1145DS-1–3 showed the same phenotypes, we used A1145DRDS-1 and A1145DS-1 for further experiments as A1145DRDS and A1145DS, respectively.
Introduction of riboB into ΔrseA-riboB2
A 4.2-kb DNA fragment, containing the intergenic region between pyroA and sac3, was amplified by PCR from the genomic DNA of A. nidulans A1145. The PCR product was cloned into pUC118 to generate the pUC-PSIG plasmid. The riboB marker cassette was then inserted into pUC-PSIG to obtain the pUC-PSIG-riboB plasmid. The DNA fragment for riboB complementation was prepared from pUC-PISG-riboB and introduced into A. nidulans ΔrseA-riboB2. The riboB complemented ΔrseA-riboB2 was designated A1145DR-1, -2, and -3 (i.e., ΔrseA mutants). The riboB integrations into the A1145DR-1–3 genomes were confirmed by Southern hybridization (Fig. S6B). Since these three strains displayed the same phenotype, we used A1145DR-1 for further experiments as A1145DR.
Introduction of pyrG and riboB into A. nidulans A1145
To obtain A1145-riboB2, pyrG was introduced into A. nidulans A1145, as previously described15 and designated as A1145-riboB2. The DNA fragment for riboB complementation was then introduced into A1145-riboB2. riboB-complemented A1145-riboB2 was designated as A1145WT-1, -2, and -3. Integrations of pyrG and riboB in the A1145WT-1–3 genomes were confirmed by Southern hybridization (Figs. S5B and S6B). We used A1145WT-1 for further experiments as A1145WT.
Construction of SMGC-1 and SMGC-2-derived strains
A pyrithiamine-resistant marker (ptrA) was amplified from pPTR I (Takara Bio, Shiga, Japan) and inserted into pUC-PSIG to generate the pUC-PSIG-ptrA plasmid. A 6.2-kb DNA fragment containing the promoter, coding region, and terminator of srdA was amplified by PCR from the genomic DNA of A. nidulans A26 and inserted into pUC-PSIG-ptrA by the SLiCE reaction53 to obtain the pUC-PSIG-AN5849 plasmid. The DNA fragment used for srdA introduction was amplified from pUC-PSIG-AN5849 and introduced into A. nidulans SMGC-1 and SMGC-2 by the protoplast-PEG method50. The srdA-introduced SMGC-1 and SMGC-2 were designated as SMGC-1-PTSR and SMGC-2-PTSR, respectively. A DNA fragment containing the ptrA marker gene (but not srdA) was amplified by PCR from the plasmid pUC-PSIG-ptrA and introduced into SMGC-1 and -2, this served as the srdA-complementation control. The spontaneous mutants in which only the prtA marker gene was introduced were designated as SMGC-1-PT and SMGC-2-PT, respectively. The introduction of the fragments into the srdA-introduced and control strains was confirmed by Southern hybridization (Fig. S6C).
Next-generation genomic sequencing of the suppressor mutants
The libraries for sequence analysis were prepared from the genomic DNA of SMGC-1, SMGC-2, and DRA, using the HiSeq SBS Kit v4 (Illumina, Inc., San Diego, USA). Genomic libraries were sequenced, using a HiSeq2500 (Illumina, Inc.), with a 100-base paired-end run. The reads were cleaned using Trimmomatic version 0.3654 and mapped to a reference genome using BWA version 0.7.1755. The total number of sample reads ranged between 22.8 and 26.9 M. In all samples, at least 92% of the bases were higher than Q30. The mapping rates of the samples ranged between 94.4 and 98.4%. The reference bases, covered at 50× depth of the samples, ranged between 90.6 and 96.6%. Using the mapping data, we carried out the variant calling of SMGC-1 and -2. The genomic sequence data of A. nidulans A4 were obtained from Aspergillus Genome Database56 and used as a reference genome for mapping cleaned reads. PCR duplicates in the mapping data were removed using Picared tools version 1.111 (http://picared.soucefoge.net/). The nucleotides altered in SMGC-1 and SMGC-2 were called using samtools, version 1.657. The variants called by samtools were further filtered using bcftools version 1.658. Finally, variants that met our filtering criteria (number of high-quality bases ≥ 10, genotype quantity ≥ 10, and allele frequency ≥ 95) were selected as the mutations found in SMGC-1 and SMGC-2.
Observation of mutants using a scanning electron microscope
Fungal strains were cultivated on MMGp agar plates at 37 °C for 60 h. The samples were fixed overnight with 0.1 M cacodylate buffer (pH 7.4) containing 2% paraformaldehyde and 2% glutaraldehyde. The samples were further fixed with 0.1 M cacodylate buffer (pH 7.4) containing 1% tannic acid for 2 h. The samples were post-fixed with 0.1 M cacodylate buffer containing 2% osmium tetroxide for 3 h. All fixation steps were performed at 4 °C. The samples were dehydrated using an ethanol gradient and dried using the tert-butyl alcohol freeze-drying method59. Dried samples were coated with a thin layer of osmium using an osmium plasma coater. The conidiophores of the samples were observed under a scanning electron microscope, JSM-7500F (JEOL Ltd., Tokyo, Japan).
Determination of conidiation efficiency
MMGp agar plates were inoculated with 5.0 × 103 sample conidia, and incubated at 37 °C for 5 days. The diameters of five colonies were measured and an average diameter was calculated. The conidia-containing colonies were harvested in 12 mL of 0.05% Tween 20, using a spreader. The conidial suspensions were filtered through Miracloth (Merck Millipore, Billerica, Massachusetts, USA). The volume of the filtrate was adjusted to 12 mL by adding 0.05% Tween 20. The number of conidia in the filtrates was counted using a hemocytometer.
Measurement of extracellular endo-xylanase production under SSC
Wheat bran was purchased from Yuutekku (Hokkaido, Japan) and prepared for SSC, as previously described15. Pre-moistened sterile wheat bran (10 g) was inoculated with 3.0 × 106 conidia and incubated at 37 °C for 3 days with a relative humidity between 90 and 100%. The procedure for the preparation of crude extract from solid-state culture (SSC) has been previously described15. Azo-xylan (birch wood) (MEGAZYME, Wicklow, Ireland) was used as the assay substrate. The xylanase assay was performed according to the manufacturer’s instructions (Lot. 30601).
Quantification of mycelia in solid-state cultures
The amount of mycelia in the SSCs was calculated as follows:
where: M(SSC) = amount of mycelia in SSCs (mg/g dry SSCs), Chi(SSC) = chitin content in dry SSCs (μg/g dry SSCs), Chi(MYC) = chitin content in dry mycelia from liquid culture (μg/mg dry mycelia).
The detailed protocols for the quantification of chitin from dry SSCs, and dry mycelia from liquid culture, have been previously reported15.
Determining growth sensitivities to cell wall perturbating agents
The growth sensitivity of the mutant strains to calcofluor white (CFW) was determined by conidia point inoculation of CFW-containing (2.5 μg/mL) MMGp agar plates. Five-fold dilution series (2.0 × 103 to 2.5 × 105) of conidial suspensions were spotted on the assay plate and incubated at 37 °C for 3 days. To determine growth sensitivity to caspofungin (CAS), 6.0 × 104 conidia from the samples were point inoculated on CAS-containing (3.0 μg/mL) MMGp agar plates and incubated at 37 °C for 3 days. The sensitivities to these cell wall perturbating agents were determined by observing the phenotype of the colonies and their diameters.
Western blot detection of phosphorylation levels of HogA and MpkA
Western blotting was used for analysis of phosphorylation of MAP kinases, HogA and MpkA. MMGp agar plate was covered with sterilized cellophane film. Sixty-thousand conidia were point-inoculated onto the cellophane film and incubated at 37 °C for 2 days. The fungal mycelia-containing cellophane film separated from the agar plate and rapidly frozen in liquid nitrogen. Preparation of crude extracts and their analysis by western blotting were performed as previously described15,48, using HogA and MpkA antibodies. The signal intensities of the western blots were quantified using ImageJ freeware (https://imagej.nih.gov/ij/).
Statistical analyses
Statistical analyses (two-sample t-tests and multiple comparison tests) of the experimental data obtained in this study were carried out using R version 4.2.0 (https://cran.ism.ac.jp/). The p-values of the multiple comparison tests were adjusted by Holm’s method.
Data availability
The data are available upon request to the corresponding author.
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Acknowledgements
We thank Keiko Takagi and Tetsuki Suzawa for their helpful discussions. Comparative genomic sequence analysis of sector mutants was performed by Eurofins Genomics K.K. This work was done by using facilities of the Agro-Biotechnology Research Center at the University of Tokyo.
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M.O. performed the experiments and analyzed the data; M.O., R.F., R.I., and H.H. conceived and designed the experiments; M.O. and H.H. wrote the manuscript; and Y.K. and H.H. supervised this study.
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M. O. is applying for a patent for the commercial use of srdA mutants (institution; Noda Institute for Scientific Research, name of inventor; Masahiro Ogawa, application number; JP 2023-11914, status of patent; patent pending, specific aspect of manuscript covered in patent application; mutations and deletion of srdA causes increasing of conidiation capacity in Aspergillus). The authors declare no conflicts of interest.
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Ogawa, M., Fukuda, R., Iwama, R. et al. srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans. Sci Rep 13, 4285 (2023). https://doi.org/10.1038/s41598-023-31363-8
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DOI: https://doi.org/10.1038/s41598-023-31363-8
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