Deletion of the col-26 Transcription Factor Gene and a Point Mutation in the exo-1 F-Box Protein Gene Confer Sorbose Resistance in Neurospora crassa

L-Sorbose induces hyperbranching of hyphae, which results in colonial growth in Neurospora crassa. The sor-4 gene, which encodes a glucose sensor that acts in carbon catabolite repression (CCR), has been identified as a sorbose resistance gene. In this study, we found that the deletion mutant of col-26, which encodes an AmyR-like transcription factor that acts in CCR, displayed sorbose resistance. In contrast, the deletion mutants of other CCR genes, such as a hexokinase (hxk-2), an AMP-activated S/T protein kinase (prk-10), and a transcription factor (cre-1), showed no sorbose resistance. Double mutant analysis revealed that the deletion of hxk-2, prk-10, and cre-1 did not affect the sorbose resistance of the col-26 mutant. Genes for a glucoamylase (gla-1), an invertase (inv), and glucose transporters (glt-1 and hgt-1) were highly expressed in the cre-1 mutant, even in glucose-rich conditions, but this upregulation was suppressed in the Δcre-1; Δcol-26a double-deletion mutant. Furthermore, we found that a dgr-2(L1)a mutant with a single amino-acid substitution, S11L, in the F-box protein exo-1 displayed sorbose resistance, unlike the deletion mutants of exo-1, suggesting that the function of exo-1 is crucial for the resistance. Our data strongly suggest that CCR directly participates in sorbose resistance, and that col-26 and exo-1 play important roles in regulating the amylase and glucose transporter genes during CCR.


Introduction
Filamentous fungi have elongated hyphae at their growing tips with hyphal branches, and undergo radial growth on agar medium. Sorbose, a rare sugar, exhibits toxic effects on several fungi, including Neurospora crassa. When 1% sorbose is added to medium in the presence of 0.2% sucrose, N. crassa propagates hyper-branched hyphae and forms compact colonies [1][2][3][4]. The formation of compact colonies in the presence of sorbose is a useful methodological tool for the isolation of mutants, allowing high-resolution genetic analyses and contributing to the establishment of N. crassa as a model organism for molecular genetics and biochemistry. Various studies have been conducted to identify the way in which sorbose induces such compact colonies; however, the underlying mechanisms remain largely unclear. Sorbose induces changes in the polysaccharide composition of the cell wall, such as a marked decrease in beta-1,3-glucan [3,4], possibly because a beta-1,3-glucan synthetase is inhibited by the sugar [5]. In contrast, a beta-1,3-glucan synthetase inhibitor, micafungin, and a GPI-anchor biosynthesis inhibitor, aminopyrifen, induced abnormal morphology on a sorbose medium in N. crassa mutant strains in the cell wall integrity MAP kinase genes mak-1 and mak-2, and chitin synthetase genes chs-5 and chs-7 [6,7]. These observations suggest that sorbose may disturb the synthesis of the fungal cell wall.
Several sorbose-resistant mutants have been isolated and characterized in fungi. Aspergillus nidulans sorA mutants show cross-resistance to a glucose analog, 2-deoxyglucose (2-DG), and are defective in sugar uptake [8]. The sorA gene has been shown to encode

Sensitivity to 2-Deoxyglucose and Mitochondrial Respiration Inhibitors
A glucose analog, 2-deoxyglucose (2-DG), and two mitochondrial complex III inhibitors, azoxystrobin and antimycin A, were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). To measure the sensitivity to 2-DG, mycelial disks that were precultured on Vogel's minimal agar medium containing 1.0% fructose were transferred onto 1% fructose Vogel medium containing 2-DG (0.1% to 0.4%), and fungal growth was assessed after 18 h of cultivation at 25 • C. Azoxystrobin and antimycin A act as specific inhibitors of the mitochondrial respiratory chain by binding to the Qo and Qi sites of the cytochrome bc1 complex, respectively. To determine the sensitivity to azoxystrobin and antimycin A, a series of conidia suspensions (1 × 10 7 to 1 × 10 3 cells/mL) was spotted on sor medium containing azoxystrobin (0.4 mg/L) and antimycin A (0.4 mg/L), and colony formation was photographed after 2 or 5 days of cultivation at 25 • C.

Gene Expression Analysis by qPCR
Total RNA was isolated as previously described by Noguchi et al. [40]. The conidia (1.0 × 10 6 cells/mL) were inoculated and precultured in Vogel's liquid medium containing 1.2% glucose for 22 h at 25 • C on a reciprocating shaker (135 rpm) and then, the growing hyphae were transferred to fresh Vogel's medium containing glucose or maltose and cultured for 2 h at 25 • C. The resultant mycelia were harvested by filtration using an aspirator, and frozen in liquid nitrogen until RNA isolation. Total RNA was isolated from each sample using a FastRNA Pro Red kit (MP Biomedicals, Tokyo, Japan). Each RNA sample (1 µg of total RNA) was subjected to cDNA synthesis and quantitative real-time reverse transcription PCR (RT-qPCR) using the LightCycler system (Roche Diagnostics, Tokyo, Japan), as described by Yamashita et al. [41]. The mRNA expression of the target genes was quantified using Universal ProbeLibrary (Roche Diagnostics, Tokyo, Japan). Primers and probes used in this study are summarized in Table S3. The relative gene expression value was calculated by comparing the threshold cycle (Cp), with actin used as the reference gene in the RT-qPCR analysis. A minimum of three biological replicates were performed for each experiment.

Glucose and Sorbose Consumption of Sorbose-Resistant Strains
To measure glucose or sorbose consumption during liquid culture, the conidia (10 6 cells/mL) were inoculated in Vogel's minimal liquid medium with 1% glucose or 1% sorbose + 0.2% glucose, and incubated at 25 • C. The culture filtrates were harvested after 18, 24, and 48 h. Glucose concentration in the medium was measured using the F-kit for glucose (J.K. International, Tokyo, Japan) in accordance with the manufacturer's protocol. The sorbose concentration was measured using the Si-Mo method as described by Katano et al. [42]. To measure the sorbose concentration, 200 µL of culture filtrate was mixed with 800 µL of reaction buffer (6 mL of 1 M Na 2 MoO 4 and 2 mL of 0.25 M Na 2 SiO 3 were mixed and adjusted to pH 4.5 using 5 M HCl) and incubated 70 • C for 30 min. The absorbance of the resultant samples was measured at 750 nm. Sorbose concentration in the cultures were calculated from the standard curves. Three biological replicates were performed.

SDS-PAGE Analysis of Extracellular Proteins
Conidia (10 6 cells/mL) were cultured in Vogel's minimal medium containing 2% glucose for 5 days on a reciprocating shaker (135 rpm) at 25 • C. The extracellular protein in culture filtrates was concentrated using a Centrifugal Ultrafiltration Filter Unit 3000 (Amicnon Tokyo, Japan), SDS-PAGE was performed with a 8-16% Mini-PROTEIN TGX Gels (BIO-RAD, Tokyo, Japan), and the gel was stained with Coomassie Brilliant Blue.

Loss of Function of Transcription Factor col-26/dgr-1 Confers Sorbose Resistance in N. Crassa
During fungicide sensitivity screening of the deletion mutants in the transcription factor library of N. crassa, we found that the col-26 deletion mutant was hypersensitive to azoxystrobin and antimycin A, which are both mitochondrial respiratory chain complex III inhibitors ( Figure S1). In N. crassa, only the sor-4 gene (alternate names of rco-3 and dgr-3), which encodes a glucose sensor protein, has been identified among sorbose resistance genes. Further characterization of the col-26 mutants revealed that the ∆col-26a strain displayed sorbose resistance, similar to sor-4 mutants ( Figure 1A). Suspensions of conidia (approximately 10 6 cells/mL) were spotted on sor medium containing 1% sorbose and 0.2% sucrose; the ∆sor-4a and ∆col-26a strains, but not the wild-type strain, followed a normal growth pattern. We also found the sorbose resistance of a 2-deoxyglucose (2-DG)-resistant strain dgr-1(BE52)A (FGSC#4326). The dgr-1 mutation has been identified as a frame shift at codon 335 of the col-26 protein ( Figure S2A). These observations are consistent with the conclusion that the loss of function of the transcription factor col-26 confers sorbose resistance in N. crassa. culture filtrates was concentrated using a Centrifugal Ultrafiltration Filter Unit 3,000 (Amicnon Tokyo, Japan), SDS-PAGE was performed with a 8-16% Mini-PROTEIN TGX Gels (BIO-RAD, Tokyo, Japan), and the gel was stained with Coomassie Brilliant Blue.

Loss of Function of Transcription Factor col-26/dgr-1 Confers Sorbose Resistance in N. Crassa
During fungicide sensitivity screening of the deletion mutants in the transcription factor library of N. crassa, we found that the col-26 deletion mutant was hypersensitive to azoxystrobin and antimycin A, which are both mitochondrial respiratory chain complex III inhibitors ( Figure S1). In N. crassa, only the sor-4 gene (alternate names of rco-3 and dgr-3), which encodes a glucose sensor protein, has been identified among sorbose resistance genes. Further characterization of the col-26 mutants revealed that the Δcol-26a strain displayed sorbose resistance, similar to sor-4 mutants ( Figure 1A). Suspensions of conidia (approximately 10 6 cells/mL) were spotted on SOR medium containing 1% sorbose and 0.2% sucrose; the Δsor-4a and Δcol-26a strains, but not the wild-type strain, followed a normal growth pattern. We also found the sorbose resistance of a 2-deoxyglucose (2-DG)resistant strain dgr-1(BE52)A (FGSC#4326). The dgr-1 mutation has been identified as a frame shift at codon 335 of the COL-26 protein ( Figure S2A). These observations are consistent with the conclusion that the loss of function of the transcription factor COL-26 confers sorbose resistance in N. crassa.  To compare the growth rates of ∆sor-4a and ∆col-26a strains on Vm medium and sor medium, mycelial disks precultured on each medium were transferred and cultured on fresh medium of each type. Both sorbose-resistant strains grew at similar rates to that of the wild-type strain (2.04 mm to 2.16 mm/h) on Vm medium ( Figure 2A). On sor medium, the growth rate of the sor-4(DS(r))A strain (1.73 ± 0.13 mm/h) was very similar to that of the sor-4(DS(r))A strain (1.69 ± 0.18 mm/h) ( Figure 2B). There was no statically significant difference in growth rate between the ∆col-26a strain and the sor-4(DS(r))A strain. These two precultured sorbose-resistant strains began linear growth on fresh medium without delay, whereas the wild-type strain did not show linear growth on this medium ( Figure 2B). When conidia of these strains were inoculated on Vm medium, filamentous growth of the ∆sor-4a and ∆col-26a strains, as well as the wild-type strain, were clearly detectable after 18 h. However, on sor medium, all strains formed compact colonies 48 h after inoculation, and only the ∆sor-4a and ∆col-26a strains began to grow at the edge of the colonies. These observations suggest that there was a transition stage between colonial growth and linear hyphal growth in the ∆sor-4a and ∆col-26a strains on sor medium. In addition, conidia of the ∆sor-4a and ∆col-26a strains, as well as the wild-type strain, did not germinate on Vm medium containing sorbose as the sole carbon source; therefore, the addition of a normal carbon source, such as sucrose or glucose, is essential for filamentous growth on medium containing 1% sorbose.
To compare the growth rates of Δsor-4a and Δcol-26a strains on Vm medium and SOR medium, mycelial disks precultured on each medium were transferred and cultured on fresh medium of each type. Both sorbose-resistant strains grew at similar rates to that of the wild-type strain (2.04 mm to 2.16 mm/h) on Vm medium (Figure 2A). On SOR medium, the growth rate of the sor-4(DS(r))A strain (1.73 ± 0.13 mm/h) was very similar to that of the sor-4(DS(r))A strain (1.69 ± 0.18 mm/h) ( Figure 2B). There was no statically significant difference in growth rate between the Δcol-26a strain and the sor-4(DS(r))A strain. These two precultured sorbose-resistant strains began linear growth on fresh medium without delay, whereas the wild-type strain did not show linear growth on this medium ( Figure 2B). When conidia of these strains were inoculated on Vm medium, filamentous growth of the Δsor-4a and Δcol-26a strains, as well as the wild-type strain, were clearly detectable after 18 h. However, on SOR medium, all strains formed compact colonies 48 h after inoculation, and only the Δsor-4a and Δcol-26a strains began to grow at the edge of the colonies. These observations suggest that there was a transition stage between colonial growth and linear hyphal growth in the Δsor-4a and Δcol-26a strains on SOR medium. In addition, conidia of the Δsor-4a and Δcol-26a strains, as well as the wild-type strain, did not germinate on Vm medium containing sorbose as the sole carbon source; therefore, the addition of a normal carbon source, such as sucrose or glucose, is essential for filamentous growth on medium containing 1% sorbose. Growing hyphae of the sor-4(DS(r))A and Δcol-26a strains on SOR medium were transferred onto fresh SOR medium. For the wild-type strain, conidia were spread on the SOR medium and incubated for 2 days, and then agar disks containing growing cells were transferred onto fresh SOR medium. Hyphal elongation (mm) from the edges of inoculation disks were measured for 3 days. Errors are expressed as standard deviation.

Sorbose Resistance, 2-DG Resistance, and QoI Sensitivity of CCR Mutants
The sorbose-resistance factors COL-26 and SOR-4 are known to be involved in CCR and their mutant strains are less sensitive to 2-DG, an analog of glucose that cannot be isomerized to fructose; therefore, it is not further metabolized and is often used to select for CCR factors in filamentous fungi [13,43]. The involvement of CCR factors in sorbose resistance prompted us to examine whether the deletion of other CCR factors was involved in sorbose resistance and/or 2-DG resistance. We examined three factors, namely a hexokinase, HXK-2, a major CCR-transcription factor, CRE-1, and an AMP-activated S/T protein kinase, PRK-10. None of these N. crassa deletion mutants displayed sorbose resistance ( Figure 1B); however, the Δhxk-2a mutant was as resistant to 2-DG as the Δsor-4a and Δcol-26a strains ( Figure 3A). In contrast, the Δprk-10a mutant was hypersensitive to 2-DG. The Δcre-1 mutant was previously reported to have 2-DG resistance when Avicel was used as a carbon source [33], but our results showed that the Δcre-1a mutant was somewhat sensitive to 2-DG on agar medium containing fructose as a carbon source. We

Sorbose Resistance, 2-DG Resistance, and QoI Sensitivity of CCR Mutants
The sorbose-resistance factors col-26 and sor-4 are known to be involved in CCR and their mutant strains are less sensitive to 2-DG, an analog of glucose that cannot be isomerized to fructose; therefore, it is not further metabolized and is often used to select for CCR factors in filamentous fungi [13,43]. The involvement of CCR factors in sorbose resistance prompted us to examine whether the deletion of other CCR factors was involved in sorbose resistance and/or 2-DG resistance. We examined three factors, namely a hexokinase, HXK-2, a major CCR-transcription factor, cre-1, and an AMP-activated S/T protein kinase, PRK-10. None of these N. crassa deletion mutants displayed sorbose resistance ( Figure 1B); however, the ∆hxk-2a mutant was as resistant to 2-DG as the ∆sor-4a and ∆col-26a strains ( Figure 3A). In contrast, the ∆prk-10a mutant was hypersensitive to 2-DG. The ∆cre-1 mutant was previously reported to have 2-DG resistance when Avicel was used as a carbon source [33], but our results showed that the ∆cre-1a mutant was somewhat sensitive to 2-DG on agar medium containing fructose as a carbon source. We sequenced the hxk-2 gene of the dgr-4(KHY7)a mutant (FGSC#8287) and found the insertion of a 139 bp fragment within the third exon of hxk-2 ( Figure S2B). This insertion resulted in a frame shift at codon 289 and immature termination at codon 292 in hxk-2. This is the first report to show the dgr-4 gene is identical to the hxk-2 gene. sequenced the hxk-2 gene of the dgr-4(KHY7)a mutant (FGSC#8287) and found the insertion of a 139 bp fragment within the third exon of hxk-2 ( Figure S2B). This insertion resulted in a frame shift at codon 289 and immature termination at codon 292 in hxk-2. This is the first report to show the dgr-4 gene is identical to the hxk-2 gene. Errors are expressed as the standard error.
As described above, Δcol-26a showed hyper-sensitivity to antimycin A and azoxystrobin, which inhibit mitochondrial complex III by binding the Qi-site and the Qosite of cytochrome b, respectively. Both the Δsor-4a and Δcol-26a mutants exhibited very similar sensitivity to these respiration inhibitors ( Figure 4A). Not only sorbose-resistant mutants, but also Δhxk-2a and Δprk-10a mutants were hypersensitive to antimycin A and azoxystrobin ( Figure 4B). In contrast, QoI-and QoI sensitivity in the Δcre-1a mutant was almost same to that of the wild-type strain. It is well known that the alternative oxidase AOD-1 reduces sensitivity to complex III inhibitors. Therefore, we analyzed aod-1 expression in the Δcol-26a mutants. The aod-1 gene was upregulated by azoxystrobin to the same level as in the wild-type strain, suggesting that QoI sensitivities of CCR mutants are independent with alternative oxidase activity. As described above, ∆col-26a showed hyper-sensitivity to antimycin A and azoxystrobin, which inhibit mitochondrial complex III by binding the Qi-site and the Qo-site of cytochrome b, respectively. Both the ∆sor-4a and ∆col-26a mutants exhibited very similar sensitivity to these respiration inhibitors ( Figure 4A). Not only sorbose-resistant mutants, but also ∆hxk-2a and ∆prk-10a mutants were hypersensitive to antimycin A and azoxystrobin ( Figure 4B). In contrast, QoI-and QoI sensitivity in the ∆cre-1a mutant was almost same to that of the wild-type strain. It is well known that the alternative oxidase AOD-1 reduces sensitivity to complex III inhibitors. Therefore, we analyzed aod-1 expression in the ∆col-26a mutants. The aod-1 gene was upregulated by azoxystrobin to the same level as in the wild-type strain, suggesting that QoI sensitivities of CCR mutants are independent with alternative oxidase activity.
col-26 and cre-1 have been shown to regulate several carbohydrate-related enzymes involved in CCR. We compared the expression pattern of relevant genes in the ∆cre-1a, ∆col-26a, and ∆col-26; ∆cre-1a mutants. First, we selected six genes, gla-1 (glucoamylase), inv (invertase), gh31-3 (alpha-glucosidase), gh13-2 (alpha-amylase), glt-1 (low-affinity glucose transporter), and hgt-1 (high-affinity glucose transporter), and compared their expression in medium with glucose or maltose as the carbon source. These genes, except for gh13-2 and glt-1, were highly induced in the wild-type strain grown on maltose medium ( Figure 5A). As previously reported [29], the deletion of the negative regulator cre-1 resulted in high constitutive expression of CCR-related genes ( Figure 5B). The inductions of gla-1, inv, gh31-3, gh13-2, and hgt-1 in the ∆cre-1a mutant was quite evident in glucose-rich conditions, but minimal in glucose-depleted conditions. In contrast, the expression profiles of all six genes were almost the same in the ∆col-26a and ∆sor-4a mutants in both conditions. The expression of gla-1, inv, gh31-3, and hgt-1 was slightly upregulated in both the ∆col-26a and ∆sor-4a mutants in glucose-rich condition, whereas the expression of gh13-2 and glt-1 was significantly downregulated in glucose-rich and glucose-depleted conditions. It should be noted that the expression pattern of the ∆col-26;∆cre-1a double mutant was almost the same as that of the ∆col-26a and ∆sor-4a mutants ( Figure 5B). These data indicated that derepression by the loss of cre-1 function might be overcome by the deletion of the col-26 gene.
COL-26 and CRE-1 have been shown to regulate several carbohydrate-related enzymes involved in CCR. We compared the expression pattern of relevant genes in the Δcre-1a, Δcol-26a, and Δcol-26;Δcre-1a mutants. First, we selected six genes, gla-1 (glucoamylase), inv (invertase), gh31-3 (alpha-glucosidase), gh13-2 (alpha-amylase), glt-1 (low-affinity glucose transporter), and hgt-1 (high-affinity glucose transporter), and compared their expression in medium with glucose or maltose as the carbon source. These genes, except for gh13-2 and glt-1, were highly induced in the wild-type strain grown on maltose medium ( Figure 5A). As previously reported [29], the deletion of the negative regulator cre-1 resulted in high constitutive expression of CCR-related genes ( Figure 5B). The inductions of gla-1, inv, gh31-3, gh13-2, and hgt-1 in the Δcre-1a mutant was quite evident in glucose-rich conditions, but minimal in glucose-depleted conditions. In contrast, the expression profiles of all six genes were almost the same in the Δcol-26a and Δsor-4a mutants in both conditions. The expression of gla-1, inv, gh31-3, and hgt-1 was slightly upregulated in both the Δcol-26a and Δsor-4a mutants in glucose-rich condition, whereas the expression Several glucose transporters have been reported to be downregulated by the ∆col-26a and ∆sor-4a mutants [35]; therefore, we measured the concentrations of extracellular glucose and sorbose during cultivation ( Figure 6). The wild-type strain consumed the most glucose after incubation for 24 h when the initial glucose concentration was 1%, meanwhile, more than 60% of glucose remained in the culture medium in the cases of the ∆col-26a and ∆sor-4a mutants, suggesting their low uptake of glucose ( Figure 6A). In contrast, after 24 h incubation, approximately 90% of sorbose remained unincorporated in the case of the ∆col-26a and ∆sor-4a mutants, as well as the wild-type strain ( Figure 6B). These data suggest that these sorbose-resistance mutants do not incorporate sorbose more than the wild type and do not assimilate it.

A Single S11L Mutation, but Not the Loss-of-function Mutation of F-box Protein exo-1, Confers Sorbose, and 2-DG Resistances in N. Crassa
A 2-DG-resistant strain in dgr-2, dgr-2(L1)a, has sorbose resistance ( Figure 7A). Recently, Gabriel et al. [36] revealed that the exo-1 gene encoded a F-box protein, and identified a S11L missense mutation within the exo-1 gene in dgr-2(L1)a strain ( Figure S4) [36]. In N. crassa, the exo-1 mutant produced the maximum extracellular glucoamylase activity in starch-supplemented medium [44]; however, the exo-1 deletion mutant did not show any sorbose resistance ( Figure 7A), indicating that the single amino-acid substitution in exo-1/DGR-2, S11L, confers sorbose resistance as well as 2-DG resistance. As shown in Figure 7B, the dgr-2(L1)a strain was resistant to 2-DG but ∆exo-1a was somewhat sensitive to the sugar. Furthermore, we confirmed that the ∆exo-1a strain displayed hypersecretion of proteins, as described previously [36] (Figure 7C), even though the dgr-2(L1)a strain did not. In glucose-rich conditions, all four genes-namely gla-1, inv, glt-1, and hgt-1-were slightly upregulated in the ∆exo-1a strain, whereas the expression pattern of the exo-1 S11L mutant resembles to that of the ∆col-26a strain ( Figure 7D). These results suggest that the phenotypes of ∆exo-1a differ from those of the exo-1 S11L strain.  Several glucose transporters have been reported to be downregulated by the Δcol-26a and Δsor-4a mutants [35]; therefore, we measured the concentrations of extracellular glucose and sorbose during cultivation ( Figure 6). The wild-type strain consumed the most glucose after incubation for 24 h when the initial glucose concentration was 1%, meanwhile, more than 60% of glucose remained in the culture medium in the cases of the Δcol-26a and Δsor-4a mutants, suggesting their low uptake of glucose ( Figure 6A). In contrast, after 24 h incubation, approximately 90% of sorbose remained unincorporated in the case of the Δcol-26a and Δsor-4a mutants, as well as the wild-type strain ( Figure 6B). These data . Six genes, gla-1 (glucoamylase), gh13-2 (alpha-amylase), gh31-3 (alpha-glucosidase), inv (invertase), glt-1 (low-affinity glucose transporter), and hgt-1 (high-affinity glucose transporter) were selected as target genes for qPCR analysis. With the exception of gh13-2 and glt-1, all genes were highly upregulated in maltose medium. (B) Comparison of gene expression patterns in the ∆col-26a, ∆sor-4a, ∆cre-1a and ∆col-26; ∆cre-1a mutants. Expression levels in each mutant strain were calculated relative to the wild-type strain grown under glucose-rich conditions (Glucose) and glucose derepression conditions (Maltose). Errors are expressed as the standard error. At least three biological replicates of each experiment were performed.
suggest that these sorbose-resistance mutants do not incorporate sorbose more than the wild type and do not assimilate it.

Discussion
L-Sorbose induces hyperbranching of hyphae and results in colonial growth on agar media in Neurospora crassa. Among the six genes identified as conferring sorbose resistance (sor-1 to sor-6), only sor-4, which encodes a glucose sensor protein, has been thoroughly investigated [14,15]. In this work, we revealed two more genes, col-26, which encodes a transcription factor, and exo-1, which encodes a F-box protein, that are likely involved in sorbose resistance (Figures 1A and 7A). Comparison of the phenotypes of the exo-1 S11L (dgr-2(L1)a) and ∆exo-1a mutants. Sorbose resistance (A) and 2-DG resistance (B) of wild-type, ∆col-26a, exo-1 S11L (dgr-2(L1)a), and ∆exo-1a strains. (C) SDS-PAGE analysis of proteins secreted by each strain. The extracellular protein in culture filtrates was concentrated and applied for SDS-PAGE analysis. (D) Gene expression of gla-1, inv, glt-1, and hgt-1 under glucose-rich conditions (see Figure 5) in each strain. Errors are expressed as the standard error.

Discussion
L-Sorbose induces hyperbranching of hyphae and results in colonial growth on agar media in Neurospora crassa. Among the six genes identified as conferring sorbose resistance the S11L mutation results in the constitutive activation of exo-1, hyper-ubiquitination, and the degradation of col-26.
As previously reported [29,30], the loss of cre-1 function leads to the overexpression of gla-1, gh13-2, gh31-3, inv, glt-1, and hgt-1, even in glucose-rich conditions ( Figure 5A). cre-1-like transcription factors are the main negative regulators in glucose repression in many fungi [18]. The expression of glt-1, which encodes a low-affinity glucose transporter, in three sorbose-resistance mutants, namely ∆col-26a, ∆sor-4a, and ∆col-26;∆cre-1a was significantly reduced, even in glucose-rich conditions, suggesting that the glucose sensor sor-4 and the transcription factor col-26 are essential for the expression of glt-1, as described elsewhere [35]. In addition, the expression of gh13-2, which encodes an alpha-amylase, in these three mutants was comparable to that of glt-1, suggesting that these two genes would be controlled under the same system. We noticed that the gene expression profiles for carbohydrate-related genes in col-26 and sor-4 were similar. Moreover, a double mutant ∆col-26;∆cre-1a had a very similar gene expression profile to ∆col-26a and ∆sor-4a. It should be noted that the upregulation found in the cre-1 strain was mostly negated in the ∆col-26;∆cre-1a strain. The col-26 transcription factor could positively control its target genes, which should be suppressed by unphosphorylated cre-1, and the loss cannot be compensated for because of the absence of the negative regulator cre-1. Indeed, the ∆col-26;∆cre-1a strain displayed a more similar phenotype to the col-26 single mutant than the cre-1 mutant. Meanwhile, the expression of gla-1, inv, gh31-3, and hgt-1 in these three sorbose-resistant mutants in glucose-rich conditions was slightly upregulated. The presence of any cre-1 and col-26 independent regulatory system (s) cannot be eliminated.
The mechanism underlying sorbose resistance in N. crassa is still unclear. In our study, we revealed that, in addition to the glucose sensor sor-4, two factors-namely col-26 and exo-1-are involved in sorbose resistance. All evidence obtained regarding sorbose resistance indicates the connection of the expression of carbohydrate-related genes in glucose-depleted conditions and the toxicity of the chemical. Sorbose might disturb signaling pathway(s) concerning carbohydrate-related gene expression by interacting with the sugar-sensing system. col-26 upregulates glucose transporter genes, including glt-1 and hgt-1 [35]; indeed, col-26 mutant uptake less glucose than the wild-type strain. We speculate that sorbose-resistant mutants hardly assimilate the sugar, as its incorporation by the col-26 mutants was only marginally different to the that of the wild-type strain ( Figure 6).
CCR is fundamental mechanism using proper energy sources; therefore, it is conserved in prokaryote and eukaryotes. In fungi, CCR is directly connected with various applications, such as the production of Japanese sake and miso by Aspergillus species, bioethanol production by T. reesei, and plant protection from plant pathogenic fungi. Recent studies indicate that catabolite repression in fungi is connected to several signaling molecules, such as cAMP-dependent protein kinase and the stress-response MAP kinase. Therefore, our results in the model fungus N. crassa will contribute to elucidation of the complex mechanism of fungal CCR.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof8111169/s1, Figure S1 Screening of transcription factor knockout library for sensitivity to the mitochondria complex III inhibitors, azoxystrobin and antimycin A, Figure S2A Physical and genetical mapping of the col-26 and dgr-1genes, and the mutation of dgr-1(BE52)A strain, S2B Physical and genetical mapping of the hxk-2 and dgr-4 genes, and the mutation of the dgr-4(KHY7)a strain, Figure S3 PCR confirmation of the deletion of cre-1 and col-26 genes in ∆col-26; ∆cre-1 double mutants, Figure S4A Amino-acid homology of N-terminal region of F-box protein exo-1 protein in fungi, and Figure S4B Alignment of exo-1 amino-acid sequences in fungi. The dgr-2(L1)a strain has S11L mutation in exo-1. Table S1 List of sequencing primers for col-26 and hxk-2 genes, Table S2 List of primers used for PCR detection of gene deletions, Table S3 List of RT-qPCR primers and universal probes.