Neurospora crassa strains used in this study are listed in Table 1. All of them were obtained from the Fungal Genetics Stock Center¹ (University of Kansas Medical Center). Vogel’s minimum medium (Vogel, 1956), supplemented with 2% (w/v) sucrose for slants or 2% glucose for plates and the liquid medium, was used for culturing strains. All cultures were grown at 28°C in the light. Antifungal compounds were added as needed.

Ketoconazole, fluconazole, and itraconazole were dissolved in dimethyl sulfoxide (DMSO) and then aseptically added to the autoclaved medium before making agar plates. The final concentrations of ketoconazole, fluconazole, and itraconazole in the agar plates were 2, 25, and 10 μg/ml, respectively. The final DMSO concentration was below 0.25% (v/v). Two microliters of conidial suspension were inoculated on plates (Φ = 9 cm) with or without antifungal drugs and incubated at 28°C for 66 h.

The MICs of three azoles for each strain were determined in 96-well microtiter plate according to National Committee for Clinical Laboratory Standards (CLSI, 2008). Briefly, 100 μl of 2× azole solution and 100 μl of conidial suspension were added into each well. The final conidial concentration was approximately 1 × 10⁶ conidia/ml. The plates were incubated at 28°C for 24 h. The MIC was determined as the lowest drug concentration without growth.

The transcriptional responses of ergosterol biosynthesis genes to ketoconazole treatment in the wild-type strain were detected by digital gene expression (DGE) profiling (Nielsen et al., 2006). Briefly, conidia of the wild-type strain was inoculated into 20-ml liquid medium in a plate (Φ = 9 cm) and incubated for 24 h at 28°C in darkness to form mycelial mat on the surface of the liquid medium. Mycelial mat then was cut into small pieces (Φ = 10 mm) and transferred to the liquid medium (2 pieces/100 ml). Cultures were incubated at 28°C with shaking at 180 rpm for 12 h. Ketoconazole was then added into the medium to reach a final concentration of 2.5 μg/ml. After 24 h of incubation, mycelia were harvested and total RNA was extracted and subjected to DGE analysis as described by Sun et al. (2011).

Sterols of dried mycelium (0.3 g) was extracted into 1.5-ml chloroform under ultrasonication for 12 h. The extracts were dried and subsequently dissolved into 150 μl methanol under ultrasonication for 1 h. After filtration with a Millipore filter, the extracts were subjected to HPLC-MS analysis following the method reported by Cañabate-Díaz et al. (2007) with some modifications.

Liquid chromatography separation was performed on an Agilent Zorbax Extend-C18 1.8 μm 2.1 mm × 50 mm column using an Agilent 1200 Series system (Agilent, USA). Total flow rate was 0.4 ml min⁻¹; mobile phase A consisted of water with 0.01% acetic acid and mobile phase B consisted of acetonitrile. Total elution program was 27 min. Gradient began with 70% mobile phase B, changed to 100% B over 2 min, maintained a constant level from 2 to 18 min at 100% B and then decreased to 70% B over 1 min prior to re-stabilization over 8 min before the next injection. The column temperature was maintained at 40°C. The injection volume was 10 μl.

Mass spectra were acquired with an Agilent Accurate-Mass-Q-TOF MS 6520 system. Analytes were detected in the positive ionization mode with an APCI probe. For Q-TOF/MS conditions, fragmentor and capillary voltages were kept at 130 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. Temperature of the drying gas and vaporizer were both set at 350°C. The corona was set to 4 μA. The flow rate of the drying gas and the pressure of the nebulizer were 3 l min⁻¹ and 50 psi, respectively. Full-scan spectra were acquired over a scan range of m/z 80–1000 at 1.03 spectra s⁻¹. The derived sterols were identified with reference molecular weight and fragmentation spectra for known standards.

Protein sequences were aligned with the Clustal X 2.1 software (Larkin et al., 2007). Then, the neighbor-joining (NJ) tree was constructed using MEGA 5 software (Tamura et al., 2011). To assess the confidence of phylogenetic relationships, the bootstrap tests were conducted with 1000 resamplings.

In order to knockout erg5 homologous genes (erg5A: FVEG_07284; erg5B: FVEG_08786) in F. verticillioides, the upstream and downstream flanking sequences of the FVEG_07284 and FVEG_08786 were amplified. The primer pairs were shown in Table 2.

The resulting PCR products were cloned into the plasmid pCX62 (Zhao et al., 2004) and results in knockout construct pCX62-ΔFv07284 and pCX62-ΔFv08786, in which the PCR products were ligated with the hygromycin phosphotransferase (hph) gene. Then, the deletion cassette was transformed into F. verticillioides 7600 and resulted in deletion mutants. Fungal transformation followed the protocol reported by Miller et al. (1985), with minor modification as described by Li et al. (2006).

Genome-wide transcriptional responses to ketoconazole treatment from two independent experiments were analyzed by DGE method. For genes involved in ergosterol biosynthesis, seven were consistently upregulated while four consistently downregulated upon ketoconazole treatment in two independent experiments (Table 3). The most dramatically upregulated genes were erg11 (NCU2624), erg6 (NCU03006), erg2 (NCU04156), and erg5 (NCU05278), which were consistently increased upon ketoconazole treatment by at least three folds in two independent experiments. The consistently downregulated genes include erg7 (NCU01119), erg8 (NCU08671), erg13 (NCU03922), and erg20 (NCU11381). However, the transcriptional changes in these downregulated genes were less dramatic than those in erg11, erg6, erg2, and erg5. None of them had a transcriptional change higher than three folds.

Six ketoconazole-responsive genes involved in ergosterol synthesis have the knockout mutants available in Fungal Genetics Stock Center. All these genes are ketoconazole-upregulated genes, including erg2, erg3, erg5, erg6, erg24, and erg25. On the solid medium without antifungal drugs, all deletion mutants had the growth rates similar to the wild-type strain and no obvious defect was observed in their asexual and sexual reproduction (the data not shown)². Drug sensitivity test showed that the deletion of only erg5 altered the sensitivity to azoles. On agar plates, all three tested azoles, including ketoconazole, itraconazole, and fluconazole, had greater inhibition to the erg5 mutants than the wild-type strain (Figure 1). MIC analysis in the liquid medium showed that MIC values of the ERG5 mutants for ketoconazole, itraconazole, and fluconazole were only 60, 50, and 40% of that of the wild-type strain, respectively (Table 4).

ERG5 catalyzes the biosynthesis of ergosta 5,7,22,24(28)-trienol, a direct precursor for ergosterol biosynthesis. ERG5 mutations caused accumulation of ergosta 5,7,24(28)-trienol and ergosta 5,7-dienol in S. cerevisiae ERG5 deletion mutant (Barton et al., 1974; Skaggs et al., 1996). Thus, it is possible that the erg5 mutant might accumulate more toxic 14α-methylated sterols than the wild-type strain. To test this possibility, a comparative analysis of sterol profiles between the erg5 mutant and the wild type was performed by HPLC-MS. Our results showed that, in the normal liquid medium without ketoconazole, the erg5 mutant produced more ergosta 5,7-dienol than the wild-type strain. As expected, ergosterol in the erg5 mutant was almost undetectable (Figure 2). When treated with ketoconazole, 4,4-dimethyl-ergosta 8,14,24(28)-trienol, the direct product catalyzed by ERG11 (lanosterol 14α-methylase), was reduced in the wild-type strain and the erg5 mutant at the similar level. Ketoconazole treatment greatly increased levels of 14α-methylated sterols, such as eburicol and 14α-methyl-ergosta-8,24(28)-dien-3β, 6α-diol, in both the wild-type strain and the erg5 mutant (Figure 2). However, the levels of these compounds were similar between the wild-type strain and the erg5 mutant, suggesting that the azole-hypersensitive phenotype in the erg5 mutant is not due to over-accumulation of 14α-methylated sterols.

To gain an insight into the sequence conservation and phylogenic relationship of ERG5 homologs in fungi, Blast and phylogenetic analysis were conducted. Blast analysis revealed that ERG5 homologs are high conserved in amino acid sequences. Phylogenetic analysis with amino acid sequences of 27 ERG5 homologs (E value = 0; Alignment coverage >90%; sequence similarity >76%) from 3 yeast fungal species (C. albicans, S. cerevisiae, Schizosaccharomyces pombe) and 16 filamentous fungal species showed that all ERG5 homologs were clustered into 2 big clades (Figure 3). The ERG5 homologs from yeast fungal species were distributed in the first clade (clade I), which represents the most ancestral group, while those from filamentous fungal species were located in the second clade (clade II).

As shown in Figure 3, ERG5 in filamentous fungi can be further categorized into two classes based on their phylogenic relationship, namely Type IIA and Type IIB, respectively. N. crassa, N. fischeri, A. niger, A. nidulans, T. reesei, M. grisea, and V. albo-atrum have only one erg5 gene, all in Type IIA. A. fumigatus, A. oryzae, and A. clavatus and all examined Fusarium species have two erg5 genes, one in Type IIA and the other in Type IIB. A. terreus is the only species that has three erg5 genes, two in Type IIA and one in Type IIB. Similar to ERG5, multiple ERG3 and ERG11 homologs were also found in Aspergillus and Fusarium species (Ferreira et al., 2005; Liu et al., 2010; Becher et al., 2011).

To see if ERG5 in other filamentous fungi also mediate azole sensitivity, gene knockout mutants for Type IIA erg5 gene erg5A (FVEG_07284) and for Type IIB erg5 gene erg5B (FVEG_08786), in the pathogenic fungus F. verticillioides were generated. As shown in Figure 4, on the medium without drug, the growth rates of the erg5 deletion mutants were similar to that of the wild-type strain and no defect in conidiation was observed in the mutants. When inoculated on the medium with 0.5 μg/ml ketoconazole, both erg5A and erg5B mutant displayed greater growth inhibition than the wild-type strain, indicating the ERG5 homologs have similar roles in azole resistance among different fungal species.