Contributions of yap1 Mutation and Subsequent atrF Upregulation to Voriconazole Resistance in Aspergillus flavus

Aspergillus flavus is the second most significant pathogenic cause of invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure of A. flavus to subinhibitory concentrations of VRC in vitro causes the emergence of a VRC-resistant mutant with a novel resistance mechanism.

ABSTRACT

Aspergillus flavus is the second most significant pathogenic cause of invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure of A. flavus to subinhibitory concentrations of VRC in vitro causes the emergence of a VRC-resistant mutant with a novel resistance mechanism. The VRC-resistant mutant shows a MIC of 16 μg/ml for VRC and of 0.5 μg/ml for itraconazole (ITC). Whole-genome sequencing analysis showed that the mutant possesses a point mutation in yap1, which encodes a bZIP transcription factor working as the master regulator of the oxidative stress response, but no mutations in the cyp51 genes. This point mutation in yap1 caused alteration of Leu558 to Trp (Yap1^Leu558Trp) in the putative nuclear export sequence in the carboxy-terminal cysteine-rich domain of Yap1. This Yap1^Leu558Trp substitution was confirmed as being responsible for the VRC-resistant phenotype, but not for that of ITC, by the revertant to Yap1^{wild type} with homologous gene replacement. Furthermore, Yap1^Leu558Trp caused marked upregulation of the atrF ATP-binding cassette transporter, and the deletion of atrF restored susceptibility to VRC in A. flavus. These findings provide new insights into VRC resistance mechanisms via a transcriptional factor mutation that is independent of the cyp51 gene mutation in A. flavus.

INTRODUCTION

Invasive aspergillosis is increasingly recognized as one of the most serious complications in patients with hematological malignancy, allogeneic hematopoietic stem cell transplantation, and various other highly immunocompromised conditions (1). Antifungal drugs have been approved to treat invasive aspergillosis and are classified into three classes: the triazoles (voriconazole, itraconazole, posaconazole, and isavuconazole), the polyenes (amphotericin B and various liposomal formulations), and the echinocandins (caspofungin, micafungin, and anidulafungin). Treatment with voriconazole (VRC) is currently the first-line therapy for treatment of aspergillosis (2). However, the efficacy rates of VRC and isavuconazole (ISA) treatment against invasive aspergillosis are currently less than 40%, and mortality remains quite high (≥20%) (3).

In addition, the emergence and increase of azole-resistant Aspergillus strains have been recognized as being medical problems over the past 2 decades (4–8). In A. fumigatus, mutations in the cyp51A gene and its promoter region have been found to be responsible for azole resistance (9). Amino acid substitutions in the Cyp51A protein at residues G54, G138, P216, F219, M220, G432, and G448 cause azole resistance (10–14). Recently, the emergence of azole-resistant A. fumigatus strains harboring a 34-bp tandem repeat (TR34) in the promoter region of cyp51A with L98H (TR34/L98H) or a 46-bp tandem repeat (TR46) with Y121F and T286A (TR46/Y121F/T286A) has been reported in many countries (15–21). These mutants show azole resistance through Cyp51A substitutions combined with upregulation of cyp51A caused by mutation of TR36 or TR46 (22, 23). Besides these target gene mutations, upregulation of multidrug efflux transporters, such as the ATP-binding cassette (ABC), and the major facilitator superfamily (MFS) are also involved in azole resistance. A. fumigatus is known to have 49 genes encoding ABC transporters and 278 genes encoding MFS transporters (24). Among these efflux transporters, Cdr1B, AtrF, AtrI, and Mdr4 have been reported to contribute to azole resistance in A. fumigatus (25–29). Furthermore, a substitution in HapE, a subunit of the CCAAT-binding transcription factor, was discovered to be responsible for azole resistance in a clinical isolated strain (30).

A. flavus is the pathogen second most frequently responsible for aspergillosis, followed by A. niger and A. terreus (31–35). A. flavus is considered more virulent than A. fumigatus based on its pathogenicity in disseminated mouse models (36). This evidence suggests that the emergence and spread of azole-resistant A. flavus is of great medical importance. Indeed, some azole-resistant A. flavus isolates are reported to have emerged that possess mutations in the cyp51 genes, such as cyp51A, cyp51B, and cyp51C (37, 38). Also, some efflux transporter genes, such as mdr2, mdr4, and atrF, were upregulated in the presence of VRC in some VRC-resistant A. flavus strains (39). However, the contribution of cyp51 substitutions and upregulation of efflux transporter genes to azole resistance have not yet been demonstrated with genetic interaction in A. flavus.

Therefore, the aims of this study are to isolate an unreported azole-resistant A. flavus mutant and to demonstrate a novel azole-resistant molecular mechanism with evidence of genetic interaction.

(This study was presented in part at ASM Microbe 2017, 1 to 5 June 2017, New Orleans, LA [40].)

RESULTS

Serial passage cultures of A. fumigatus and A. flavus in the presence of azole drugs.

To monitor the emergence of azole-resistant A. flavus, in vitro serial passages of cultures of A. fumigatus and A. flavus were performed in the presence of azoles, as previously described (41), with some modifications (strains used are described in Table 1). For A. fumigatus exposed to itraconazole (ITC), the conidium harvest concentration (CHC) after the fifth passage culture reached 16 μg/ml, which was 128-fold higher than that prior to passage culture (Fig. 1A). However, in A. fumigatus exposed to VRC, the CHC after the fifth passage culture was 0.25 μg/ml, which was only 2-fold higher than that prior to passage culture (Fig. 1A). A. fumigatus conidia from the fifth ITC passage culture showed a MIC of 16 μg/ml for ITC and of 4 μg/ml for VRC and harbored a single-nucleotide mutation in the cyp51A coding region (G1294A; exon based), causing a Gly432Ser substitution in Cyp51A (Table 2). This substitution has previously been reported in a clinical isolate obtained from a patient with aspergillosis (12). In A. flavus exposed to VRC, the CHC after the fifth passage reached 16 μg/ml, which was 32-fold higher than that prior to passage culture (Fig. 1B), whereas only moderate elevation of CHC was observed in A. flavus exposed to ITC. The A. flavus conidia from the fifth VRC passage culture showed a MIC of 16 μg/ml for VRC and of 0.5 μg/ml for ITC (Fig. 1B). This VRC-resistant A. flavus isolate did not harbor a mutation in the cyp51A gene (Table 2).

TABLE 1.

Strains used or obtained in homologous gene replacement studies

Strain	Relevant genotype	Growth ratea	Reference or source
A1421	A. flavus ΔpyrG, Δku70		64
SR-A	A1421, yap1 (T1673G)	Normal	This study
SR-B	SR-A, pyrG	Normal	This study
SR-C	SR-A, yap1::pyrG	Normal	This study
SR-D	SR-A, ΔatrF::pyrG	Normal	This study
SR-E	A1421, pyrG	Normal	This study
SR-F	A1421, yap1::pyrG	Normal	This study
SR-G	A1421, yap1(T1673G)::pyrG	Normal	This study
SR-H	A1421, ΔatrF::pyrG	Normal	This study

^aGrowth rate was compared with that of strain A1421.

FIG 1.

Serial passage cultures in the presence of azole drugs. After 7 days of culture in the presence of itraconazole (ITC) or voriconazole (VRC), newly formed conidia were harvested from the highest concentration at which more than 1 × 10⁷ CFU conidia formed. The harvested conidia were then inoculated to fresh RPMI 1640 agar medium containing ITC or VRC. This cycle was repeated five times, and conidia harvested from the fifth passage cultures were used for further experiments.

TABLE 2.

Azole MICs and Cyp51A substitution of A. fumigatus and A. flavus

Strain and passage	MIC (μg/ml)		Cyp51A amino acid substitutiona
	Itraconazole	Voriconazole
A. fumigatus ATCC 204305
Parent	0.5	0.5
5th ITC passage	>16	4	Gly432Ser
5th VRC passage	1	1	None
A. flavus ATCC 204304
Parent	0.5	0.5
5th ITC passage	1	2	None
5th VRC passage	0.5	16	None

^aSubstitutions were determined by comparing serially passaged strains to each parental strain.

Whole-genome sequencing analysis of VRC-resistant A. flavus.

To determine the molecular mechanism for VRC resistance in A. flavus, we conducted a whole-genome sequencing analysis to identify the VRC resistance-related mutation in the ATCC 204304^Vr strain compared with the sequence of the parental strain (ATCC 204304). A. flavus ATCC 204304^Vr showed a MIC of 16 μg/ml for VRC and of 0.5 μg/ml for ITC (see Table S1 in the supplemental material) and showed no impaired growth in RPMI medium; this was also the case for six other single clones (data not shown). Raw Illumina whole-genome sequencing reads (Table S2) were aligned using the two reference genomes, NRRL3357 and AF70. The candidates of point mutation in ATCC 204304^Vr are shown in Table 3. Results show that the T1673G point mutation (exon based) in AFLA_129340, which encodes the bZIP transcription factor Yap1, was identified as the only common single-nucleotide mutation when aligned with sequence of both NRRL3357 and AF70. This point mutation discovered in ATCC 204304^Vr was also confirmed with Sanger sequencing, and that for another six single clones also was confirmed by Sanger sequencing (Table S1). This indicates that the emergence of the yap1 T1673G point mutation occurred mainly during the serial VRC passage culture of A. flavus in this study.

TABLE 3.

Candidates of point mutation in the VRC-resistant A. flavus

Locus tag	Substitution		Reference genome
	Nucleotide	Amino acid
AFLA_129340	T1673G	L558W	NRRL3357
AFLA70_107g002681			AF70
AFLA_086460	C1340T	T447I	NRRL3357
AFLA_086460	A1342C	I448L	NRRL3357
AFLA_118220	G520A	V174I	NRRL3357
AFLA70_204g001611	G399A	W133STOP	AF70

Homologous gene replacement of the mutated yap1 gene in A. flavus.

To investigate whether the T1673G point mutation in yap1 was responsible for VRC resistance, homologous gene replacement with the mutated yap1 was performed in the azole-susceptible A. flavus strain A1421. We first obtained a mutated yap1 transformant by VRC selection (SR-A) (Fig. 2A). This mutated yap1 recombinant, after pyrG complementation (SR-B), showed a MIC of 8 μg/ml for VRC (Table 4). The revertant of mutated yap1 to wild-type (WT) yap1 (SR-C) showed a MIC of 0.5 μg/ml for VRC (Table 4). These results suggest that the T1673G point mutation in yap1 is responsible for the VRC-resistant phenotype. Also, the other recombinant-introduced yap1 mutation, which was selected by complementation of uracil auxotrophy (SR-G) (Fig. 2C), showed a MIC of 2 μg/ml for VRC. Although this MIC value was 4-fold lower than that when VRC selection was used (SR-B) (Table 4), this result is also evidence which corroborates the responsibility of the yap1 T1673G point mutation for a VRC-resistant phenotype. This T1673G point mutation in yap1 causes alteration of Leu558 to Trp in the carboxy-terminal cysteine-rich domain (c-CRD) of Yap1 (Fig. 3A). Leu558 in A. flavus Yap1 is conserved among other molds and yeasts, such as A. fumigatus, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae (Fig. 3B).

FIG 2.

Construction schemes of gene mutation strains. Double-crossover recombination schemes in A. flavus A1421. (A, left) Replacement of wild-type (WT) yap1 in A1421 with T1673G mutated yap1 fragment. (Right) Transformants were selected in the presence of VRC. (B) Replacement of yap1 T1673G mutation in SR-A with WT yap1 containing pyrG fragment. Transformants were selected in the absence of uracil. (C) Replacement of WT yap1 in A1421 with WT yap1 or the T1673G mutated yap1 cassette containing a pyrG fragment. Transformants were selected in the absence of uracil.

TABLE 4.

Azole MICs of A. flavus strains with several homologous gene replacements

Strain and background	Relevant characteristic	MIC (μg/ml)
		Itraconazole	Voriconazole
Yap1L558W (VRC selection)
SR-B	pyrG	0.5	8
SR-C	Revertant to Yap1WT::pyrG	0.5	0.5
SR-D	ΔatrF::pyrG	0.5	1
A1421
SR-E	pyrG	0.5	0.5
SR-F	Yap1WT::pyrG	0.5	0.5
SR-G	Yap1L558W::pyrG	0.5	2
SR-H	ΔatrF::pyrG	0.5	0.5

FIG 3.

Yap1 substitution in the voriconazole-resistant A. flavus. (A) Schematic of the Yap1-substituted protein. The Cys residue in c-CRD is shown in red, and the putative NES is shown in blue. (B) Amino acid sequence alignment in Yap1 c-CRDs in A. fumigatus, A. flavus, C. albicans, C. glabrata, and S. cerevisiae with Clustal Omega.

Gene expression levels of efflux transporters and possible Yap1 regulating genes.

Yap1 has been known as the major regulator of responses to reactive oxygen species (ROS) stress in S. cerevisiae and pathogenic fungi (42–51). Based on previous studies of S. cerevisiae (52–57), the Leu558 residue in the A. flavus Yap1 is considered a key residue of the nuclear export signal (NES) that controls nuclear localization of Yap1. Therefore, we hypothesized that Yap1^L558W constitutively localizes in the nucleus and subsequently upregulates the expression of genes related to VRC resistance.

We focused on the promoter regions of efflux transporter genes, such as mdr1, mdr2, mdr3, atrF, and mfs1, the upregulation of which has been reported to be related to VRC resistance in A. flavus (39). Among them, a putative Yap1 response element [YRE; TTA(G/C/T)TAA] (42, 47), which binds to the bZIP domain of Yap1, was observed only in the atrF promoter region from −462 to −456 relative to the start codon, TTAGTAA (Table S5). Moreover, we focused on oxidative stress response-related genes which have putative YREs in their promoter regions, such as dsba, gst, cat1, and glr1 (Table S5). The transcriptional levels of yap1, cyp51A, efflux transporters, and oxidative stress response-related genes in ATCC 204304^Vr then were evaluated by real-time quantitative PCR (RT-qPCR). The expression levels of atrF, dsba, and gst were 62-, 27-, and 15-fold higher, respectively, than those of the parental ATCC 204304 (Fig. 4A). These upregulations were also observed in the transformed Yap1^L558W strain by VRC selection (SR-B) and were cancelled out in the revertant Yap1^WT strain (SR-C) (Fig. 4B). These results suggest that Yap1^L558W caused marked upregulations of atrF, dsba, and gst. Although the promoter region of cyp51A possesses a putative YRE (Table S5), marked upregulation of cyp51A was not observed in the Yap1^L558W recombinant (Fig. 4B). This indicates that the change of cyp51A expression level was not involved in VRC resistance caused by Yap1^L558W in A. flavus.

FIG 4.

Comparison of the transcriptional expression levels in several Yap1 mutants. Strains were grown for 24 h in RPMI medium in the absence of azole drugs. Four replicates in each strain were performed. Total RNA was prepared from mycelia and reverse transcribed. The cDNAs were subjected to RT-qPCR and analyzed for mRNA levels of yap1, cyp51A, efflux transporters, and oxidative stress response-related genes. Error bars represent standard deviations. (A) Fold changes between transcriptional levels of ATCC 204304^Vr and ATCC 204304. (B) Fold changes of transcriptional levels of SR-B, SR-C, SR-D, and SR-G compared to those of SR-E.

Contribution of marked atrF upregulation to VRC resistance in A. flavus.

To elucidate the contribution of atrF upregulation to reduced susceptibility to VRC, we constructed null mutants of atrF. As expected, null mutation of atrF sensitized the Yap1^L558W strain with VRC selection to VRC, which showed an 8-fold lower MIC for VRC (SR-D) (Table 4). Meanwhile, null mutation of atrF did not sensitize the Yap1^WT strain to VRC (SR-H) (Table 4). These results suggest that the marked upregulation of atrF plays a central role in VRC resistance in the Yap1^L558W strain with VRC selection, whereas the basal expression level of atrF did not affect VRC susceptibility in A. flavus.

DISCUSSION

Most of the resistance mechanisms in A. flavus against antifungal drugs have not yet been elucidated in detail. In this study, we were able to isolate a characteristic VRC-resistant A. flavus mutant, which was ITC susceptible and did not harbor any mutations in the cyp51 gene, through serial exposure to VRC (Table 2). Conversely, VRC-resistant A. fumigatus did not emerge after serial exposure to VRC, whereas an ITC-resistant A. fumigatus strain carrying the G432S substitution in Cyp51A was isolated after exposure to ITC (Table 2). The G432S-substituted A. fumigatus was isolated from a patient with aspergillosis and showed a MIC of 16 μg/ml for ITC (12). Although we have not found any reports on anecdotal clinical cases of VRC-resistant and ITC-susceptible A. flavus strain so far, we are concerned with whether the VRC-resistant A. flavus obtained in this study can emerge and prevail in clinical settings.

We indicated the presence of a novel VRC resistance mechanism caused by a mutant of the Yap1 transcriptional factor, which has been studied as a major regulator of the oxidative stress response in S. cerevisiae, C. albicans, C. glabrata, Cryptococcus neoformans, and A. fumigatus (42–51). We found that genes having the putative YRE, such as atrF, dsba, and gst, were upregulated in the Yap1^L558W strain. These results suggest that the Yap1^L558W substitution is a gain-of-function mutation causing constitutive accumulation of Yap1 in the nucleus. In A. fumigatus, ITC exposure caused ROS production via mitochondrial complex I, and the inhibition of ROS production led to 2-fold elevation of the MIC for ITC (58). In this study, even when oxidative response genes, such as dsba and gst, were upregulated in the A. flavus Yap1^L558W strain, the MIC for ITC was not changed (Table 4). Therefore, oxidative stress responses might not play a role in ITC resistance in A. flavus.

We demonstrated that the upregulation of atrF plays a central role in the VRC-resistant phenotype (Fig. 4B). Although the upregulation of the efflux transporters, especially when exposed to VRC, was previously observed in some VRC-resistant A. flavus strains (39), the efflux transporters that play a key role in the VRC resistance mechanism remain unknown. The demonstrated relationship between atrF upregulation and VRC resistance in this study promotes a better understanding of the roles of the efflux transporters in azole resistance in A. flavus. For example, it has recently been reported that the upregulation of A. fumigatus atrF in S. cerevisiae leads to a VRC-resistant and ITC-susceptible phenotype (27), which is the same phenotype as that found in Yap1^L558W A. flavus in this study. These results suggest that overproduced AtrF exports VRC more efficiently than ITC in both A. flavus and A. fumigatus. Moreover, the C-terminally truncated Yap1, which might cause a gain of function of Yap1, leads to a VRC-resistant and ITC-susceptible phenotype in A. fumigatus (59). As A. fumigatus atrF has a putative YRE (TTACTAA), at −391 to −385 relative to the start codon, a gain-of-function mutant of Yap1 in A. fumigatus may lead to the upregulation of atrF and may subsequently cause VRC resistance, as is the case with Yap1^L558W in A. flavus.

A similar event possibly occurs in C. albicans. A C-terminal truncation of Yap1 in C. albicans leads to the upregulation of the ABC transporter MDR1 gene (60), which has a putative YRE (TTATTAA) at −166 to −160 relative to the start codon. In addition, by a gain-of-function mutation in MRR1, which encodes a zinc cluster transcription factor, the upregulated MDR1 causes fluconazole (FLC) resistance (61). These observations suggest that a gain-of-function mutant of Yap1 in C. albicans, like Yap1^L558W in A. flavus, leads to upregulation of MDR1 and subsequently causes FLC resistance. Indeed, the C. albicans gene MDR1 is an ortholog of FLR1 which is regulated by Yap1 and plays a key role in FLC resistance in S. cerevisiae and C. glabrata (62, 63).

Interestingly, we observed that when the yap1 mutation was introduced by VRC selection, the MIC value for VRC and the expression level of atrF were significantly higher than those of the yap1 mutant when VRC selection was not employed (Fig. 4B). These results reveal that exposure to VRC is required for the acquisition of a highly VRC-resistant phenotype caused by marked upregulation of atrF via the yap1 mutation. Therefore, the elucidation of the molecular mechanism for the upregulation of atrF by any factors other than the yap1 mutation is required for better understanding of VRC resistance in A. flavus.

MATERIALS AND METHODS

Strains and media.

A. fumigatus ATCC 204305 and A. flavus ATCC 204304 were obtained from the American Type Culture Collection. A. flavus A1421 (CA14, Δku70, and ΔpyrG), obtained from the Fungal Genetics Stock Center, was used for the gene mutation and deletion strains. Conidia were grown on potato dextrose agar (PDA).

MIC test.

The MIC was determined by the broth microdilution method according to the CLSI M38-A2 standard (64). Conidium concentration was adjusted to approximately 1 × 10⁴ CFU/ml, and conidia were incubated for 48 h at 35°C in RPMI 1640 with 0.2% glucose buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS). The MIC was defined as the minimal concentration of antifungal agents that resulted in no visible growth. VRC and ITC were obtained from Tokyo Chemical Industry.

Serial passage culture in the presence of azole drugs.

Serial passage culture studies were performed based on previously described methods, with some modifications (41). The conidium concentration of A. fumigatus or A. flavus was adjusted to 1 × 10⁶ CFU/ml, and 100 μl of conidium suspension was inoculated to 10 ml of RPMI 1640 agar medium with 0.2% glucose buffered with 0.165 M MOPS (pH 7.0) containing a 2-fold serial dilution of VRC or ITC (0.125 μg/ml to 16 μg/ml) in 70-ml flasks with filter caps. After 7 days of culture at 35°C, the conidia were harvested from the highest drug concentration at which more than 1 × 10⁷ CFU conidia could be obtained, and the concentration in this experiment was defined as the CHC. For serial passage culture, the harvested conidia were inoculated to fresh RPMI 1640 agar medium containing 2-fold serial dilution of VRC or ITC and were cultured for 7 days. At the end of serial passage culture, the MIC test was conducted using the conidia from the fifth passage culture. From the conidia from the fifth VRC passage culture of A. flavus, seven single clones were isolated, and single clone 1 was named ATCC 204304^Vr (see Table S1 in the supplemental material).

Preparation of DNA and RNA.

For DNA extraction, the A. fumigatus or A. flavus mycelia were cultured for 20 h in 250-ml flasks containing 50 ml of potato dextrose broth (PDB) with 0.5% yeast extract. The mycelia were frozen in liquid nitrogen and disrupted by a TissueRuptor homogenizer (Qiagen). Genomic DNA was extracted using a DNeasy plant minikit (Qiagen) according to the manufacturer's instructions. For RNA extraction, the A. flavus mycelia were cultured in 250-ml flasks containing 50 ml of PDB with 0.2% glucose buffered with 0.165 M MOPS (pH 7.0) for 20 h from 1 × 10⁵ CFU conidia. The mycelia were frozen in liquid nitrogen and disrupted by a TissueRuptor homogenizer (Qiagen). Total RNA was extracted and genomic DNA was removed using an RNeasy plant minikit (Qiagen) according to the manufacturer's instructions.

DNA sequencing of PCR products.

Primers used are listed in Table S3. Primer sets P1-P2 and P3-P4 were used to amplify the entire cyp51A gene of A. fumigatus and A. flavus, respectively. The PCR products were used as the template to sequence the entire A. fumigatus and A. flavus cyp51A gene. Primer set P5-P6 was used to amplify a partial A. flavus yap1 gene, and the PCR product was used as the template to confirm the T1673G mutation.

Whole-genome sequencing analysis.

Unique index-tagged libraries from the genomic DNA of ATCC 204304 and ATCC 204304^Vr were generated using a Nextera XT DNA sample prep kit (Illumina) and AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions. Whole-genome sequencing was performed using the MiSeq system (Illumina) with 300-base paired-end reads. The sequences determined in this study have been deposited in the GenBank/ENA/DDBJ Sequence Read Archive database (accession no. DRA007143). The raw FASTQ reads were quality checked, trimmed, and aligned against the two reference genomes, NRRL3357 (AAIH02) and AF70 (JZDT01), using CLC Genomics workbench 9.5.2 (Qiagen). Aligned data sets were processed to single-nucleotide variant (SNV) calling, using the Fixed Ploidy Variant Detection tool of CLC Genomics Workbench 9.5.2. To identify VRC-resistant strain-specific SNVs, resulting candidate SNVs were compared to the parent strain ATCC 204304 and the VRC-resistant mutant ATCC 204304^Vr when aligned to each reference genome using the Pipeline pilot workflow tool (Biovia). Finally, the common SNVs on the gene-coding region when aligned with both NRRL3357 and AF70 were selected for VRC resistance-related SNV candidates and functional validation.

Construction of the gene mutation and deletion strains.

Primers used for gene mutation and deletion strains are listed in Table S4. Gene replacement fragments or cassettes were generated to construct the gene mutation and deletion strains. To generate a replacement fragment for yap1, a WT or mutated yap1 fragment containing approximately 1,000 bp of 5′- and 3′-nontranslated regions (NTR) was amplified with primer set P1-P2 from genomic DNA of ATCC 204304 or ATCC 204304^Vr, respectively. Transformation was performed by generating protoplasts, as described previously, with some modifications (65). Resulting transformants with the mutated yap1 fragment were selected after at least 48 h of growth at 35°C on PDA medium containing 2.5 mg/ml uracil, 1.2 M sorbitol, and 2 μg/ml VRC. These selected transformants were regrown on PDA containing uracil and VRC to remove untransformed conidia derived from heterokaryon protoplasts after transformation, as described previously (66). Targeted replacement of the yap1 mutation in its locus was confirmed by the sequencing of the yap1 T1673G mutation in the transformants. Gene replacement cassettes having the pyrG selectable marker for gene mutation or deletion were constructed by overlap extension PCR. For WT or mutated yap1 replacement cassettes, primer set P3-P4 was used for the upstream flanking regions (approximately −700 to +300 bp from the stop codon), and primer set P5-P6 was used for the downstream flanking regions (approximately +300 to +1,000 bp from the stop codon). The pyrG fragment containing 44 bp of the 3′ end of yap1 upstream flanking region and 5′ end of yap1 downstream flanking region was amplified with primer set P7-P8 from genomic DNA of ATCC 204304 and used for the selectable marker in replacement cassettes. Primer set P9-P10 was used in the second PCR to overlap the first three PCR fragments. For the atrF deletion cassette, the pyrG fragment was amplified with primer set P11-P12 from genomic DNA of ATCC 204304 and used as the selectable marker in the replacement cassette. Primer set P13-P14 was used for the upstream flanking region (approximately 700 bp of the 5′-NTR) containing 44 bp of the 5′ end of the pyrG fragment. Primer set P15-P16 was used for the downstream flanking regions (approximately 800 bp of 3′-NTR) containing 44 bp of 3′ end of the pyrG fragment. Primer set P17-P18 was used in the second PCR to overlap the three first PCR fragments. After transformation using these PCR fragments, resulting transformants were selected after at least 48 h of growth at 35°C on uracil-deficient yeast nitrogen base (YNB) agar medium with 2% glucose. These selected transformants were regrown on uracil-deficient YNB agar medium to remove untransformed conidia derived from heterokaryon protoplasts after transformation. Targeted yap1 gene replacement was confirmed through PCR by detecting the presence of the pyrG marker in the 3′-NTR of the yap1 locus. Targeted atrF gene deletion was confirmed by PCR by detecting the presence of the pyrG marker in the open reading frame of the atrF locus.

RT-qPCR.

Total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). RT-qPCR was conducted with the 7500 Fast System and Fast SYBR green master mix (Thermo Fisher Scientific). The primer sets are listed in Table S6. The relative expression levels were calculated by the comparative cycle threshold (ΔΔC_T) method. The beta-tubulin gene was used as the internal standard.