Characterizing the role of Zn cluster family transcription factor ZcfA in governing development in two Aspergillus species

Filamentous fungi reproduce asexually or sexually, and the processes of asexual and sexual development are tightly regulated by a variety of transcription factors. In this study, we characterized a Zn2Cys6 transcription factor in two Aspergillus species, A. nidulans (AN5859) and A. flavus (AFLA_046870). AN5859 encodes a Zn2Cys6 transcription factor, called ZcfA. In A. nidulans, ΔzcfA mutants exhibit decreased fungal growth, a reduction in cleistothecia production, and increased asexual reproduction. Overexpression of zcfA results in increased conidial production, suggesting that ZcfA is required for proper asexual and sexual development in A. nidulans. In conidia, deletion of zcfA causes decreased trehalose levels and decreased spore viability but increased thermal sensitivity. In A. flavus, the deletion of the zcfA homolog AFLA_046870 causes increased conidial production but decreased sclerotia production; these effects are similar to those of zcfA deletion in A. nidulans development. Overall, these results demonstrate that ZcfA is essential for maintaining a balance between asexual and sexual development and that some roles of ZcfA are conserved in Aspergillus spp.


Introduction
The genus Aspergillus consists of approximately 350 accepted species that are closely related [1][2][3]. Several species are useful for fermented food, enzyme production, and pharmaceutical purposes [4]. However, some Aspergillus species have detrimental effects on humans or plants [5]. Several fungi can also produce harmful secondary metabolites called mycotoxins [6,7]. Aspergillus flavus is one of the key Aspergillus species and an agent of invasive aspergillosis in immunocompromised patients [8,9]. A. flavus produces harmful secondary metabolites, called aflatoxins, that are potent carcinogens [10,11]. The contamination of crops such as maize and peanuts with A. flavus causes significant economic loss [12]. Therefore, the control of A. flavus growth and aflatoxin production is crucial to the agricultural industry. To use Aspergillus species for the benefit of humanity, we must understand its biology. A. nidulans is a model organism for studies in fungal developmental biology and gene regulation; therefore, it is one of the best characterized Aspergillus species [13,14]. The reproductive modes of A. nidulans can be divided into two types, sexual and asexual [15]. After the germ tube is formed from spores, it grows into hyphae, forming the mycelium, a web-like mass of fungal hyphae [16,17]. After acquiring developmental competence, fungi can produce asexual or sexual developmental structures depending on environmental conditions [18,19]. During the formation of conidiophores (asexual structures) or sexual fruiting bodies, a variety of genes and proteins participate in these processes [20,21].
Transcription factors (TFs) are DNA-binding proteins that regulate transcription [22]. These proteins recognize specific DNA sequences in the vicinity of genes and induce or repress mRNA transcription. In the fungal genome database, 80 transcription factor families are found, and various TFs coordinate gene expression during growth and developmental processes [15,23]. In asexual developmental processes, BrlA is a key TF for the initiation of conidiation [24,25]. BrlA contains a C 2 H 2 zinc finger DNA-binding domain that recognizes brlA response elements (BREs) in the promoter regions of certain genes, including abaA [26]. AbaA, a TEF1 family TF, activates mRNA expression of wetA and other genes in the middle phase of conidiogenesis [27,28]. In asexual spores, a spore-specific TF WetA coordinates the mRNA expression of genes associated with spore maturation [29]. TFs BrlA, AbaA, and WetA play key roles in asexual development and are considered the core regulators of conidiation [30]. Together with these three TFs, other TFs play a vital role in the initiation of conidiation. For instance, upstream developmental activators, such as FlbB, FlbC, FlbD, and FlbE, induce brlA expression [31], whereas three key TFs, NsdD, SfgA, and VosA, repress conidiation [32]. In sexual developmental processes, NsdC and NsdD are key TFs that positively regulate sexual developmental processes [33][34][35]. With these genes, a variety of TFs are reported to be involved in sexual reproduction [21].
The Zn cluster family (Zcf) is a fungal-specific family of TFs and is the largest family of TFs known in eukaryotes [23]. Zcf TFs contain several DNA binding motifs, such as the C 2 H 2 zinc finger, the C 4 zinc finger, and the C 6 zinc finger, and are involved in a variety of cellular processes in fungi [36]. For example, CrzA is a C 2 H 2 zinc finger TF that governs calcium homeostasis, fungal growth, and detoxification in A. nidulans [37,38]. AflR contains a Cys 6 Zn 2 binuclear cluster motif that activates the sterigmatocystin biosynthesis gene cluster in A. nidulans [39,40]. Although several Zcf TFs have been characterized, the functions of many other Zcf TFs have not been elucidated. Previous studies have found putative target genes of VosA, a key transcription factor for conidial maturation, in A. nidulans conidia [41]. We screened mRNA levels of putative VosA target genes in conidia and found that one gene (AN5859) affected the mRNA expression of these target genes when vosA or velB were deleted in conidia (S1 Fig). In this study, we characterized the AN5859 gene, which encodes the Zcf protein ZcfA in two Aspergillus species, A. nidulans, and A. flavus.

Strains, media, and culture conditions
The fungal strains used in this study are listed in Table 1. A. nidulans strains were grown on liquid or solid minimal media with 1% glucose (MMG) for general purposes or sexual medium (SM) for sexual development [42,43]. To confirm the effects of overexpression of the A. nidulans zcfA (AnizcfA) mRNA under the alcA promoter [44], tested strains were inoculated on MMG, MM with 100 mM threonine as the sole carbon source (MMT), or YLC (0.1% yeast extract, 1.5% lactose, 30 mM cyclopentanone) at 37˚C for 5 days [45,46]. A. flavus strains were grown on MMG with 0.1% yeast extract (MMGY) for general tests. Escherichia coli DH5α cells were grown in Luria-Bertani medium with ampicillin (100 μg/mL) for plasmid amplification.
For mRNA isolation, samples were collected as previously described [47]. For conidia, WT and mutant conidia were inoculated onto solid MMG and incubated for two days at 37˚C. Conidia were then collected using Miracloth and stored at −80˚C. For mycelia samples, WT and mutant conidia were inoculated into liquid MMG and incubated at 37˚C for the indicated times. Cultured mycelia were collected, squeezed to remove moisture, and stored at -80˚C. For developmental samples, submerged culture mycelia were filtered, washed, and spread in a monolayer on solid MMG, and the plates were incubated under light condition for asexual developmental induction or cultured under dark condition for sexual developmental induction.

Construction of the AnizcfA mutant strains
The oligonucleotide primers used in this study are listed in Table 2. To generate deletion mutant strains, the double-joint PCR (DJ-PCR) method was used [51]. The 5'-and 3'-flanking regions for AnizcfA were amplified with primer pairs OHS220:OHS222 and OHS221:OHS223, respectively, using A. nidulans FGSC4 (wild type, WT) genomic DNA as a template. The A. fumigatus pyrG (AfupyrG) marker was amplified with the primer pair OHS089:OHS090 using A. fumigatus AF293 genomic DNA as a template. The final PCR construct for the AnizcfA deletion cassette was amplified with the OHS224:OHS225 primer pair using the three DNA fragments from the first round of PCR (the 5'-and 3'-flanking regions and the AfupyrG marker) as the template. The AnizcfA deletion cassette was introduced into A. nidulans RJMP1.59 protoplasts generated by the Vinoflow FCE lysing enzyme (Novozymes) [52,53]. Success was confirmed by PCR followed by restriction enzyme digestion (S2 Fig).
To generate the ΔAnizcfA complemented strain, the WT AnizcfA gene region, including its predicted promoter, was amplified with the primer pair OHS395:OHS396, digested with NotI, and cloned into pHS13

Construction of the AnizcfA overexpression strain
To generate the alcA(p)::AnizcfA fusion construct, the AnizcfA open reading frame (ORF) derived from genomic DNA was amplified using the primer pair OHS741:OHS742. The PCR product was then digested with BamHI and cloned into pHS3, which contains the A. nidulans alcA promoter and the trpC terminator [54]. The resulting plasmid pYE2.1 was then introduced into TNJ36 [49]. Strains that overexpress AnizcfA were selected from among the transformants and screened by qRT-PCR after induction of the promoter (S4 Fig).

Construction of the AflzcfA deletion mutant strain
To produce the AflzcfA deletion cassette, the 5' and 3' flanking regions of the AflzcfA gene were amplified using the primer pairs OHS0475:OHS0476 and OHS0477:OHS0478, respectively, using A. flavus NRRL3357 genomic DNA as a template. The AfupyrG gene was used as a selective marker. The AflzcfA deletion cassette was amplified with primer pair OHS479:OHS480 and introduced into the recipient strain NRRL3357.5 [50]. Multiple (at least three) mutants were isolated and confirmed by PCR followed by restriction enzyme digestion (S5 Fig).

Nucleic acid isolation and qRT-PCR analysis
To isolate genomic DNA, approximately 10 6 conidia of WT and mutant strains were inoculated in 2 ml liquid MMG + 0.5% yeast extract medium and allowed to grow in stationary Table 2. Oligonucleotides used in this study.

Name
Sequence (5 0 ! 3 0 ) a Purpose OHS0220 CCAATGAGTAGCAGCAACCTTG 5 0 AnizcfA DF culture at 37˚C for 24 h. The mycelial mat was collected, squeeze-dried, and genomic DNA was isolated as described [53]. For qRT-PCR analyses, total RNA isolation was carried out as previously described [55,56]. Briefly, fresh conidia were collected and homogenized using a Mini-Bead Beater (BioSpec Products, USA) in the presence of 1 ml of TRIzol reagent (Invitrogen, USA) and 0.3 ml of zirconia/silica beads (RPI Corp., USA). The supernatant was mixed with an equal amount of cold isopropanol. After centrifugation, the supernatant was removed, and the pellet washed with 70% DEPC (diethyl pyrocarbonate)-ethanol. cDNA was synthesized from total RNA using reverse transcriptase (Promega, USA). The qRT-PCR procedure was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, USA) and CFX96 Touch Real-Time PCR (Bio-Rad, USA). For an endogenous control, β-actin gene was used.

Conidial viability assay
To test conidial viability, conidia from two-day-old and ten-day-old cultures of WT and mutant strains were collected using ddH 2 O with 0.01% Triton X-100 (Sigma, USA) [47]. After counting the number of conidia with a hematocytometer, approximately 100 conidia were inoculated onto solid MMG and incubated at 37˚C for 48 h in triplicate. After incubation, colony-forming units were counted.

Conidia trehalose analysis
The conidia trehalose assay was performed as previously described [57]. Briefly, conidia (2 × 10 8 ) from two-day-old cultures of WT and mutant strains were collected, washed with ddH 2 O, resuspended in 200 μl of ddH 2 O, and incubated at 95˚C for 20 min. The supernatant was collected after centrifugation and was transferred to a new tube. An equal volume of 0.2 M sodium citrate (pH 5.5) was added, and the sample was incubated at 37˚C for 8 h with or without (as a negative control) 3 mU of trehalase (Sigma, USA). The amount of glucose generated from the trehalose was assayed with a Glucose Assay Kit (Sigma, USA) in triplicate.

Thermal stress response assay
Thermal tolerance tests were carried out as previously described [57,58]. Briefly, approximately 10 3 conidia per ml were incubated for 15 min at 55˚C. The conidial suspension was then diluted with ddH 2 O, and the conidia were inoculated onto solid MMG. After incubation at 37˚C for 48 h, colony numbers were counted and calculated as a survival ratio relative to counts obtained from the untreated control.

Sterigmatocystin (ST) extraction and thin-layer chromatography (TLC) analysis
Briefly, 10 5 conidia of each strain were inoculated into 5 ml liquid complete medium (CM) and cultured at 30˚C for 7 days. Secondary metabolites were extracted by adding 5 ml of CHCl 3 , and the organic phase was separated by centrifugation and transferred to new glass vials. The organic phase was evaporated in an oven, and the residue was resuspended in 100 μl of CHCl 3 . Secondary metabolites were spotted onto a TLC silica plate that included a fluorescence indicator (Kieselgel 60, 0.25 mm; Merck) and resolved in chloroform:ethyl acetate (9:1, v/v). The images of TLC plates were captured following exposure to ultraviolet illumination at 366 nm. The TLC plate was then treated with 1% aluminum hydroxide hydrate (Sigma, USA). Quantification of ST spot intensity (366 nm on the TLC plates) was calculated using ImageJ software. Experiments were performed in triplicate per strain.

Microscopy
Photographs of colonies were taken with a Pentax MX-1 digital camera. Photomicrographs were taken with a Zeiss Lab.A1 microscope equipped with an AxioCam 105c camera and AxioVision (Rel. 4.9) digital imaging software.

ZcfA homologs in Aspergillus species
Previous studies reported that the Zn cluster family TFs are major TFs in fungi and are involved in various biological processes [23,36]. In this study, we characterized the Zcf TF ZcfA (AN5859) in the model fungus A. nidulans. AN5859 encodes a protein that contains a GAL4-like Zn 2 Cys 6 binuclear cluster DNA-binding domain at the N-terminus and a fungal transcription factor regulatory middle homology region (MHR). To find homologs in other Aspergillus species, we screened the Aspergillus genome database. Interestingly, although most Aspergillus species contain ZcfA homologs, a ZcfA homolog is not seen in Aspergillus strains belonging to the section Fumigati, such as A. fumigatus (Fig 1A). To characterize the zcfA gene in A. nidulans, we first investigated the expression of AnizcfA mRNA during the fungal lifecycle. AnizcfA mRNA levels increase 12 h after induction of asexual development and decrease in conidia (Fig 1B).

The role of AnizcfA in fungal growth and asexual development
To further characterize AnizcfA, an AnizcfA deletion (ΔAnizcfA) mutant and complemented strains were generated, and their phenotypes examined. WT, ΔAnizcfA, and complemented (C' AnizcfA) strains were point-inoculated onto MM solid media, incubated under light and dark conditions, and then colony growth and production of asexual spores were assessed (S6 Fig). As shown in Fig 2, the colony diameter of the ΔAnizcfA strain under both light and dark conditions was less than those of WT and complemented strains. However, the ΔAnizcfA strain produced more conidia per plate under both dark and light conditions. These results indicate that ZcfA is required for proper growth and conidiation in A. nidulans.
To further test the role of ZcfA in developmental processes, the ΔAnizcfA strain was inoculated onto SM and incubated under dark conditions. The WT and complemented strains produced more sexual fruiting bodies under these conditions, but the ΔAnizcfA strain produced asexual spores predominantly and a negligible number of sexual fruiting bodies (Fig 3A). To test whether the deletion of AnizcfA affects the expression of genes involved in asexual and sexual development, submerged culture mycelia of WT, ΔAnizcfA, and C' AnizcfA strains were spread onto solid MMG and cultured under dark conditions to induce sexual development (Fig 3B). After cultivation, the samples were collected, and the mRNA expression of α-1,3-mutanase (mutA), a gene that is specifically expressed in Hülle cells [59], and brlA, a key gene in conidial initiation [24] were measured. As shown in Fig 3C, deletion of AnizcfA leads to reduced (or delayed) mRNA expression of mutA. In contrast, mRNA expression of brlA is induced in the ΔAnizcfA strain. Taken together, these results suggest that ZcfA is critical for fungal growth and maintaining a balance between asexual and sexual development in A. nidulans.

Role of AniZcfA in conidial viability and trehalose content
To investigate the role of AniZcfA in conidia, we first examined the viability of conidia produced by the ΔAnizcfA strain. As shown in Fig 4A, the viability of ΔAnizcfA conidia decreased at ten days compared with that at two days. We then measured the amount of trehalose produced by WT, ΔAnizcfA, and C' strains and found that trehalose levels in ΔAnizcfA conidia Role of zcfA during development of A. nidulans and A. flavus were slightly lower than those in WT and complemented conidia (Fig 4B). A previous study reported that conidial trehalose affects tolerance to thermal stresses in A. nidulans conidia [57]. To determine whether the lower level of trehalose in ΔAnizcfA conidia alters their stress response, thermal tolerance was assayed. The ΔAnizcfA conidia were more sensitive to heat stress than WT and C' conidia ( Fig 4C). Although the phenotype of the ΔAnizcfA conidia is only slightly changed, these results suggest that ZcfA plays a key role in conidial viability and trehalose levels in A. nidulans.

Overexpression of AnizcfA leads to increased conidia production
To further investigate the role of AnizcfA in fungal development, a strain that overexpresses AnizcfA was developed. Control and AnizcfA overexpression mutant (OEzcfA) strains were inoculated under non-inducing and inducing conditions (Fig 5). Under conditions that induce the alcA promoter (MMT medium), overexpression of AnizcfA results in increased production of asexual spores. In YLC media (another alcA-inducing condition), AnizcfA overexpression strains produced fewer sexual fruiting bodies than WT. These results support the idea that AniZcfA is essential for proper developmental processes.

Deletion of AnizcfA alters sterigmatocystin production
Because fungal development is associated with secondary metabolism [60], we hypothesized that zcfA might be involved in secondary metabolism. To test whether zcfA affects ST production in A. nidulans, secondary metabolites were extracted from WT, ΔAnizcfA, and C' strains. Three independent samples were extracted, spotted, and loaded onto TLC plates. The ΔAnizcfA strain produced more ST than WT or complemented strains (Fig 6). However, the overexpression of zcfA did not affect ST production (S7 Fig). This suggests that ZcfA influences the production of ST in A. nidulans.

ZcfA homolog is required for development in A. flavus
As mentioned above, ZcfA is conserved in most Aspergillus species and plays a crucial role in fungal development in the model organism, A. nidulans. Therefore, we hypothesized that ZcfA homologs might play similar roles in other Aspergillus species and tested this hypothesis in A. flavus. We generated an A. flavus zcfA deletion mutant strains (ΔAflzcfA) and examined their developmental phenotypes (S8 Fig). WT and ΔAflzcfA mutant strains were inoculated onto MMGY, and the plates were incubated under light and dark conditions (Fig 7A). The deletion of AflzcfA causes increased production of asexual spores in both light and dark conditions ( Fig  7B). In addition, the ΔAflzcfA mutant cannot produce sclerotia, or sexual structures, in this condition (Fig 7C and 7D). These results suggest that ZcfA is essential for proper fungal Role of zcfA during development of A. nidulans and A. flavus development and that its function is similar among Aspergillus species that contain a homolog of ZcfA.

Discussion
Zcf proteins are a family of fungal-specific TFs that are the largest known family of TFs among eukaryotes [36]. In the A. nidulans genome, approximately 50 proteins that contain C 6 zinc finger motifs have been identified, and these Zcf proteins regulate the expression of genes associated with primary and secondary metabolism. For example, AflR acts as an activator for the ST biosynthesis gene cluster [39]. AmyR is also a Zn 2 Cys 6 transcription activator that regulates amylolytic gene expression [61,62]. In this study, we characterized the Zn 2 Cys 6 transcription factor ZcfA in two Aspergillus species. In both species, ΔzcfA mutant strains exhibited Role of zcfA during development of A. nidulans and A. flavus increased conidial production and decreased formation of sexual fruiting bodies, suggesting that ZcfA may act as an asexual development repressor or a sexual development activator. To further examine the role of ZcfA, phenotypes, zcfA overexpression strains were examined in A. nidulans. Contrary to our expectation, overexpression of zcfA increased asexual spore production but decreased the production of sexual fruiting bodies. This result implies that ZcfA may not be an asexual development repressor or a sexual development activator. Our results suggest that ZcfA appears to be essential for proper fungal development in A. nidulans.
As shown in Fig 1A, zcfA mRNA expression in A. nidulans increases during asexual development, suggesting that zcfA expression might regulate other regulators of asexual development, such as BrlA or AbaA. We examined the zcfA promoter region and found several AbaA response elements (AREs, 5'-CATTCY-3') [28] but no BrlA response elements (BRE) [26]. Further zcfA expression analysis will be conducted to elucidate how zcfA expression is regulated during asexual development. In conidia, mRNA expression of zcfA was decreased (Fig  1). It is possible that transcription factors (e.g., WetA, VosA, and VelB) that are important for spore maturation are involved in the expression of zcfA mRNA. We have measured zcfA transcript levels in ΔwetA, ΔvosA, and ΔvelB mutant conidia. Our preliminary data (S1 Fig) and published data [29] show that zcfA mRNA levels in conidia from these mutants are increased compared to WT. Overall, these results indicate that zcfA mRNA levels may be regulated by certain asexual regulators in A. nidulans.
In conidia, ZcfA affects spore viability, trehalose contents, and thermal tolerance in A. nidulans. It appears that these roles of ZcfA are conserved in the development of two Aspergillus species; therefore, we hypothesize that the roles of ZcfA might be conserved in conidia. We examined the role of ZcfA in A. flavus conidia and found that trehalose contents, conidial viability, and stress response of ΔAflzcfA mutant conidia were similar to those of WT conidia. These results suggest that the roles of ZcfA role in conidia are not conserved among Aspergillus species.
In summary, we characterized the Zn cluster family transcription factor ZcfA in the model organism A. nidulans and A. flavus. In both species, ZcfA affects fungal differentiation. The deletion of zcfA causes a decrease (or lack of) in the formation of sexual fruiting bodies in both Aspergillus species. ZcfA does not act as a repressor or activator of fungal development, yet it is required for proper asexual and sexual development in Aspergillus species. In A. nidulans,