The Putative C2H2 Transcription Factor VadH Governs Development, Osmotic Stress Response, and Sterigmatocystin Production in Aspergillus nidulans

The VosA-VelB hetero-dimeric complex plays a pivotal role in regulating development and secondary metabolism in Aspergillus nidulans. In this work, we characterize a new VosA/VelB-activated gene called vadH, which is predicted to encode a 457-amino acid length protein containing four adjacent C2H2 zinc-finger domains. Mutational inactivation of vosA or velB led to reduced mRNA levels of vadH throughout the lifecycle, suggesting that VosA and VelB have a positive regulatory effect on the expression of vadH. The deletion of vadH resulted in decreased asexual development (conidiation) but elevated production of sexual fruiting bodies (cleistothecia), indicating that VadH balances asexual and sexual development in A. nidulans. Moreover, the vadH deletion mutant exhibited elevated susceptibility to hyperosmotic stress compared to wild type and showed elevated production of the mycotoxin sterigmatocystin (ST). Genome-wide expression analyses employing RNA-Seq have revealed that VadH is likely involved in regulating more genes and biological pathways in the developmental stages than those in the vegetative growth stage. The brlA, abaA, and wetA genes of the central regulatory pathway for conidiation are downregulated significantly in the vadH null mutant during asexual development. VadH also participates in regulating the genes, mat2, ppgA and lsdA, etc., related to sexual development, and some of the genes in the ST biosynthetic gene cluster. In summary, VadH is a putative transcription factor with four C2H2 finger domains and is involved in regulating asexual/sexual development, osmotic stress response, and ST production in A. nidulans.


Introduction
Aspergillus spp. are widely distributed in nature and closely related to human life. public health, the fermentation and food processing industry including A. oryzae and A. niger, as well as some plant and human pathogens such as A. flavus and A. fumigatus [1]. The production of conidia is the primary mode of reproduction in the genus Aspergillus. Conidial formation is an elaborate process, which involves cell differentiation, gene expression, and signal transduction [2]. Conidia are an important carrier for the transmission and infection of Aspergillus, and its developmental process is also related to mycotoxin biosynthesis [3]. Therefore, elucidating the regulatory network of conidial development and exploring the functions of critical regulatory proteins are crucial for understanding the law of growth and development of Aspergillus, rational utilization of beneficial industrial strains and effective control of pathogens.
Aspergillus nidulans is an important model organism for studying fungal development, secondary metabolism, and its regulatory pathway. The mechanism of conidiation has been well studied in A. nidulans [4,5]. In A. nidulans, BrlA, AbaA, and WetA constitute the central regulatory pathway of conidial formation, which can sequentially activate conidial development and control the expression of specific genes involved in asexual development [2]. The activation of BrlA is a fundamental step to initiate conidiation [4,6]. Then, BrlA can activate AbaA, which controls the differentiation and function of phialide [7]. In the late stage, WetA is activated by AbaA, which regulates the expression of several spore-specific genes and conidial wall synthesis [8]. In addition, it is reported that velvet family proteins also play an essential role in the asexual development of A. nidulans [9,10].
The VosA-VelB protein complex has a DNA-binding domain similar to that of mammalian NF-κB transcription factor, which can recognize the cis-acting motif specifically in the promoter region of its target genes [15]. Chromatin immunoprecipitation (ChIP) showed that VosA-VelB could bind to promoter sequences of more than 150 genes, including genes activated by VosA-VelB, such as tpsA and treA genes related to trehalose synthesis [15]. Another VosA/VelB-activated gene, vadA, is a novel bifunctional regulatory factor in A. nidulans, which is involved in regulating conidial germination, trehalose synthesis, β-glucan synthesis, oxidative stress, and sterigmatocystin synthesis [16]. In addition, VosA-VelB also has negative regulatory effects on many genes, including brlA and wetA in the central regulatory pathway of conidia, and it can also inhibit β-glucan synthesis in conidia and ascospores by directly binding to the promoter region of fksA gene encoding β-1, 3-glucan synthase [17].
While several target genes of VosA-VelB have been functionally characterized, many remain to be investigated. In this study, we have identified another VosA-VelB target gene vadH (VosA/VelB-Activated Developmental gene H; AN6503 AspGD). The promoter region of vadH is bound by both VosA and VelB in conidia. The vadH gene is predicted to encode a highly conserved transcription factor (VadH) harboring four C 2 H 2 zinc finger domains. The homologous proteins of VadH are universal in the fungal kingdom, and some have been well characterized. Saccharomyces cerevisiae Azf1, a homolog of VadH, participates in activating the genes related to carbon and energy metabolism when glucose exists, and switches to maintaining cell wall integrity when glucose is depleted [18]. In Magnaporthe oryzae, Cos1 is involved in regulating the development of conidiophores and melanin biosynthesis [19]. The VadH homolog CgAzf1 coordinates melanin production, conidium development, appressorium formation and virulence in Colletotrichum gloeosporioides [20]. In this work, the biological functions of vadH have been characterized by gene knockout, overexpression, and transcriptome analyses.

Strains and Culture Conditions
All fungal strains used in this study are listed in Table S1 [13,21,22], and media are prepared as previously described [9]. Briefly, minimal media with glucose (MMG) and MMG with 0.5% yeast extract (MMYE) were used for general purposes, and sexual medium (SM) was used for enhancing sexual development. To determine the number of conidia and cleistothecia, the wild type (WT: A. nidulans FGSC4), mutants, and complemented strains were inoculated and cultured on solid MMG, MMYE or SM for seven days at 37 • C. Micrographs were taken by a Zeiss M2Bio microscope. For the overexpression of vadH under the niiA promoter, strains were inoculated on a non-inducing medium (MMG containing 0.2% ammonium tartrate as a nitrogen source) or an inducing medium (MMG containing 0.6% sodium nitrate as a nitrogen source) and incubated at 37 • C for three days.

Nucleic Acid Isolation and Manipulation
Total RNA isolation was performed as previously described [23]. For asexual and sexual development, cultures from mutants and WT were collected and transferred on solid MMG and SM, respectively. Then, the plates were exposed to air for asexual developmental induction or tightly sealed and blocked from light for sexual developmental induction. For asexual development, samples were collected at 18 and 36 h for RNA isolation. Samples were harvested at 36 and 72 h after transfer for sexual development. For vegetative growth, one milliliter of conidial suspension (10 6 conidia/mL) was inoculated in 100 mL liquid MMG and incubated at 37 • C. The mycelium was collected at 18 and 36 h postinoculation (hpi) for RNA isolation. Quantitative reverse transcription-PCR was used to analyze the expression levels of vadH. The primers were listed in Table S2. The quantitative reverse transcription PCR (qRT-PCR) was carried out by the Fast SYBR Green Master Mix. Gene expression levels were normalized using the endogenous control gene actin. The average normalized expression level was calculated using the 2 −∆∆Ct method [24]. All the experiments were repeated three times.

Target Deletion of VadH
Genomic DNA extraction was performed as previously described [25,26]. The primers used in this section are listed in Table S2. The vadH-deletion mutant strain (∆vadH) was generated by double-joint PCR (DJ-PCR) as previously described [23]. The up-and downstream sequences of the vadH gene were amplified from A. nidulans FGSC4 genomic DNA the by PCR using the primer pairs OXL-1/OXL-2 and OXL-3/OXL-4. The A. fumigatus pyrG marker was amplified using the primer pair OHS-694/OHS-695 from A. fumigatus AF293 genomic DNA. The vadH deletion cassette was amplified with primer pair OXL-5/OXL-6 and introduced into A. nidulans RJMP1.59 [21]. Protoplasts were generated from A. nidulans RJMP1.59 by the Vinoflow FCE lysing enzyme (Novozymes) [25]. For the complementation of ∆vadH, the vadH gene sequence, including its predicted promoter, was amplified with the primer pair OXL-15/OXL-16 and attached to a pHS13 vector [13]. To generate the overexpressing strain, the vadH ORF derived from the genomic DNA was amplified using the primer pair OXL-31/OXL-32. The PCR product was then attached into pHS11 and introduced into A. nidulans RJMP1.59. The vadH-overexpressing strains were screened by qRT-PCR.

Spore Viability Determination
To test spore viability, conidia obtained from two-day-old cultured WT, mutant and complementary strains were spread on solid MMG and cultured at 37 • C. Then, conidia were collected after culturing for seven days. About 100 conidia were coated onto solid MMG and incubated at 37 • C for 48 h in triplicate. Survival rates were determined as the ratio of the number of viable colonies to the number of conidia inoculated. rate (%) = (D ck − D t )/D ck × 100%. Where D ck is the colony diameter of the strain in the control, and D t is the colony diameter of the strain in the treatment group.

Sterigmatocystin (ST) Determination
ST extraction and determination were performed as previously described [16]. Briefly, 10 6 conidia from the strains were inoculated in 2 mL liquid MMG and cultured at 37 • C for seven days. ST was extracted by adding 2 mL of CHCl 3 . The organic phase (CHCl 3 ) was separated through centrifugation and transferred to a new glass bottle. The extracting solution was evaporated in a fume hood and dissolved in 1 mL acetonitrile:methanol (50:50, v/v). After filtering through a millipore filter (0.45 µm), the samples were analyzed by high-performance liquid chromatography with diode-array detection (HPLC-DAD, Agilent Technologies, Waldbronn, Germany).

RNA Sequencing (RNA-Seq)
The isolation of RNA samples was performed as previously described [23]. RNA-Seq analyses of VadH include three aspects: vegetative growth, asexual and sexual development. The preparation of samples in three stages was conducted as previously described [9]. Samples for vegetative growth were collected at 18 hpi, and samples for asexual and sexual development were obtained at 18 and 36 hpi, respectively. A MGISEQ-2000 platform (BGI, Shenzhen, China) was used to analyze the samples. The genome of A. nidulans FGSC A4 from AspGD (http://www.aspergillusgenome.org/, accessed on 1 Septemper 2020) was used as a reference. Data processing and analyses were performed as described previously [27]. The results of RNA-Seq were verified by qRT-PCR according to the published method [27].

Statistical Analysis
Statistical differences between WT and mutant strains were evaluated by Student's unpaired t-test. Data are reported as mean ± SD, and statistical significance was defined as p < 0.05.

Characterization of VadH
The gene vadH (AN6503) is predicted to encode a 457-amino acids (aa) protein, which contains four adjacent C 2 H 2 zinc-finger domains from the position 239 to 352 ( Figure 1A). The homologous proteins of VadH are ubiquitous in fungi, and all of them harbor four C 2 H 2 -type domains ( Figure 1A,B). VadH is homologous with CgAzf1 in Colletotrichum gloeosporioides [20], Cos1 in Magnaporthe oryzae [19], and Azf1 in Saccharomyces cerevisiae [18].
To evaluate the effects of VosA and VelB on the expression of vadH, mRNA levels of vadH were determined in the ∆vosA and ∆velB strains. As shown in Figure 2A, the expression levels of vadH in ∆vosA and ∆velB are significantly lower than those of WT in different stages, suggesting that VosA and VelB can positively regulate the expression of vadH. In the wild type, the expression levels of vadH during asexual and sexual development are apparently higher than those in the stage of vegetative growth ( Figure 2B).

VadH Balances Asexual and Sexual Development
To study the function of vadH, we generated the vadH-deletion mutant (∆vadH) and the complemented strain (C'vadH), and the results related to the verification of the mutant were shown in Figure S1. For colony growth, no significant difference was found between ∆vadH and the wild type on MMG and MMYE, whereas the colony color of ∆vadH was much lighter than that of strain WT ( Figure 3A,B). ∆vadH produced fewer conidia than WT on both MMG and MMYE ( Figure 3C), and the spore viability of ∆vadH was also slightly lower than that of the WT ( Figure 3D). Compared with WT, the mutant ∆vadH exhibited significantly decreased conidial germination ( Figure S2). For sexual development, WT, ∆vadH and C'vadH were inoculated on SM, and the number of cleistothecia was determined ( Figure 3E). As shown in Figure 3F, the mutant ∆vadH produced significantly more cleistothecia than WT and ∆vadH on MMG, and MMYE, suggesting that VadH is involved in regulating sexual development in A. nidulans. However, there was no significant difference in cleistothecia yields on SM between WT and ∆vadH.  To evaluate the effects of VosA and VelB on the expression of vadH, mRNA levels of vadH were determined in the ΔvosA and ΔvelB strains. As shown in Figure 2A, the expression levels of vadH in ΔvosA and ΔvelB are significantly lower than those of WT in different stages, suggesting that VosA and VelB can positively regulate the expression of vadH. In the wild type, the expression levels of vadH during asexual and sexual development are apparently higher than those in the stage of vegetative growth ( Figure 2B).

VadH Balances Asexual and Sexual Development
To study the function of vadH, we generated the vadH-deletion mutant (ΔvadH) and the complemented strain (C'vadH), and the results related to the verification of the mutant were shown in Figure S1. For colony growth, no significant difference was found between ΔvadH and the wild type on MMG and MMYE, whereas the colony color of ΔvadH was much lighter than that of strain WT ( Figure 3A,B). ΔvadH produced fewer conidia than WT on both MMG and MMYE ( Figure 3C), and the spore viability of ΔvadH was also slightly lower than that of the WT ( Figure 3D). Compared with WT, the mutant ΔvadH exhibited significantly decreased conidial germination ( Figure S2). For sexual development, WT, ΔvadH and C'vadH were inoculated on SM, and the number of cleistothecia was determined ( Figure 3E). As shown in Figure 3F, the mutant ΔvadH produced significantly more cleistothecia than WT and ΔvadH on MMG, and MMYE, suggesting that VadH is involved in regulating sexual development in A. nidulans. However, there was no significant difference in cleistothecia yields on SM between WT and ΔvadH.

Disruption of VadH Led to Elevated Sensitivity to Osmotic Stress
For osmotic stress assays, the strains were inoculated on the solid MMG including sorbitol (1.0 M), glycerol (1.0 M) and NaCl (1.0 M), and cultured at 37 °C for 7 days. As shown in Figure 4A,B, ΔvadH was more sensitive to hyper-osmotic stress than WT, and it

Disruption of VadH Led to Elevated Sensitivity to Osmotic Stress
For osmotic stress assays, the strains were inoculated on the solid MMG including sorbitol (1.0 M), glycerol (1.0 M) and NaCl (1.0 M), and cultured at 37 • C for 7 days. As shown in Figure 4A,B, ∆vadH was more sensitive to hyper-osmotic stress than WT, and it had an obvious difference in colony color on the stress plates.

Deleting VadH Leads to an Increase in ST Production
To test the effect of vadH on ST production, ST was extracted from WT, ΔvadH and C'vadH and analyzed using HPLC. As shown in Figure 5, the ΔvadH mutant produced more ST than WT and C'vadH, suggesting that VadH may negatively regulate ST production in A. nidulans.

Deleting VadH Leads to an Increase in ST Production
To test the effect of vadH on ST production, ST was extracted from WT, ∆vadH and C'vadH and analyzed using HPLC. As shown in Figure 5, the ∆vadH mutant produced more ST than WT and C'vadH, suggesting that VadH may negatively regulate ST production in A. nidulans.

Overexpression of VadH Suppresses Sexual Development
As mentioned above, the deletion of vadH leads to enhanced sexual development on MMG and MMYE. To further validate the function of VadH in fungal development, we generated the vadH-overexpression strain (OEvadH) and analyzed its phenotypes ( Figure  6A). On the non-inducing medium, there was no significant differences in the number of conidia and cleistothecia between OEvadH and WT ( Figure 6B,C). However, when induced, overexpression of vadH led to significantly reduced production of cleistothecia ( Figure 6C). The expression level of vadH in OEvadH on the inducing medium was verified by qRT-PCR ( Figure S3). Collectively, these results suggest that VadH is essential for sexual development, and it may act as a suppressor of sexual development.

Overexpression of VadH Suppresses Sexual Development
As mentioned above, the deletion of vadH leads to enhanced sexual development on MMG and MMYE. To further validate the function of VadH in fungal development, we generated the vadH-overexpression strain (OEvadH) and analyzed its phenotypes ( Figure 6A). On the non-inducing medium, there was no significant differences in the number of conidia and cleistothecia between OEvadH and WT ( Figure 6B,C). However, when induced, overexpression of vadH led to significantly reduced production of cleistothecia ( Figure 6C). The expression level of vadH in OEvadH on the inducing medium was verified by qRT-PCR ( Figure S3). Collectively, these results suggest that VadH is essential for sexual development, and it may act as a suppressor of sexual development.

Transcriptomic Analyses of VadH
Genome-wide expression analyses of WT and ΔvadH under three stages w formed by employing RNA-Seq. The related data have been submitted to G (PRJNA905844). For the vegetative growth stage, 895 DEGs were obtained, in wh DEGs were upregulated and 567 DEGs were downregulated. Nevertheless, for and sexual development stages, more than 1200 DEGs were identified, suggesti VadH is involved in regulating more genes in the developmental stages than t vegetative growth ( Figure 7A). The Venn diagram indicates that 126 DEGs, inclu up-regulated and 63 down-regulated DEGs, participate in the vegetative growth, and sexual development stage simultaneously ( Figure 7B). The RNA-Seq result three stages were verified by qRT-PCR, and the expression levels of selected DEG three stages showed the same trend as those in RNA-Seq with all the correlation cients being more than 95% ( Figure S4).

Transcriptomic Analyses of VadH
Genome-wide expression analyses of WT and ∆vadH under three stages were performed by employing RNA-Seq. The related data have been submitted to GenBank (PRJNA905844). For the vegetative growth stage, 895 DEGs were obtained, in which 328 DEGs were upregulated and 567 DEGs were downregulated. Nevertheless, for asexual and sexual development stages, more than 1200 DEGs were identified, suggesting that VadH is involved in regulating more genes in the developmental stages than those in vegetative growth ( Figure 7A). The Venn diagram indicates that 126 DEGs, including 63 up-regulated and 63 down-regulated DEGs, participate in the vegetative growth, asexual and sexual development stage simultaneously ( Figure 7B). The RNA-Seq results in the three stages were verified by qRT-PCR, and the expression levels of selected DEGs from three stages showed the same trend as those in RNA-Seq with all the correlation coefficients being more than 95% ( Figure S4). Based on the RNA-Seq results, we further analyzed the expression of DEGs related to sporulation and ST biosynthesis. As shown in Figure 7C, brlA, abaA and wetA in the central regulatory pathway are downregulated significantly in the asexual development stage of ΔvadH, especially abaA. In the process of sexual development, the genes mat2 and ppgA related to sexual sporulation are upregulated, and lsdA, vadJ and vadZ are downregulated in ΔvadH; some of the genes (stuA, stuL, stuO, stuQ, stuS, stuU) in the gene cluster of ST biosynthesis are also upregulated to varying degrees.
Then, we performed the KEGG pathway analysis for DEGs in three stages (Figure 8). In the vegetative growth stage, six significant enrichment pathways were obtained, which were mainly related to amino acid metabolism and aflatoxin biosynthesis ( Figure  8A). Regarding asexual and sexual development, more biological pathways were affected by VadH, including some pathways related to the metabolism of fatty acid, nitrogen and carbohydrate ( Figure 8B,C). The numbers of significant enrichment pathways in the asexual and sexual development are more than twice as much as those in the vegetative growth stage. Based on the RNA-Seq results, we further analyzed the expression of DEGs related to sporulation and ST biosynthesis. As shown in Figure 7C, brlA, abaA and wetA in the central regulatory pathway are downregulated significantly in the asexual development stage of ∆vadH, especially abaA. In the process of sexual development, the genes mat2 and ppgA related to sexual sporulation are upregulated, and lsdA, vadJ and vadZ are downregulated in ∆vadH; some of the genes (stuA, stuL, stuO, stuQ, stuS, stuU) in the gene cluster of ST biosynthesis are also upregulated to varying degrees.
Then, we performed the KEGG pathway analysis for DEGs in three stages (Figure 8). In the vegetative growth stage, six significant enrichment pathways were obtained, which were mainly related to amino acid metabolism and aflatoxin biosynthesis ( Figure 8A). Regarding asexual and sexual development, more biological pathways were affected by VadH, including some pathways related to the metabolism of fatty acid, nitrogen and carbohydrate ( Figure 8B,C). The numbers of significant enrichment pathways in the asexual and sexual development are more than twice as much as those in the vegetative growth stage. Cells 2022, 11, x FOR PEER REVIEW 12 of 16

Discussion
The velvet proteins are fungal-specific transcript factors that coordinate both development and secondary metabolism [11,15]. Previous studies indicated that the target genes regulated by VosA/VelB complex could be divided into VosA/VelB-activated de-

Discussion
The velvet proteins are fungal-specific transcript factors that coordinate both development and secondary metabolism [11,15]. Previous studies indicated that the target genes regulated by VosA/VelB complex could be divided into VosA/VelB-activated developmen-tal genes (VADs) and VosA/VelB-inhibited developmental genes (VIDs), and many VADs and VIDs are regulatory factors involved in the development process of A. nidulans [15,17]. Here, we characterized a new VAD gene vadH in A. nidulans, which encodes a zinc finger protein with four adjacent C 2 H 2 -type domains. The expression levels of vadH were noticeably decreased in the mutants ∆vosA or ∆velB, suggesting that both VosA and VelB have a positive regulatory effect on vadH. It is observed that the homologous proteins of VadH can play various roles in different fungi [18][19][20]. In A. nidulans, VadH is involved in asexual and sexual development, osmotic stress response and ST production.
Our study indicated that VadH could balance the asexual and sexual development of A. nidulans. VadH could inhibit conidial production and stimulate sexual development on MMY and MMYE, and overexpression of vadH obviously suppresses the formation of cleistothecia. VadH can negatively regulate the expression of two genes related to sexual development, mat2 and ppgA, which may affect cleistothecium formation to a certain extent. It has been reported that Mat2 and PpgA can act as activators in the sexual development of A. nidulans [28]. In addition, VadH can also positively regulate the expression of lsdA, vadJ and vadZ, which have proved to function as repressors in sexual sporulation [29][30][31]. In A. nidulans, the central regulatory pathway activates conidial development and regulates the expression of specific genes in conidiation. In the mutant ∆vadH, the genes brlA, abaA, and wetA are all downregulated in the asexual development stage, which will inhibit the conidial production of ∆vadH. Especially abaA related to the differentiation of phialide is downregulated dramatically, which may affect the formation of conidia to a great extent. Therefore, VadH may function as a positive regulator in asexual sporulation and a negative one in sexual development. It is reported that VadA (AN5709), a member of VADs, also participates in the balance between asexual and sexual development [16]. The ∆vadA mutant exhibits increased production of cleistothecia, and overexpression of vadA leads to increased conidial production. Similar to vadH, the other two VADs, VadJ (AN3214, a histidine kinase) and VadZ (AN8774, a C 6 transcription factor) also act as activators of asexual development and repressors of sexual development [30,31]. Interestingly, one member of VIDs, VidA (AN2498) proved to be essential for proper asexual and sexual development in A. nidulans. Deletion of vidA can lower the production of conidia, and slightly increases the yield of cleistothecia [32]. These reported genes regulated directly by the VosA-VelB complex all have a function of balancing asexual and sexual development, suggesting that the complex does play a crucial role in the development process of A. nidulans.
VadH participates in the osmotic pressure response of A. nidulans. Deletion of vadH leads to elevated sensitivity to hyperosmotic stress, and the asexual sporulation of ∆vadH is also suppressed significantly on the hyperosmotic media. The KEGG analysis shows that the expression of more than 60 genes in the mitogen-activated protein kinase (MAPK) signaling pathway is influenced by VadH, and many of them are involved in cell wall integrity and high osmolarity pathways. We speculate that deleting vadH may change cell wall integrity, subsequently affecting the osmotic pressure response of A. nidulans. In S. cerevisiae, Azf1 also have the function of maintaining cell wall integrity when glucose is depleted [18]. However, the VadH orthologues, Cos1 and CgAzf1 are not related to osmotic pressure response in M. oryzae and C. gloeosporioides, respectively [19,20]. Further study will be needed to elucidate the precise mechanism of VadH coordinating the hyperosmotic stress. Regarding secondary metabolism, VadH proves to be involved in regulating ST production. Disruption of vadH leads to elevated ST production, and the RNA-Seq results also indicate that VadH can negatively regulate the expression of several genes related to the biosynthesis of ST. It has been found that Cos1 and CgAzf1 are also relevant to secondary metabolism, and both of them can regulate melanin production [19,20]. Similar to vadH, the VADs, vadA, vadJ and vadZ, also exhibit the function of suppressing ST production [16,30,31], whereas the VID gene vidA is not involved in the biosynthesis of ST [32].

Conclusions
Taken together, we propose a working model for VadH regulating asexual/sexual development and secondary metabolism (Figure 9). The VosA-VelB complex can positively regulate the expression of vadH. Then, VadH functions as a positive regulator for asexual sporulation through the central regulatory pathway, and it also acts as a negative regulator for the production of cleistothecia and sterigmatocystin. In summary, a newly identified VAD gene vadH is predicted to encode a C 2 H 2 -type transcription factor, which is involved in balancing asexual/sexual development and regulating osmotic stress and sterigmatocystin production. These findings provide further evidence for the crucial roles of VADs in the development and secondary metabolism of A. nidulans. Understanding the mechanism of VadH will contribute to revealing the precise regulatory networks of the VosA-VelB complex.

Conclusions
Taken together, we propose a working model for VadH regulating asexual/sexual development and secondary metabolism (Figure 9). The VosA-VelB complex can positively regulate the expression of vadH. Then, VadH functions as a positive regulator for asexual sporulation through the central regulatory pathway, and it also acts as a negative regulator for the production of cleistothecia and sterigmatocystin. In summary, a newly identified VAD gene vadH is predicted to encode a C2H2-type transcription factor, which is involved in balancing asexual/sexual development and regulating osmotic stress and sterigmatocystin production. These findings provide further evidence for the crucial roles of VADs in the development and secondary metabolism of A. nidulans. Understanding the mechanism of VadH will contribute to revealing the precise regulatory networks of the VosA-VelB complex. Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1. Figure S1: Verification of the gene-knockout mutant and complementary strain; Figure S2: Conidial germination of WT, ΔvadH and the complemented strain; Figure S3: Verification of the mRNA level of vadH in OEvadH; Figure S4: The qRT-PCR verification of RNA-Seq data in three stages; Table S1: Aspergillus strains used in this study; Table S2: Primers used in this study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors have no conflicts of interest to declare. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells11243998/s1. Figure S1: Verification of the geneknockout mutant and complementary strain; Figure S2: Conidial germination of WT, ∆vadH and the complemented strain; Figure S3: Verification of the mRNA level of vadH in OEvadH; Figure S4: The qRT-PCR verification of RNA-Seq data in three stages; Table S1: Aspergillus strains used in this study; Table S2: Primers used in this study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.