The novel spore-specific regulator SscA controls Aspergillus conidiogenesis

ABSTRACT A major group of fungi produces asexual spores (conidia) for dissemination and propagation and, in pathogenic fungi, for infection. Despite the critical role of conidia, the underlying mechanism of spore formation, integrity, and viability is not fully elucidated. In this study, we have identified and investigated the roles of the spore-specific transcription factor (TF) SscA in three Aspergillus species, including A. nidulans, A. flavus, and A. fumigatus, which is a model system, toxin producer, or a prevalent human pathogen, respectively. Comparative transcriptomic analyses have revealed that 25 TF encoding genes showed higher mRNA levels in conidia than in hyphae in three species. Functional analyses of the 25 genes have identified SscA as a key TF for conidial formation, maturation, germination, integrity, amino acid production, and secondary metabolism in Aspergillus nidulans conidia. Importantly, the roles of SscA are conserved in other Aspergillus species. Altogether, our study demonstrates that SscA is a spore-specific TF that governs the production of intact and functional conidial formation in Aspergillus species. IMPORTANCE Filamentous fungi produce myriads of asexual spores, which are the main reproductive particles that act as infectious or allergenic agents. Although the serial of asexual sporogenesis is coordinated by various genetic regulators, there remain uncharacterized transcription factors in Aspergillus. To understand the underlying mechanism of spore formation, integrity, and viability, we have performed comparative transcriptomic analyses on three Aspergillus species and found a spore-specific transcription factor, SscA. SscA has a major role in conidial formation, maturation and dormancy, and germination in Aspergillus nidulans. Functional studies indicate that SscA coordinates conidial wall integrity, amino acid production, and secondary metabolism in A. nidulans conidia. Furthermore, the roles of SscA are conserved in other Aspergillus species. Our findings that the SscA has broad functions in Aspergillus conidia will help to understand the conidiogenesis of Aspergillus species.

F ungi are ubiquitous microorganisms present throughout the world and live in various nonliving organic materials or living organisms (1,2).Aspergillus species are saprophytic ascomycetous filamentous fungi that are commonly found in a wide range of environmental conditions, including air, animals, and humans (3).Among them, Aspergillus nidulans is a model organism that has been used in basic science research such as genetics and cell biology for more than 70 years (4,5).While A. nidulans has been explored as a versatile fungal cell factory to produce organic acids and enzymes, this fungus can cause invasive aspergillosis as an opportunistic fungal pathogen in immune-deficient people (6,7).Aspergillus flavus is an opportunistic fungal pathogen of agricultural plants producing foods and feeds.A. flavus spreads in the air in the form of asexual spores (conidia) and most can produce aflatoxins, the most potent carcinogen found in nature, which cause adverse effects on human and animal health and cause economic losses worldwide (8,9).Moreover, A. flavus is the second leading cause of aspergillosis infection in immunocompromised patients, often resulting in the death of the patient (10).Aspergillus fumigatus, the most prevalent airborne fungal pathogen, causes cystic fibrosis (CF) allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis in humans (11,12).The conidia of A. fumigatus infect the host, adapt, and proliferate by resisting host defenses.A recent study showed that the mortality rate by invasive aspergillosis was augmented to 50-100% (13).
Asexual development (conidiation) is the primary reproductive strategy of Aspergillus species.The long and branching vegetative structures (hyphae) emerge as specialized foot cells extending to stalks and forming a specialized asexual sporulation structure called conidiophore bearing thousands of conidia.On the tip of the phialide, radiat ing conidia transform from an immature state to a mature state by modulating the cellular and physiological properties of conidia.In conidial maturation, conidial trehalose biosynthesis is commonly accomplished for tolerance to external stresses, and conidial wall compositions are modified for adaptation to desiccation and various damages (14)(15)(16)(17)(18).Then, the maturated conidia stay in a dormant stage for a certain duration, during which the conidial viability is maintained through the delicate control of cellular activities and active blockage of conidial germination.For instance, dormant conidia maintain low-level respiratory metabolism and cease ATP-consuming cellular activities (14,19).They also modify transcription and translation activities, and the production of cellular primary metabolites and secondary metabolites for survival and protection (20)(21)(22)(23)(24).When the dormant conidia encounter the appropriate environment, they germinate by altering the conidial contents and structure.
Active research about conidiation for a few decades has demonstrated that a series of growth and developmental stages are systemically and delicately regulated by a variety of transcription factors (TFs) (14,25).Most TFs have DNA-binding domains that can recognize specific nucleotide sequences and regulate the expression of target genes.Representatively, the temporal and spatial activation of the BrlA-AbaA-WetA central regulatory cascade causes cessation of hyphal growth and formation of conidiophores (26,27).VelB and VosA, members of the velvet family, are highly expressed in conidia and play important roles in conidial maturation and dormancy in Aspergillus species (28)(29)(30)(31).SclB, one of the Zn(II) 2 Cys 6 TFs repressed by VosA, affects proper conidiation and spore viability in A. nidulans (32).Another Zn(II) 2 Cys 6 TF, inhibited by VosA, ZcfA, is essential for stress tolerance and long-term viability of A. nidulans conidia (33).The bZIP TFs AtfA and AtfB are required for appropriate stress resistance to oxidative stresses in A. nidulans conidia (34,35).
Although various genetic regulators have been investigated, there is still limited understanding on the TFs that regulate the formation, maturation, and dormancy of conidia.This study aims to discover novel TFs that are specifically expressed in asexual spores of A. nidulans, A. flavus, and A. fumigatus and to investigate the functions of these novel TFs in Aspergillus conidia.Our findings demonstrated that SscA is indispensable for conidial formation, maturation and dormancy, and germination in A. nidulans.

Comparative transcriptomic analyses identify 25 spore-specific TFs in 3 Aspergillus species
Conidial formation and maturation require precise spatial and temporal regulation in Aspergillus species (36).To understand conidiogenesis in Aspergilli, we investigated differentially expressed genes (DEGs) between the elongated hyphae and resting conidia of three Aspergillus species using transcriptomic analyses (Fig. S1).We have identified 4,754, 5,471, and 4,851 DEGs in A. nidulans (Ani), A. flavus (Afl), and A. fumigatus (Afu), respectively (Tables S1 to S3).Analysis employing the transcript expression profiles of orthologous groups of genes (orthogroups) (37) has revealed that 1,030 genes were commonly up-and down-regulated in the conidia of three Aspergillus species.We further performed gene ontology (GO) enrichment analyses and found that the up-regulated genes in conidia were significantly enriched in heterocycle biosynthesis, transcription, small molecule catabolism, and asexual spore wall assembly, and the down-regula ted genes were associated with the processes of anatomical structure development, reproduction, cell differentiation, cell cycle, and fungal-type cell wall organization (Fig. 1A; Table S4).These findings are consistent with previous studies showing that hyphae repeatedly reproduced for vegetative filamentous growth by modulating actin filaments and cytoskeletons and differentiated into asexual structures by mediating cell integrity and cell cycle associated proteins (38).Moreover, conidia are transcriptionally active, produce various secondary metabolites, and modify the cell wall integrity for maintain ing spore viability (39,40).
The cell wall components of hyphae and conidia are different (39).Thus, we further analyzed the expression patterns of genes contributing to the conidial structures as shown in Fig. 1B (Table 1; Table S5).Although some differences existed in the three Aspergillus species, mRNA levels of trehalose biosynthetic genes were elevated in conidia (Fig. 1C).Transcript levels of chitin (Fig. 1D) and β-glucan (Fig. 1E) biosynthetic genes were lowered, whereas those of melanin biosynthetic genes were increased in conidia compared with those in hyphae (Fig. 1F).Collectively, the transcriptomic data suggest that fungal growth, development, cell wall organization, and cellular metabolism involve diverse differentially expressed genes in conidia or hyphae.

Functional analyses of 24 genes predicted to encode TFs in A. nidulans
To pin-point novel TFs regulating conidiogenesis, we analyzed the total TFs in the three Aspergillus species based on the protein family database (Pfam) and previous research (41,42).We have identified 574, 576, and 492 putative TFs in A. nidulans, A. flavus, and A. fumigatus (Fig. S2), respectively, and more than half of TFs were Zn(II) 2 Cys 6 TFs.Using transcriptomic data, we have identified significantly differently expressed TFs in the three Aspergillus species (Fig. 2A through C).Next, we investigated commonly up-regulated TFs in the conidia of the three Aspergillus species.We found 25 TFs that were specifically expressed in the conidia of Aspergillus species ( are also identified.One of the velvet family members, VosA, was confirmed in a list of up-regulated TFs in the conidia of the three Aspergillus species reported in previous studies (30,43,44).
To explore the functions of the novel 24 TFs in A. nidulans, excluding the previously studied VosA, we first confirmed the mRNA levels of 24 genes.As shown in Fig. S3, all the 24 genes were up-regulated in the last stage of asexual development and the conidia of A. nidulans.We then generated individual null mutant for each of the novel 24 genes in A. nidulans (45) and examined the phenotypes of each null (Δ) mutant (Fig. S4).Distinct from other 23 mutants, the sscA null mutant exhibited unusual colored colonies in A. nidulans.To further understand the functions of each spore-specific TF in conidia, we examined all null mutants for conidiation, conidial viability, conidial trehalose content, and conidial stress tolerance under thermal and oxidative stresses.Although other strains exhibited slightly different phenotypes compared with wild-type (WT), the deletion of sscA substantially affected all the conidial phenotypes (Fig. 2D).These findings led us to conduct in-depth studies on the biological roles of SscA in A. nidulans conidia.

Key role of SscA in conidial formation
SscA was highly conserved in Ascomycota as well as Basidiomycota (Fig. 3A), and the domain alignment of SscA in Aspergillus species showed that one C 2 H 2 zinc finger domain was conserved in the N-terminal end (Fig. 3B).The analyses of syntenic genes around sscA in three Aspergillus species showed that expression patterns of syntenic genes around the sscA gene were mostly different, and the homologs of sscA were commonly up-expressed in conidia of three Aspergillus species (Fig. S5).To confirm the effects of sscA deletion on asexual development, WT, ΔsscA, and sscA-complemented (Cʹ sscA) strains were point-inoculated and incubated for 5 days.As shown in Fig. 3C, the ΔsscA mutant exhibited abnormal point-phenotypes compared to WT and C' sscA strains.The deletion of sscA led to alter colony color and conidial pigmentation (Fig. S6A and B).When analyzed the relative expression levels of pigment synthetic genes (yA and wA), the abundance of yA and wA was significantly decreased in ΔsscA strains along the asexual development stage (Fig. S6C and D).Next, we quantitatively analyzed the plates shown in Fig. 3C and confirmed that the conidial production in ΔsscA mutant had considerably reduced in A. nidulans (Fig. 3D).To further check the morphology of the conidiophore, we observed the conidiophores of ΔsscA strain using the microscope, and found that the conidiophores of ΔsscA strain were sparsely present and those morphologies were small and incomplete compared with those of WT and Cʹ sscA strains (Fig. 3E).Quantitatively analyses indicated that the number of conidiophores of the ΔsscA strain was significantly reduced (Fig. 3F) and the size of ΔsscA conidiophore was considerably smaller compared with those of WT and C' sscA conidiophores (Fig. 3G).We then checked mRNA expression levels of two asexual developmental genes brlA and abaA, and found that mRNA levels of two genes were decreased in the ΔsscA strain after asexual induction (Fig. 3E and F).Taken together, these findings suggest that SscA plays a vital role in conidiophore and conidia formation in A. nidulans.

SscA is required for proper conidial dormancy and germination
As shown earlier in Fig. 2D, ΔsscA caused a decrease in the amount of trehalose, which is a key spore component for stress resistance (46).To further explore the multiple roles of SscA in conidial dormancy and germination, we evaluated the long-term viability and conidial germination of the ΔsscA conidia.We first observed the conidia of each strain grown for 10 days by transmission electron microscopy (TEM).As shown Fig. 4A, conidia of WT and complemented strains exhibited full cytoplasm and normal organelle structures, termed intact conidia, but the conidia of ΔsscA strain exhibited loss of cytoplasm and deficient organelles.The phenotype of the 10-day-old ΔsscA conidia closely resembled those of the ΔvosA mutant (29).Quantitative analysis revealed that <10% of the ΔsscA mutant conidia were intact (Fig. 4B).To determine the long-term viability of the 10-day-old ΔsscA conidia, we checked the ability of 10-day-old conidia to form germ tubes and found that the survival rate of the ΔsscA conidia was dramatically decreased compared to those of WT and complemented strains (Fig. 4C).Overall, these results demonstrate that SscA is required for proper conidial viability, cellular and metabolic integrity, and dormancy.Next, we investigated conidial germination of the 2-day-old ΔsscA conidia.We inoculated the fresh conidia of WT and mutant strains into agar media with or without glucose and quantified the germination rates.The germination rates of the WT, ΔsscA, and Cʹ sscA conidia were 15%, 55%, and 27%, respectively, at 5 h of incubation in the presence of 1% glucose (Fig. 4D).Furthermore, the germination rate of the ΔsscA conidia without glucose was higher than those of the WT and complemented conidia.Altogether, these results suggest that SscA is necessary for proper conidial germination in A. nidulans.

The absence of sscA affects trehalose biosynthesis and stress response
To elucidate the molecular functions of SscA in conidia, we performed genome-wide expression analyses of the conidia of WT and ΔsscA strains using RNA-sequencing (Table S6).Total of 4,569 genes (|Log 2 FC|≥1; P-value < 0.05) were differently expressed between WT and ΔsscA conidia (Fig. S7A).In the ΔsscA conidia, 1,874 DEGs were down-regulated and 2,695 DEGs were up-regulated.As shown in Fig. S7B, the deletion of sscA did not affect mRNA expression of genes next to sscA gene, but some of genes more than about 4 kb away from the sscA gene in genome were affected by the deletion of sscA.GO enrichment analysis of the up-regulated DEGs revealed the enrichment of genes involved in cell wall macromolecule metabolic process, glucan metabolic process, cellular amino acid catabolic process, secondary metabolite biosynthetic process, and sterigmatocystin metabolic process.GO enrichment analysis of the down-regulated DEGs showed the enrichment of genes related to response to stimulus, chemicals, and stress, gene expression, RNA metabolic process, and ncRNA metabolic process (Fig. 5A; Table S7).Among the DEGs affected by SscA, we correlated gene expression with phenotype.First, we found that mRNA levels of trehalose biosynthesis genes, including tpsA, tpsC, and orlA, were decreased in the ΔsscA conidia (Fig. 5B).Similar to the abovedescribed screening results (Fig. 2D), amount of trehalose in the sscA null conidia was reduced compared to WT and complemented strains (Fig. 5B).Second, mRNA levels of genes associated with DNA repair and stress response were decreased (Fig. 5C), implying that the sscA null conidia are likely sensitive to various stresses.In fact, when the ΔsscA conidia were tested for the sensitivity to UV, thermal, and oxidative stresses, as depicted in Fig. 5D, the conidia of sscA null mutant were more sensitive to UV, thermal, and H 2 O 2 stresses than those of WT and complemented strains.

SscA is essential for proper conidial wall integrity in A. nidulans
RNA-seq data showed that mRNA levels of genes related to the cell wall macromolecule metabolic process were increased in the ΔsscA conidia (Fig. 5A).In particular, mRNA levels of genes associated with glucan biosynthesis were highly increased in the ΔsscA conidia (Fig. 5E).We confirmed this result by qRT-PCR assay and found that expression of two beta-glucan biosynthetic genes fksA and gelA was increased in the ΔsscA conidia (Fig. 5F).We then examined the amount of β-glucans in the conidia of WT, ΔsscA, and Cʹ sscA strains and asked whether the changes in gene expression direct the phenotype.
As shown in Fig. 5G, the amount of β-glucan in the ΔsscA conidia was high compared to that in the conidia of WT and Cʹ sscA strains, suggesting that SscA is crucial for proper β-glucan levels in the conidia.Considering the augmented β-glucan contents in ΔsscA strain, we examined the resistance to echinocandins, blocking synthesis of 1,3-β-glucans.As a result, the absence of sscA exhibited slight resistance to caspofungin, one of the echinocandins (Fig. S8A and B).Furthermore, the transcript levels of genes related to chitin and hydrophobin biosynthesis were up-regulated in the ΔsscA conidia (Fig. S8C  through E).Overall, these results indicate that SscA is a key regulator of conidial wall structure and integrity in A. nidulans.

SscA down-regulates the production of sterigmatocystin (ST) and other secondary metabolites in A. nidulans conidia
In the ΔsscA conidia, mRNA levels of genes associated with the secondary metabo lite biosynthetic process and sterigmatocystin (ST) metabolic process were increased, implying that SscA inhibits the production of various secondary metabolites in A. nidulans conidia.To test this hypothesis, we analyzed expression of the ST gene cluster in WT and the ΔsscA conidia.As shown in Fig. 6A, the expression of most ST biosynthetic genes was increased in the ΔsscA conidia.The results of RNA-seq were further confirmed by qRT-PCR analysis (Fig. 6B).Amount of ST in the ΔsscA conidia was increased compared to that in the conidia of WT and complemented strains (Fig. 6C).Moreover, the amount of ST production in the ΔsscA mycelia was also increased compared to those of WT and Cʹ sscA strains (Fig. S9).To further comprehend the roles of SscA in ST biosynthesis, we generated the sscA-overexpressed strain (OEsscA) by expressing sscA under the alcA promoter.As shown in Fig. 6D, OEsscA strain produces much less amount of ST than the control strain under noninducing as well as inducing conditions.These data suggest that SscA act as a repressor of ST biosynthesis.We also checked expression patterns of 25 secondary metabolite gene clusters (Table S8).In particular, the transcript levels of asperthecin, emericellamide, and terrequinone biosynthetic genes were all elevated in the ΔsscA conidia compared to WT conidia (Fig. S10).Altogether, these results suggest that SscA plays as a master-controller for the production of various secondary metabo lites in A. nidulans conidia.

SscA contributes to the regulation of amino acid catabolism in A. nidulans conidia
Transcriptomic data revealed that mRNA levels of amino acid catabolism genes were altered in the ΔsscA conidia.The amino acid biogenesis pathway is controlled by various molecular regulators, and SscA affected mRNA levels of those genes (Fig. 7A).
To corroborate this, we evaluated the levels of amino acids in WT conidia and the ΔsscA conidia.The ΔsscA conidia exhibited considerably diminished levels of free amino acids, excluding leucine, compared to those of WT conidia.Especially, amounts of three basic amino acids were markedly decreased in the ΔsscA conidia (Fig. 7B).Collectively, these results indicate that SscA governs appropriate amino acid biosynthesis in A. nidulans conidia.

The role of SscA is conserved in the genus Aspergillus
As mentioned earlier, mRNA level of sscA were high in the conidia of three Aspergillus spp.To assess whether the role of SscA is conserved in the three Aspergillus species, we generated complemented strains that expressed AflsscA or AfusscA in the background of AnisscA deletion.As shown in Fig. 8, the changes in colony color and the number of conidia affected by the absence of AnisscA were fully restored back to WT phenotype by heterologously expressing AflsscA or AfusscA.The decrease in the amount of trehalose and stress sensitivity caused by AnisscA deletion was also restored to WT levels in AflsscAor AfusscA-expressed strains.These results strongly indicate that SscA plays conserved roles in conidial production, trehalose biosynthesis, and stress response in the genus Aspergillus.

DISCUSSION
Aspergillus conidia, the primary propagules, are adapted for long-term viability, stress tolerance.During conidial maturation, cellular transcription and translation are activated, and the accumulated transcriptome and proteome allow resting conidia to break and resume spore germination and reproduction (14,(47)(48)(49).Although studies on Aspergillus conidia have been conducted for decades, there are limited functional studies on TFs that are crucial for conidial maturation and dormancy in Aspergillus spp.In this study, we have investigated specifically expressed TFs in the conidia of three Aspergillus species and constructed a gene knockout collection in the model organism A. nidulans.This collection provided an opportunity to explore the roles of 24 TFs in conidiogenesis and conidial properties, and we found a TF that is essential for proper conidial physiology.We explored, in detail, the functions of a spore-specific C 2 H 2 zinc finger TF, AN5003 (sscA), in conidial properties.The deficiency of sscA resulted in pleiotropic phenotypes, includ ing impaired asexual spore formation and conidial long-term viability and abnormal germination.Furthermore, transcriptomic analyses revealed that the absence of sscA contributed to decreased stress response, transformations in cell wall integrity, and abnormal metabolism activities in A. nidulans (Fig. 9).A preliminary clue into the functions of SscA in fungal physiology was obtained from the result that the absence of sscA led to formation of abnormal conidiophores with apricot-colored conidia.And the conidial production was significantly decreased in the absence of sscA (Fig. 3; Fig. S6).Based on these results, we propose that an appropriate expression of SscA is required for proper asexual development in the lifespan of A. nidulans.The homologs of SscA also contribute to pleiotropic fungal morphology and development.In C. albicans, Bcr1 (biofilm and cell wall regulator 1) affects morphogene sis by differently expressing between yeast and hyphal form (50,51).In C. neoformans, the absence of usv101 influences the production of melanin (41,52).In F. graminearum, Nsf1 (nutrient and stress factor 1) is highly expressed in conidia compared to that in mycelium similar to SscA in A. nidulans (53).The disruption of FgNsf1 causes inhibi ted hyphal growth, defective asexual development, and fungal pigmentation.Further more, Gcf6 (growth and conidiation regulatory factor 6) contributes to fungal growth, conidiation, and conidial germination in Magnaporthe oryzae (54).In addition, we have shown in the present study that AflsscA and AfusscA exerted conserved roles in asexual development and conidial physiology in A. nidulans (Fig. 8).Nevertheless, it is necessary to investigate whether SscA commonly contributes to fungal development in the plant pathogen A. flavus and the human pathogenic fungus A. fumigatus in future research.
As shown in Fig. S3, abundance of sscA mRNAs increases during asexual develop ment, implying that they might be regulated by central regulators of asexual develop ment (BrlA or AbaA).We analyzed the sscA promoter region and found three BREs (BrlA-response element, 5′-(C/A)RAGGGR-3′) and one ARE (AbaA-response element, 5′-CATTCY-3′).Previous studies also suggested that Bcr1 is activated by Tec1 (homolog of AbaA) and regulates cell-surface-associated genes in C. albicans (51).Based on our results, we hypothesize that SscA expression is activated by AbaA, causing a gradual increase in mRNA levels of SscA following asexual development and asexual spore formation.To elucidate the genetic relationship between abaA and sscA, we measured levels of the sscA transcript in the asexual developmental stages of WT and the ΔabaA mutant.As shown in Fig. S11, mRNA levels of sscA are considerably decreased in the absence of abaA.Next, we further evaluated whether SscA is coordinated by WetA, VosA, and VelB in A. nidulans.Our results have revealed that SscA is essential for normal conidial viability (Fig. 4A through C) and WetA, VosA, and VelB are the primary regula tors of conidial maturation in A. nidulans conidia (44).Hence, we analyzed previously published multiomics data (44) and confirmed that the transcript levels of sscA are not affected in the ΔwetA, ΔvosA, and ΔvelB conidia.Overall, these results suggest that SscA expression is dependent on AbaA, but not on WetA, VosA, and VelB, for the proper asexual development of A. nidulans.
Our RNA-seq data showed that SscA is closely involved in the response to stresses and chemicals (Fig. 4A).Also, we observed that the deletion of sscA led to lower trehalose contents and reduced resistance to UV, thermal, and oxidative stresses in conidia (Fig. 5D).Namely, SscA is essential for stress tolerance in the conidia of A. nidulans.Previous study has demonstrated that usv1 null mutant is more sensitive to osmotic stress in S. cerevisiae (55).In C. albicans, the sensitivity of Δbcr1 to caffeine, lithium chloride, and copper is higher than that of WT (42).In C. neoformans, the deletion of usv101 causes sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, menadione, dithiothreitol, and amphotericin B (41).However, the absence of Fgnsf1 causes resistance to osmotic, oxidative, and metal cation stresses in F. graminearum (56).Collectively, the functions of SscA homolog in the stress resistance are species-specific.
Phenotypic and transcriptomic analyses of the sscA mutant have revealed that SscA is important for controlling ST biosynthesis (Fig. 6).Moreover, our analysis of the transcripts of the gene clusters of 23 secondary metabolites has revealed that SscA negatively regulates the production of other secondary metabolites in A. nidulans conidia.A previous study showed that FgNsf1 is not required for the production of the toxin zearalenone (42) but is required for the biosynthesis of the trichothecene mycotoxin, deoxynivalenol in F. graminearum (56).Therefore, the functions of SscA in secondary metabolism may be species-specific.
Genome-wide expression analyses have revealed that SscA affects cellular amino acid catabolic process in A. nidulans conidia.In fact, most genes related to amino acid catabolism in the ΔsscA mutant are highly expressed compared to that in WT strain, and consequently, the abundance of amino acids in the conidia of the ΔsscA mutant is less than those of WT (Fig. 7).Although it is not yet clear how these metabolic changes affect fungal development and conidial physiology, previous studies have reported that the amount of cellular amino acids was related to cell longevity in Saccharomyces cerevisiae (57,58).Based on our result that the deletion of sscA causes deficient long-term spore viability, we suggest that SscA regulates spore longevity in part by coordinating the metabolism of amino acids.
The Cys 2 His 2 zinc finger protein is one of the largest families of TFs in eukaryotes and the second largest TFs in A. nidulans genome (59,60).This type of TF is commonly composed of two or three β-sheets and one α-helix, enabling specific DNA-binding.
As trans-acting factors, C 2 H 2 TFs regulate certain gene expression related to cellular development, differentiation, stress response, and metabolism in eukaryotes.Previous studies have demonstrated that C 2 H 2 proteins also have pleiotropic effects on A. nidulans development, stress response, and metabolism: AslA, BrlA, FlbC, NsdC, SteA, NrdA, CreA, and MtfA.BrlA is a central regulator of asexual development; AslA and FlbC also affect asexual development by activating brlA expression (15,61).NsdC and SteA regulate normal sexual development (62).NrdA is required for oxidative stress resistance as well as normal sexual development (63).MtfA plays important roles for proper asexual and sexual development and secondary metabolism in A. nidulans (64).CreA is a wellknown C 2 H 2 TF for carbon catabolite repression, which regulates expression of various hydrolytic enzymes (65).In this study, we characterize a novel C 2 H 2 -type zinc finger TF in A. nidulans.SscA has one Cys 2 His 2 zinc finger domain composed of two β-sheets and one α-helix, patterned as C-X 5 -C-X 12 -H-X 3 -H.Like other C 2 H 2 TF, SscA also act as a multifunctional regulator for asexual spore formation, conidial stress tolerance, and metabolism.Zinc-binding proteins can act as activators, repressors, or both activators and repressors for certain genes.When considering the number of up-regulated (2,695) and down-regulated (1,874) genes in deletion of sscA, SscA might function primarily as a transcriptional repressor.However, further studies are required to understand the molecular functions of SscA as a TF.
To summarize, we investigated the DEGs between conidia and hyphae and generated a collection of TFs that are highly expressed in the conidia of three Aspergillus species.Through various phenotypic analyses, we identified a Cys 2 His 2 TF, SscA, in A. nidulans.Based on our in-depth studies, we conclude that SscA plays an essential role in the regulation of conidial formation, maturation, dormancy, germination, and metabolism in A. nidulans.Our results also suggest that the functions of AflsscA and AfusscA are conserved in A. nidulans conidia.

Generation of TF deletion mutants
Double-joint PCR (DJ-PCR) was used for the generation of the TF deletion mutant collection, and oligonucleotides used in this study are described in Table S10 (45).Briefly, the 5ʹ-and 3ʹ-flank regions of each TF were amplified using the primer pairs 5ʹ DF/3ʹ tail and 5ʹ tail/3ʹ DR of each TF gene, respectively, with A. nidulans FGSC4 genomic DNA (gDNA) as a template.The A. fumigatus pyrG marker was amplified from A. fumigatus Af293 gDNA using the primer set OHS1542/OHS1543.Three fragments, including 5ʹ-flank, 3ʹ-flank, and pyrG + , were fused through second and third PCR with the primer set 5ʹ NF/3ʹNR of each TF gene, and the deletion cassette was transformed into the A. nidulans strain RJMP 1.59.

Construction of sscA-complemented strains
For constructing the sscA-complemented strain in A. nidulans, the AnisscA gene region with its promoter was amplified using the primer pair OHS1711/OHS1713, digested with NotI, and cloned into pHS13 (28).The resulting plasmid pYE7.1 was then introduced into the recipient ΔAnisscA strain TSH31.1 to produce TYE51.1~2.
For the complementation of the ΔAnisscA strain with A. flavus, the ORF, and its upstream promoter region were amplified using A. flavus NRRL 3357 gDNA and the primer set OHS1876/OHS1780, digested with NotI, and cloned into pHS13.The resulting plasmid pYE9.1 was transformed into the recipient ΔAnisscA strain TSH31.1 to produce TYE57.1~2.
For the complementation of the ΔAnisscA strain with A. fumigatus, the ORF, and its upstream promoter region were amplified using A. fumigatus Af293 gDNA and the primer set OHS1763/OHS1764, digested with NotI, and cloned into pHS13.The resulting plasmid pYE10.1 was transformed into the recipient ΔAnisscA strain TSH31.1 to produce TYE58.1.All the complemented strains were selected among the transformants and screened by PCR and quantitative reverse transcription-PCR (qRT-PCR).

Construction of sscA-overexpressed strain
For constructing the sscA-overexpressed strain in A. nidulans, open reading frame region of the AnisscA was amplified using the primer pair OHS1712/OHS1713, digested with NotI, and cloned into pHS3, which has A. nidulans alcA promoter (28,33).The resulting plasmid pYE8.1 was then introduced into the TNJ36 to produce TYE54.1.The sscA-over expressed strains were selected among the transformants and verified by qRT-PCR by inducing the promoter (Fig. S12).

Transcriptomic analyses and GO analyses
For transcriptomic analyses of hyphae, the conidia (10 6 /mL) of three Aspergillus species were inoculated into 100 mL of liquid MM or MMYE and incubated at 37°C for 14 h.The cultured samples were filtered, washed, squeeze-dried, and stored at −80°C.For transcriptomic analyses of conidia, conidia grown on solid MM for 2 days were suspen ded with 0.02% Triton X-100 (ThermoFisher, Waltham, MA, USA) and filtered through Miracloth (Calbiochem, San Diego, CA, USA) (67).The collected conidia were resuspen ded in with 0.02% Triton X-100 and subject to RNA extraction immediately to avoid breaking of dormancy in conidia.
Total RNA isolation was performed as previously described (68).Each sample was homogenized using a Mini-Bead beater (BioSpec Products Inc., Bartlesville, OK, USA) with 0.3 mL of glass beads (Daihan Scientific, Wonju, South Korea) and 1 mL of TRIzol reagent (Invitrogen, Waltham, MA, USA) and centrifuged.The supernatant was mixed with cold isopropanol and centrifuged.The RNA pellets were washed with 70% ethanol and then dissolved in DEPC-DW (Bioneer, Daejeon, South Korea).The extracted RNA samples were treated with DNase I (Promega, Madison, WI, USA) and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), after which library construction and Illumina platform sequencing were performed by Theragen Bio (Suwon, South Korea).Briefly, for pairedend sequencing, RNA integrity was evaluated on an Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA), and the RNA library was constructed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA).Each sample with sequencing adaptors was sequenced in a single lane on the Illumina Novaseq 6000 system.Recorded Fast files were analyzed with FastQC and trimmed using Trimmomatic.Filtered reads were mapped to the A. nidulans FGSC A4, A. flavus NRRL3357, and A. fumgiatus Af293 transcriptome using the aligner STAR v.2.3.0esoftware.Based on read counts, the DEGs were identified, and normalized using the DESeq2 method.All RNA-seq experiments were performed with at least two biological replicates.
For the characterization of DEGs, GO analyses were conducted using R package and FungiDB (https://fungidb.org/fungidb/app).DEGs, which were identified based on two-fold change and P-value < 0.05, were subjected to Fisher's exact test, and all processed data were plotted using R studio (v.3.6.3).

Quantitative real-time PCR
For the induction of asexual development, cultured mycelia were transferred to solid MM and incubated at 37°C for indicated time points under light condition (69).From the isolated total RNA, cDNA was synthesized using GoScript Reverse transcriptase (Promega, Madison, WI, USA), and real-time PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on CFX96 Touch Real-Time PCR (Bio-Rad) (68).The primers used for qRT-PCR are listed in Table S6.The fold expression was computed using the 2 −ΔΔCt method, and the expression was normalized to that of β-actin, which was used as a control.

Conidial production, viability, and germination assay
To quantify the production of conidia, each strain was point-inoculated onto solid MM media and incubated at 37°C for 5 days.Then, the conidia were harvested using distilled water containing 0.02% Triton X-100 and counted using a hematocytometer under a Leica DM500 microscope (Leica, Wetzlar, Germany).
For spore viability assay, conidia were grown for 2 or 10 days and collected using DW containing 0.02% Triton X-100.Then, approximately 1 × 10 2 conidia were spread onto solid MM and incubated at 37°C for 2 days.The viability of conidia was calculated as the ratio of the number of viable colonies from 10-day-old conidia to the number of viable colonies from 2-day-old conidia in triplicate (33,67).
For the assay of conidial germination, approximately 10 7 conidia grown for 2 days were spread onto MM agar with or without glucose and cultured at 37°C.The germina tion rate was calculated as the ratio of the number of germinated spores to that of total spores every hour (68).

Spore trehalose, stress tolerance, and β-glucan assay
For the assay of spore trehalose content, conidia (2 × 10 8 ) from 2-day-old cultures were collected, resuspended in ddH 2 O, and incubated at 95°C for 20 min.The supernatant was mixed with an equal volume of 0.2 M sodium citrate (pH 5.5) and incubated at 37°C for 8 h with or without (as a negative control) trehalase (Sigma).The amount of glucose generated from trehalose was assayed using a Glucose Assay Kit (Sigma) in triplicate (33,69).
For evaluating the spore tolerance against thermal and oxidative stresses, approxi mately 10 3 conidia were incubated at 50°C in 0.1 M H 2 O 2 for 30 min.Then, the samples were diluted, spread onto solid MM, and incubated at 37°C for 2 days.For the conidial UV stress test, approximately 10 2 conidia were spread onto solid MM and UV-irradiated using a UV crosslinker (Spectrolinke XL-1,000 UV crosslinker, Thomas Scientific, Swedesboro, NJ, USA).Then, the plates were incubated at 37°C for 2 days.The survival rates were calculated as the ratio of the number of viable colonies in the treated sample to that of viable colonies in the untreated control (68,70).
To examine β-1,3-glucan analysis, approximately 10 3 -10 4 conidia grown for 2 days were resuspended in 25 µL, mixed with Glucatell reagent, and incubated at 37°C for 30 min.Then, the reaction was stopped using diazo coupling reagents, and the optical density of the samples was read at 540 nm (43).

Transmission electron microscopy
TEM analysis was performed with a modification of a previously described method (43).Briefly, 10-day-old conidia (5 × 10 8 ) were collected and fixed in Karnovsky's fixative at 4°C overnight.The samples were then fixed with 1% osmium tetraoxide (Sigma), dehydra ted in graded ethanol, and resuspended in propylene oxide (Sigma) and poly/bed 812 (Polyscience, Warrington, PA, USA), sequentially.For embedding, the samples were added to poly/bed 812 with 2% DMP-30 and solidified in an oven (65°C) overnight.Polymerized samples were sectioned on a Leica EM UC7 cryo-ultramicrotome equipped with a diamond knife and stained with UranyLess (EMS, Hatfield, PA, USA) and lead citrate (EMS).The stained sections were viewed under a Hitachi HT7700 transmission electron microscope (Hitachi, Soto-Kanda, Chiyoda-ku, Tokyo).

Amino acid analysis in conidia
For quantifying the amount of amino acids in conidia, approximately 2 × 10 9 conidia grown for 2 d were prepared, mixed with 0.5 mL of HPLC-grade acetonitrile-methanolwater (40:40:20, vol/vol/vol) and 0.3 mL glass beads, and homogenized using a Mini-Bead beater (BioSpec Products Inc.) (44).The lysates were centrifuged, and the supernatant was filtered using a 0.45-µm hydrophobic PTFE filter (Falcon, Corning, NY, USA).The purified samples were pretreated with methanol:acetonitrile:DW (2:2:1, vol/vol/vol) and analyzed using the amino acid analyzer L-8900 (Hitachi) consisting of 4.6 mm ID × 60 mm column packed with ion exchange resin.The amount of amino acids was measured using the colorimetric ninhydrin method (Wako, Tokyo, Japan) and calculated using the mixture of amino acid standard solution type ANII and type B (Wako).

Sterigmatocystin production analysis in conidia
Sterigmatocystin (ST) was extracted from conidia as described previously (68), with some modifications.Approximately 2 × 10 8 conidia were homogenized using a Mini-Bead beater (BioSpec Products Inc.) with 0.3 mL of glass beads (Daihan Scientific) and 1 mL of chloroform (Sigma).After centrifugation, the organic phase was mixed with DW for refining secondary metabolites and centrifuged.The purified organic phase was transferred to a new glass vial and dried in an oven (65°C) overnight.The samples were resuspended with 0.1 mL of chloroform, spotted in thin-layer chromatography (TLC) silica plate coated with the fluorescent indicator F254 (Merck Millipore, Burlington, MA, USA), and resolved in TLC plates containing toluene:ethyl acetate:acetic acid (8:1:1, vol/vol/vol).The thin-layer chromatography (TLC) plates were treated with 1% aluminum hydroxide hydrate (Sigma), and images were captured under UV exposure (366 nm).The relative intensities of ST were calculated using the ImageJ software.

Microscopy
Photographs of colonies were taken using a Pentax MX-1 digital camera.Photomicro graphs were taken using a Leica DM500 microscope equipped with Leica ICC50 E and Leica Application Suite X software.

Statistical analysis
Statistical differences between the control and target strains were evaluated using Student's unpaired t-test.Data are reported as mean ± standard deviation.P-values < 0.05 were considered significant.

FIG 1
FIG 1 The transcriptional profiles of conidia are different from those of hyphae in three Aspergillus species.(A) Gene ontology (GO) enrichment analysis of commonly differentially expressed genes in the hyphae and conidia of A. nidulans, A. flavus, and A. fumigatus.Left: down-expressed genes in conidia, Right: up-expressed genes in conidia.(B) The structure of conidial wall.(C-F) Heat map showing the mRNA expression of genes associated with cell wall compositions.(C) Trehalose biosynthesis, (D) chitin biosynthesis, (E) β-glucan biosynthesis, (F) melanin biosynthesis.The dot indicates that the corresponding homologous gene is absent.

FIG 2
FIG 2 The twenty-five putative spore-specific transcription factors in three Aspergillus species.(A-C) Volcano plots showing up-and down-expressed TFs encoded by A. nidulans (A), A. flavus (B), and A. fumigatus (C).Pink, orange, and yellow points are TFs with P < 0.05 and Log 2 FC ≥1.Ultramarine blue, green, and blue points are TFs with P < 0.05 and Log 2 FC ≤ −1.Up: up-expressed in conidia, Down: down-expressed in conidia, N.S.: not significantly expressed in conidia.(D) Phenome heat map showing the results of screening 24 spore-specific transcription factor deletion strains against conidiation, long-term viability, amount of trehalose, and thermal, and oxidative stress tolerance.Right color key represents reduction (blue) and enhancement (red), respectively, compared with the wild-type strain.

FIG 3 (FIG 4
FIG 3 (Continued) homologs in Aspergillus species.Protein sequences were aligned with Clustal W and Mega X software.(C) Growth phenotypes of wild-type (WT), ΔsscA, and complementary (Cʹ sscA) strains grown on solid minimal media (MM) at 37°C for 5 days under light and dark conditions.(D) The bar plots showed quantitative examination of conidial production per square centimeter shown in (C).Error bars indicate standard deviations of three biological replicates (***P < 0.001, **P < 0.01).(E) Microscopic images of wild-type (WT), ΔsscA, and complementary (Cʹ sscA) strains grown on solid minimal media (MM) at 37°C for 5 days under light.Below panels exhibited the enlarged conidiophores of each strain (bar = 0.25 mm).(F) Bar plat showing the size of conidiophores shown in (E).Error bars indicate standard deviations of three biological replicates (***P < 0.001).(G) Bar plat shown in (E).Error bars indicate standard deviations of three biological replicates (***P < 0.001).(H and I) Quantitative real-time RT-PCR analysis of brlA (H) or abaA (I), gene in WT, ΔsscA, C' sscA strains after inducing asexual development.The mRNA expression was normalized to that of the endogenous control β-actin gene.The control group was expression levels of wild-type 0 h after inducing asexual development (***P < 0.001, **P < 0.01, *P < 0.05).

FIG 5
FIG 5 SscA is essential for stress tolerance and conidial wall integrity.(A) Gene ontology (GO) analysis of significantly up-regulated (2,695) and down-regulated (1,874) genes in the conidia of sscA-deleted strain compared to that in the conidia of wild-type strain (biological process).(B) Transcript levels of trehalose biosynthetic genes in WT and ΔsscA conidia.Below histograms indicate the amount (μg) of trehalose in the conidia of WT, ΔsscA, and Cʹ sscA strains grown for 2 days (***P < 0.001).(C) Transcription levels of 14 genes involved in DNA repair system, which were down-regulated in the sscA deletion mutant.(D) Bar graphs showing the relative survival rate of WT, ΔsscA, and Cʹ sscA conidia stressed by ultraviolet radiation, heat, and oxidative reagents.The viable rate was calculated as the ratio of the number of survived colonies to that of the untreated control.Error bars indicate standard deviations of three biological replicates (***P < 0.001).(E) Transcription pattern of β-glucan biosynthetic genes in WT and ΔsscA conidia.(F) Bar plots showing the relative expression levels of β-glucan biosynthetic genes (fksA and gelA) in the designated strains (***P < 0.001, **P < 0.01).(G) The quantified β-glucan levels (pg) in WT, ΔsscA, and Cʹ sscA conidia (**P < 0.01).

FIG 6
FIG 6 SscA contributes to sterigmatocystin biosynthesis process in conidia.(A) A diagram of sterigmatocystin (ST) biosynthesis gene cluster.Right heat map shows the relative transcript abundance of the ST gene cluster.(B) Bar plots indicate the mRNA expression levels of ST biosynthetic genes (aflR, stcA, and stcE) in the designated strains (**P < 0.01).(C) Thin-layer chromatography (TLC) image of ST extracted from WT, ΔsscA, and Cʹ sscA conidia.Right bar plot shows the relative band intensity of ST analyzed in panel C (**P < 0.01).(D) TLC analysis of sterigmatocystin (ST) produced by control and OEsscA under liquid noninducing and inducing media.The right bar graph represented the relative band intensity of ST (***P < 0.001).

FIG 7
FIG 7SscA contributes to the regulation of primary metabolites in conidia.(A) Heat map plot representing significantly differentially expressed genes involved in amino acid catabolism in A. nidulans.(B) Free amino acid profiling of wild-type and ΔsscA conidia using the amino acid analyzer.The relative levels were calculated as the amount of amino acids in ΔsscA conidia to that of amino acids in WT conidia (***P < 0.001, **P < 0.01, *P < 0.05).

FIG 8
FIG 8 SscA of A. flavus and A. fumigatus has conserved roles in the physiology of A. nidulans.(A) Colony morphology of wild-type, ΔAnisscA, Cʹ AnisscA, Cʹ AflsscA, and Cʹ AfusscA strains grown on solid minimal media (MM) at 37°C for 5 days under light and dark conditions.(B) Quantitative data of conidial production shown in panel A (***P < 0.001, **P < 0.01).(C) Bar plot indicating the amount (μg) of trehalose in the conidia of the designated strains (***P < 0.001).(D and E) Histograms representing the survival rate of the conidia of the designated strains against thermal (D, 50°C) and oxidative (E, 0.1 M H 2 O 2 ) stresses.The survival rates were calculated as the ratio of the number of viable colonies to that of the untreated control.Error bars indicate standard deviations of three biological replicates (***P < 0.001, **P < 0.01).

FIG 9 A
FIG9 A proposed model depicting the functions of SscA in A. nidulans conidial formation, maturation, dormancy, and germination.SscA, which is highly expressed in conidia, has multiple functions in conidial properties.First, SscA affects normal conidial formation.Second, SscA influences conidial maturation and dormancy, by regulating mRNA levels of genes related to stress tolerance, DNA repair, trehalose biosynthesis, and cell wall integrity.Third, SscA controls conidial germination.Finally, SscA affects amino acids catabolism and sterigmatocystin biosynthesis in conidia.

TABLE 1
Differentially expressed genes (DEGs) associated with cell wall integrity in three Aspergillus species' conidia versus hyphae

TABLE 2
List of transcription factors specifically up-regulated in three Aspergillus species' conidia compared with hyphae