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. 2014 Feb 14;197(1):159–173. doi: 10.1534/genetics.114.161430

NsdD Is a Key Repressor of Asexual Development in Aspergillus nidulans

Mi-Kyung Lee *,†,1, Nak-Jung Kwon *,1,2, Jae Min Choi ‡, Im-Soon Lee †, Seunho Jung ‡, Jae-Hyuk Yu *,3
PMCID: PMC4012476  PMID: 24532783

Abstract

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (−) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (−) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.

Keywords: Aspergillus, asexual development, negative regulator, mycotoxin, GATA factor

THE filamentous ascomycete Aspergillus nidulans has served as an excellent model system for studying cell biology, asexual development (conidiation), and secondary metabolism (Timberlake 1990; Martinelli 1994; Yu and Keller 2005). The A. nidulans asexual reproductive cycle can be divided into two distinct phases: growth and development. The growth phase involves germination of an asexually derived spore called a conidium and formation of an undifferentiated network of interconnected hyphal cells that form the mycelium. After a certain vegetative growth period, under appropriate conditions, some of the hyphal cells stop normal growth and begin development by forming complex structures called conidiophores that bear multiple chains of conidia (Adams et al. 1988; Park and Yu 2012a).

A key and essential step for conidiophore development in Aspergillus is the activation of brlA, which encodes a C2H2 zinc-finger transcription factor (TF) (Figure 1A) (Adams et al. 1988; Chang and Timberlake 1993). Further genetic and biochemical studies identified the additional key regulators abaA and wetA that function during the middle and late stages of conidiation, respectively (Figure 1A) (Sewall et al. 1990; Andrianopoulos and Timberlake 1991; Marshall and Timberlake 1991). These three genes have been proposed to define a central regulatory pathway that acts in concert with other genes to control the spatial and temporal specificity of gene expression during conidiophore development and spore maturation (Mirabito et al. 1989; Yu 2010; Park and Yu 2012a).

Figure 1.

Figure 1

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Genetic model of conidiation and multicopy screening. (A) A genetic model for upstream and downstream developmental regulators of conidiation in A. nidulans. (B) Strategy and summary for multicopy screening. The pRG3-AMA1-based A. nidulans WT (FGSC4) genomic DNA library was introduced into ∆sfgA strains (veA+: TNJ30 and veA1: TNJ134). Six genes with the number of transformants are indicated. (C) Phenotypes of multicopy mutants. WT-veA+ (TNJ36.1), WT-veA1 (TNJ36.4), ∆sfgA M-AN1652 (TM1652), ∆sfgA M-AN2009 (TM2009), ∆sfgA M-AN7507 (TM7507), ∆sfgA M-AN3152 (TM3152), ∆sfgA M-AN5833 (TM5833), and ∆sfgA M-AN9141 (TM9141) strains were point inoculated on solid MMG and incubated at 37° for 3 days. For the colony morphologies of ∆sfgA with veA+ and ∆sfgA with veA1 strains, please see Figure 3B.

Activation of brlA requires activities of various upstream elements including fluG, flbA, flbB, flbC, flbD, and flbE. Mutations in any of these genes result in “fluffy” colonies that are characterized by undifferentiated cotton-like masses of vegetative cells (Adams et al. 1988). FlbA is a regulator of G-protein signaling (RGS) protein, and flbA mutants are distinguished from the others by the fluffy autolytic phenotype (Lee and Adams 1994b; Yu et al. 1996). FlbC is a putative TF with two C2H2 zinc-finger DNA-binding domains and is thought to directly control brlA expression (Kwon et al. 2010a). FlbE is a 201-aa-length polypeptide with two conserved yet uncharacterized domains and colocalizes with FlbB, a basic leucine zipper (b-zip) TF, at the hyphal tip in an actin cytoskeleton-dependent manner (Garzia et al. 2009). FlbE physically interacts with FlbB, and they activate FlbD interdependently (Garzia et al. 2009; Kwon et al. 2010b). FlbD (a cMyb-type TF) and FlbB then cooperatively function in activating expression of brlA (Garzia et al. 2010). Both of these two independent conidiation activation cascades (Figure 1A) are necessary for full activation of brlA expression and proper conidiation (Wieser and Adams 1995; Garzia et al. 2010; Kwon et al. 2010a). Importantly, loss of fluG or flbA function results in the blockage in both conidiation and production of the carcinogenic mycotoxin sterigmatocystin (ST), the penultimate precursor of the better-known potent carcinogen aflatoxins (Lee and Adams 1994a; Hicks et al. 1997).

FluG is required for the synthesis of the extracellular sporulation-inducing factor (a diorcinol-dehydroaustinol adduct) that triggers the commencement of conidiation in A. nidulans (Lee and Adams 1994a; Rodriguez-Urra et al. 2012). The two genetic responses to FluG activity were thought to be (1) activation of the FLBs → BrlA development-specific regulatory cascades and (2) positive modulation of FlbA, which in turn inhibits vegetative growth signaling mediated by a heterotrimeric G protein composed of FadA and SfaD::GpgA (Lee and Adams 1994a; Yu et al. 1996; Yu 2006). Our studies have revealed that both processes might involve the removal of a key repression of conidiation imposed by SfgA, which is a putative TF with a Gal4-type Zn(II)2Cys6 binuclear DNA-binding domain (Seo et al. 2003, 2006). SfgA acts as an important upstream negative regulator of conidiation functioning downstream of FluG, but upstream of FlbA, FlbB, FlbC, FlbD, and BrlA (Seo et al. 2006). The deletion of sfgA causes hyperactive conidiation and eliminates the need for fluG in conidiation and ST production. These led us to propose that the developmental transition from vegetative growth in A. nidulans occurs via removal of the negative regulator SfgA, which inhibits precocious activation of brlA during proliferation, thereby allowing proper vegetative growth.

During conidiogenesis, the spatial and temporal expression of brlA, abaA, and wetA is tightly controlled (Boylan et al. 1987; Mirabito et al. 1989; Adams et al. 1998; Ni et al. 2010). For instance, transcripts of brlA and abaA specifically accumulate during the early-to-middle phases of conidiophore formation and quickly disappear upon the formation of conidia. Our gain-of-function (multicopy) genetic screen has identified VosA as a key negative feedback regulator of brlA (Ni and Yu 2007). In conjunction with the differentiation of conidia, vosA is activated by AbaA (and WetA). This, in turn, represses brlA and, together with WetA, promotes maturation of conidia and trehalose biogenesis in spores (Figure 1A) (Ni and Yu 2007). VosA localizes in the nucleus of mature conidia and contains the velvet DNA-binding domain recognizing 11 nucleotide sequences and a transcriptional activation domain, indicating it is a TF (Ni and Yu 2007; Ahmed et al. 2013).

In the present study, we specifically aimed to identify key negative regulators of conidiation that acted downstream of FluG-SfgA and likely upstream of brlA. Employing two types of sfgA deletion strains (veA+ or veA1) as recipients, we screened a pRG3-AMA1-based wild-type (WT) genomic DNA library (Osherov et al. 2000) for the genes inhibiting conidiation when present in multiple copies. Among 100,000 transformants screened, 61 candidates were isolated and 6 responsible genes, AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141, were identified. Notably, the previously reported GATA-type TF NsdD activating sexual development in A. nidulans (Han et al. 2001) was identified by 10 independent transformants. Further studies indicate that NsdD is a key negative controller of conidiation, likely acting at the brlA level in A. nidulans, and also influences ST biosynthesis.

Materials and Methods

Aspergillus strains and culture conditions

Aspergillus strains used in this study are listed in Table 1. Individual strains were inoculated into liquid or on solid 1% glucose minimal medium (MMG) with appropriate supplements and incubated at 37°. If needed, 0.5% yeast extract (YE) was used (Pontecorvo et al. 1953; Kafer 1977). For liquid-submerged culture, 5 × 105 conidia⋅ml−1 were inoculated into 100 ml liquid MMG and incubated at 37°, 220 rpm. Samples were taken at 1-day intervals for up to 6 days of cultivation. Standard A. nidulans transformation techniques were used as described previously (Szewczyk et al. 2006; Park and Yu 2012b). Characterization of phenotype was performed by point inoculating each strain on solid MMG at 37° for up to 4 days. Entire colonies and close-up views of the middle zone of individual colonies were examined and photographed under a microscope. Expression of target genes under the control of the niiA promoter was controlled by nitrate source: repression on MMG plus 0.2% ammonium tartrate or induction on MMG plus 0.6% sodium nitrate (Arst et al. 1979). To examine the effect of the overexpression (OE) of fluG and sfgA by the ectopic copy of the gene under control of the alcA promoter (Waring et al. 1989), strains were grown through stationary culture in noninducing liquid MMG (MMG plus 0.5% YE) and inducing liquid MMG (MMT: 1.1% threonine plus 0.5% YE) and incubated at 37° for 3 days. Northern blot analyses during vegetative growth and postdevelopmental induction were carried out as described in Seo et al. (2003; Ni and Yu 2007). Briefly, 2 × 106 conidia⋅ml−1 were inoculated into 100 ml liquid MMG and cultured at 37° for 18 hr. The mycelium was collected at designated time points from liquid-submerged cultures and squeeze-dried. For sexual and asexual developmental induction, cultured mycelia were harvested and transferred on solid MMG and the plates were air exposed for asexual developmental induction or tightly sealed and blocked from light for sexual developmental induction. Samples for RNA isolation were collected at designated time points after transfer. The Escherichia coli DH5α strain was cultured in Luria–Bertani medium with 50 µg/ml of ampicillin (Sigma-Aldrich) for plasmid amplification.

Table 1. A. nidulans strains used in this study.

Strain Genotype Source
FGSC4 A. nidulans WT FGSCa
FGSC26 biA1; veA1 FGSC
RJMP1.59b pyrG89; pyroA4 Shaaban et al. (2010)
RNIW3 pyrG89; pyroA4; veA1 Kwon et al. (2012)
TNJ36.1 pyrG89; AfupyrG+; pyroA4 Kwon et al. (2010b)
TNJ36.4 pyrG89; AfupyrG+; pyroA4; veA1 This study
TNJ30 pyrG89; ∆sfgA::pyroA+; pyroA4 This study
TNJ57 pyrG89; ∆sfgA::AfupyrG+; pyroA4 Kwon et al. (2012)
TNJ134 pyrG89; ∆sfgA::pyroA+; pyroA4; veA1 This study
TNJ135 pyrG89; ∆sfgA::AfupyrG+; pyroA4; veA1 This study
TM7507 pyrG89; ∆sfgA::pyroA+; pyroA4; pRG-AMA1::AN7507 This study
TM2009 pyrG89; ∆sfgA::pyroA+; pyroA4; pRG-AMA1::AN2009 This study
TM1652 pyrG89; ∆sfgA::pyroA+; pyroA4; pRG-AMA1::AN1652 This study
TM3152 pyrG89; ∆sfgA::pyroA+; pyroA4; veA1; pRG-AMA1::AN3152 This study
TM5833 pyrG89; ∆sfgA::pyroA+; pyroA4; veA1; pRG-AMA1::AN5833 This study
TM9141 pyrG89; ∆sfgA::pyroA+; pyroA4; veA1; pRG-AMA1::AN9141 This study
TNJ160 pyrG89; ∆sfgA::pyroA+; pyroA4; pRG-AMA1::AN3152 This study
TNJ173 pyrG89; AfupyrG+; pyroA4; pRGA-MA1::AN3152 This study
TNJ174 pyrG89; AfupyrG+; pyroA4; veA1; pRGA-MA1::AN3152 This study
TNJ98c pyrG89; AfupyrG+; pyroA4:: niiA(p)::AN1652::FLAG::3/4pyroA+ This study
TNJ104c pyrG89; AfupyrG+; pyroA4; niiA(p)::AN2009::FLAG::3/4pyroA+ This study
TNJ122c pyrG89; AfupyrG+; pyroA4; niiA(p)::AN7507::FLAG::3/4pyroA+ This study
TNJ110c pyrG89; AfupyrG+; pyroA4; niiA(p)::AN3152::FLAG::3/4pyroA+ This study
TNJ116c pyrG89; AfupyrG+; pyroA4; niiA(p)::AN5833::FLAG::3/4pyroA+ This study
TNJ128c pyrG89; AfupyrG+; pyroA4; niiA(p)::AN9141::FLAG::3/4pyroA+ This study
TNJ79 pyrG89; ∆fluG::AfupyrG+; pyroA4 This study
TNJ133 pyrG89; ∆fluG::AfupyrG+; pyroA4; veA1 This study
TNJ70c pyrG89; pyroA4; alcA(p)::sfgA::FLAG::3/4pyroA+ Kwon et al. (2012)
TNJ208c pyrG89; pyroA4; alcA(p)::fluG::FLAG::3/4pyroA+ This study
TNJ96 pyrG89; pyroA4; ∆AN1652::AfupyrG+ This study
TNJ97 pyrG89; ∆fluG::pyroA+; pyroA4; ∆AN1652::AfupyrG+ This study
TNJ102 pyrG89; pyroA4; ∆AN2009::AfupyrG+ This study
TNJ103 pyrG89; ∆fluG::pyroA+; pyroA4; ∆AN2009::AfupyrG+ This study
TNJ108 pyrG89; pyroA4; ∆nsdD::AfupyrG+ This study
TNJ109 pyrG89; ∆fluG::pyroA+; pyroA4; ∆nsdD::AfupyrG+ This study
TNJ111 pyrG89; pyroA4; ∆nsdD::AfupyrG+; veA1 This study
TNJ112 pyrG89; ∆fluG::pyroA+; pyroA4; ∆nsdD::AfupyrG+; veA1 This study
TNJ114 pyrG89, ∆AN5833::AfupyrG+; pyroA4 This study
TNJ115 pyrG89, ∆AN5833::AfupyrG+; ∆fluG::pyroA+; pyroA4 This study
TNJ120 pyrG89; ∆AN7507::AfupyrG+, pyroA4 This study
TNJ121 pyrG89; ∆fluG::pyroA+; ∆AN7507::AfupyrG+, pyroA4 This study
TNJ126 pyrG89; pyroA4; ∆AN9141::AfupyrG+ This study
TNJ127 pyrG89; ∆fluG::pyroA+; pyroA4; ∆AN9141::AfupyrG+ This study
TNJ32 pyrG89; pyroA4; ∆flbE::AfupyrG+ Kwon et al. (2010b)
TNJ179 pyrG89; pyroA4; ∆nsdD::pyroA+; ∆flbE::AfupyrG+ This study
TNJ45 pyrG89; pyroA4, ∆flbB::AfupyrG+ This study
TNJ175 pyrG89; pyroA4, ∆flbB::AfupyrG+; ∆nsdD::pyroA+ This study
TNJ177 pyrG89; pyroA4; ∆flbD::AfupyrG+ This study
TNJ178 pyrG89; pyroA4; ∆nsdD::pyroA+; ∆flbD::AfupyrG+ This study
TNJ31 pyrG89; pyroA4; ∆flbC::AfupyrG+ Kwon et al. (2010a)
TNJ176 pyrG89; pyroA4; ∆nsdD::pyroA+; ∆flbC::AfupyrG+ This study
TNJ182 pyrG89, ∆flbA::AfupyrG+; pyroA4 This study
TNJ183 pyrG89, ∆flbA::pyroA+; pyroA4; ∆nsdD::AfupyrG+ This study
TNJ37 pyrG89; pyroA4; ∆abaA::AfupyrG+ Kwon et al. (2010a)
TNJ187 pyrG89; pyroA4; ∆nsdD::pyroA+; ∆abaA::AfupyrG+ This study
TNJ38 pyrG89; pyroA4; ∆brlA::AfupyrG+ Kwon et al. (2010a)
TNJ186 pyrG89; pyroA4; ∆nsdD::pyroA+; ∆brlA::AfupyrG+ This study
TNJ63 pyrG89; pyroA4; ∆rgsA::AfupyrG+ This study
TNJ185 pyrG89; pyroA4; ∆rgsA::pyroA+; ∆nsdD::AfupyrG+ This study
THS15.1 pyrG89; pyroA4; ∆vosA::AfupyrG+ Park et al. (2012)
TNJ181 pyrG89; pyroA4; ∆nsdD::pyroA+; ∆vosA::AfupyrG+ This study
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a

Fungal Genetics Stock Center (University of Missouri, Kansas City).

b

Strains contain the veA+ allele unless otherwise indicated as veA1.

c

The 3/4 pyroA marker in pHS11 and pHS3 causes the targeted integration at the pyroA4 locus.

Nucleic acid isolation and manipulation

The oligonucleotides used in this study are listed in supporting information, Table S1. Genomic DNA isolation was carried out as described in Yu et al. (2004). About 106 ml−1 conidia of WT and mutant strains were inoculated into 2 ml liquid MMG with 0.5% YE and stationary cultured at 37° for 1 day. The hyphal mat was collected and squeeze-dried and genomic DNA was isolated. Total RNA samples were prepared at various time points after liquid-submerged culture and postdevelopmental induction as described in Han et al. (2004; Mah and Yu 2006). For Northern blot analysis, ∼10 μg of total RNA was separated by electrophoresis, using a 1% agarose gel containing 6% formaldehyde, and ethidium bromide and the nucleic acids were transferred to the Hybond-N+ membrane (0.45 μm, GE Healthcare Life Sciences). The DNA probes were prepared by PCR amplification of the coding regions of individual genes with appropriate primer pairs (all primers listed in Table S1) from A. nidulans WT (FGSC4) genomic DNA. Individual PCR amplicons were labeled with 32P-dCTP and used for Northern blot hybridization as described in Yu and Leonard (1995).

Multicopy screening of pRG3-AMA1-based genomic DNA library

For multicopy screening, two types of sfgA deletion (∆) mutants were generated and used. Briefly, the 5′- and 3′-flanking regions of sfgA with the pyroA+ marker tails were amplified with the primer pairs of oNK397;oNK398 and oNK399;oNK400, and the A. nidulans pyroA+ gene was amplified with primer pair oNK395;oNK396 from A. nidulans WT (FGSC4) genomic DNA. The joined sfgA deletion construct was amplified with the nested primer pair oNK401;oNK402, and the final PCR amplicon was introduced into RJMP1.59 (pyrG89; pyroA4; veA+) and RNIW3 (pyrG89; pyroA4; veA1). The generated sfgA deletion strains TNJ30 (pyrG89; ∆sfgA::pyroA+; pyroA4; veA+) and TNJ134 (pyrG89; ∆sfgA::pyroA+; pyroA4; veA1) were then transformed with the pRG3-AMA1-NotI genomic DNA library with the Neurospora crassa pyr4+ marker (Osherov et al. 2000; Park and Yu 2012b). Sixty-one transformants showing fluffy or repressed conidiation were isolated. Genomic DNA was isolated from individual transformants and electroporated into E. coli DH5α to rescue the total 36 recombinant plasmids. Inserts of these plasmids were then directly sequenced with the primer pair oMN33 and oMN35, and the sequence data were matched with the A. nidulans genome [Broad Institute (Cambridge, MA) and AspGD]. Thirteen genes were defined by sequencing the inserts, and each recombinant plasmid with the insert was introduced back to the recipient A. nidulans ΔsfgA strains to verify their ability to inhibit conidiation when present in multicopy. Collectively, the six potential multicopy repressors of conidiation were identified.

Construction of overexpression strains

To generate the fusion constructs under the niiA promoter, i.e., niiA(p)::AN1652, niiA(p)::AN2009, niiA(p)::AN7507, niiA(p)::AN3152, niiA(p)::AN5833, and niiA(p)::AN9141, each ORF derived from A. nidulans WT genomic DNA was PCR amplified using the primer pairs oNK754; oNK755 (OE1652 with EcoRI and NotI), oNK872; oNK747 (OE2009 with BamHI and NotI), oNK867; oNK739 (OE7507 with HindΙΙΙ and NotI), oNK892; oNK893 (OE3152 with BamHI and HindIII), oNK890; oNK891 (OE5833 with EcoRI and HindIII), and oNK932; oNK925 (OE9141 with HindIII and NotI). The PCR products were then double digested with the enzymes shown above and cloned into pHS11, which contains the A. nidulans niiA promoter and the trpC terminator (Park et al. 2012). For the alcA(p)::fluG and alcA(p)::sfgA fusion constructs, each ORF derived from A. nidulans WT genomic DNA was amplified using the primer pairs oNK126; oNK127 (ORF of fluG with BamHI and NotI) and oNK47; oNK991 (ORF of sfgA with BamHI and NotI). The PCR product was then double digested with the enzymes shown above and cloned into pHS3, which contains the A. nidulans alcA promoter and the trpC terminator (Kwon et al. 2010a). The resulting recombinant DNA was then introduced into TNJ36.1. The overexpression strains among transformants were screened by Northern blot analysis, using each ORF probe (primer pairs to generate overexpression constructions), followed by PCR confirmation (Yu et al. 2004).

Generation of deletion mutants

The 5′- and 3′-flanking regions of each gene were amplified from genomic DNA of FGSC4, using designated primer pairs. For the deletion mutants of AN1652, AN2009, AN7507, nsdD (AN3152), AN5833, and AN9141, the flanking regions were amplified using primer pairs oNK748;oNK749 (5′ AN1652 with AfupyrG tail), oNK750;oNK751 (3′ AN1652 with AfupyrG tail), oNK740;oNK741 (5′ AN2009 with AfupyrG tail), oNK742;oNK743 (3′ AN2009 with AfupyrG tail), oNK732;oNK733 (5′ AN7507 with AfupyrG tail), oNK734;oNK735 (3′ AN7507 with AfupyrG tail), oNK12;oNK913 (5′ nsdD with AfupyrG tail), oNK914;oNK915 (3′ nsdD with AfupyrG tail), oNK906;oNK907 (5′ AN5833 with AfupyrG tail), oNK908;oNK909 (3′ AN5833 with AfupyrG tail), oNK896;oNK897 (5′ AN9141 with AfupyrG tail), and oNK898;oNK899 (3′ AN9141 with AfupyrG tail). The A. fumigatus pyrG gene was amplified from A. fumigatus WT (AF293) genomic DNA with the primer pair oJH84;oJH85. The final PCR fragments were amplified using the nested primer pairs oNK752;oNK753 (AN1652), oNK744;oNK745 (AN2009), oNK736;oNK737 (AN7507), oNK916;oNK917 (nsdD), oNK910;oNK911 (AN5833), and oNK900;oNK901 (AN9141). The deletion cassettes were introduced into RJMP1.59 protoplasts generated by VinoTaste Pro lysing enzyme (Novozymes) (Szewczyk et al. 2006; Park and Yu 2012b). The recipient deletion mutants were used to generate double-deletion mutants with ∆fluG with the pyroA+ allele by subsequent transformation. For the deletion mutants of fluG, sfgA, flbA, flbB, flbD, and rgsA with A. fumigatus pyrG+ as the marker, each flanking region was PCR amplified using primer pairs oNK788;oWS7 (5′ fluG region), oWS8;oNK791 (3′ fluG region), oNK397;oNK612 (5′ sfgA region), oNK613;oNK400 (3′ sfgA region), oNK142;oNK1032 (5′ flbA region), oNK1033;oNK145 (3′ flbA region), oNK522;oNK523 (5′ flbB region), oNK524;oNK525 (3′ flbB region), oNK1017;oNK1018 (5′ flbD region), oNK1019;oNK1020 (3′ flbD region), oNK540;oNK1030 (5′ rgsA region), and oNK1031;oNK543 (3′ rgsA region), using A. nidulans WT genomic DNA as a template. The final PCR constructs were amplified with the nested primer pairs oNK792;oNK793 (∆fluG), oNK401;oNK402 (∆sfgA), oNK146;oNK147 (∆flbA), oNK526;oNK527 (∆flbB), oNK1021;oNK1022 (∆flbD), and oNK544;oNK545 (∆rgsA). The deletion cassettes were introduced into RJMP1.59 protoplasts. The flbB, flbD, flbE (TNJ32), flbC (TNJ31), abaA (TNJ37), brlA (TNJ38), and vosA (THS15.1) deletion mutants were used to generate double-deletion mutants with ∆nsdD, using the pyroA+ marker, by subsequent transformation. To generate ∆flbA ∆nsdD and ∆rgsA ∆nsdD double-deletion mutants, the flbA or rgsA gene was deleted from TNJ108, using the pyroA+ marker (pyrG; pyroA4; ∆nsdD::AfupyrG+) strain.

Determination of alamarBlue reduction and dry weight

Fungal cell viability was determined by the percentage of reduction of alamarBlue (AB) (AbD Serotec). Conidia (106 ml−1) of each strain were inoculated into 100 ml MMG and cultured from 1 to 6 days at 37°, 240 rpm. Then, 0.5 ml of mycelial aggregates was aliquoted into 1 ml of fresh liquid medium containing 150 µl of the AB reagent. The samples were incubated for 6 hr at 37° in the dark as described in Shin et al. (2009). The supernatant was placed into the 96-well plates excluding mycelial aggregates for analysis of absorbance at A570 and A600. The percentage of AB reduction was detected by Synergy HT (BIO-TEK), using KC4 v3.1 software, and was calculated by a formula, (117,216 × A570 of sample – 80,586 × A600 of sample)/(155,677 × A600 of media – 14,652 × A570 of media) × 100 (Shin et al. 2009). After getting the sample for the AB reduction, the remaining cultures were collected by filtering to determine the mycelial mass. The samples were dried in a 75° oven for 4 hr and subjected to dry weight determination.

Sterigmatocystin extraction and TLC analysis

Briefly, 106 ml−1 conidia of each strain were inoculated into 2 ml liquid complete medium (CM) and cultured at 37° for 3–5 days as described (Yu and Leonard 1995). ST was extracted by adding 2 ml of CHCl3, and the organic phase was transferred to 1.5-ml microcentrifuge tubes and centrifuged at 700 × g for 5 min. The CHCl3 layer was collected, dried, and resuspended in 50 µl of CHCl3. Approximately 5 µl of each sample was loaded onto a thin-layer chromatography (TLC) silica plate including a fluorescence indicator (Kiesel gel 60, 0.25 mm thick; Merck). ST standard (5 µg; Sigma, St. Louis) was applied onto the TLC plate. The plate was then developed with toluene:ethyl acetate:acetic acid (80:10:10, v/v/v), where the Rf value of ST was ∼0.65. Aluminum chloride (20% w/v in 95% ethanol) was sprayed on the TLC plate to enhance the detection of ST and the plate was baked at 70° for 5 min (Stack and Rodricks 1971). The TLC plate was exposed to UV of 320 nm, and ST levels were measured. This experiment was performed in triplicate.

Light and fluorescence microscopy

The colony photographs were taken using a Sony (Parkridge, NJ) DSC-F828 camera. Photomicrographs were taken using a Zeiss (Thornwood, NY) M2Bio microscope equipped with AxioCam and AxioVision (Rel. 4.8) digital imaging software.

Results

Strategy and results of multicopy screening for negative regulators of conidiation

A ΔsfgA strain with the veA+ or veA1 allele was transformed with the pRG3-AMA1 (NotI)-based A. nidulans WT genomic DNA library (Osherov et al. 2000), and of >100,000 transformants, 61 candidate colonies that clearly exhibited reduced conidiation were isolated. All transformants were subjected to genomic DNA isolation followed by plasmid rescue through electroporation to E. coli. Of 61 candidates, genomic DNA samples of 31 transformants led to the successful rescue of plasmids in E. coli. Direct sequencing of each plasmid insert using the oMN33 and oMN35 primers (Park and Yu 2012b) followed by genome search identified 13 different genes, where 5 genes were identified ∼2–10 times, and 8 genes were present in one transformant each. Rescued plasmids with varying inserts were then reintroduced into the recipient strains and 6 clones/genes are confirmed to cause changes in conidiation (summarized in Figure 1B).

From the ΔsfgA veA+ screen AN1652, AN2009, AN7507, and AN3152 were present in six, three, two, and eight transformants, respectively. From the ΔsfgA veA1 screen AN3152, AN5833, and AN9141 were identified by two, three, and one transformants, respectively. Only AN3152 was identified in both screens, suggesting that the genetic screens did not reach a saturation, or visual screening based on the colony morphologies of transformants would have missed some of the candidates. The AN7507 locus encodes a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain protein. AN2009 encodes a homeodomain protein, and AN1652 encodes the C2H2-type zinc-finger protein MsnA (Han and Prade 2002). AN5833 encodes a protein with an acyl-CoA synthetase domain, and AN9141 encodes a protein with the GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain. Finally, the most represented AN3152 locus encodes the GATA-type TF (NsdD) that is known to activate sexual development (Han et al. 2001). In summary, five genes encode putative TFs, whereas one (AN5833) encodes a putative metabolic enzyme.

To verify the repressive role of these genes in conidiation, each gene region (5′-flanking-ORF-3′ flanking) was cloned into the pRG3-AMA1 multicopy vector and introduced into ΔsfgA veA+ or ΔsfgA veA1 strains. As shown in Figure 1C, multicopy (M) of AN1652 and AN9141 resulted in the fluffy phenotypes in the absence of sfgA, suggesting these putative TFs might be associated with stimulating hyphal growth while inhibiting development. Both vegetative growth and development of the fungus were restricted by M-AN2009, suggesting that proper expression of this homeodomain protein is important for the normal life cycle of A. nidulans. Multicopy of AN7507 and AN5833 caused reduced conidiation without distinctly enhanced vegetative growth. Finally, M-AN3152 (NsdD) resulted in the near absence of conidiation with the elevated accumulation of hyphal mass and enhanced formation of Hülle cells (specialized cells assisting sexual fruiting body formation), similar to those caused by enhanced expression of NsdD reported in in the previous study (Han et al. 2001).

Overexpression and expression analyses of the six candidate genes

To further check their potential roles in repressing conidiation, the ORF of each of six genes was cloned under the inducible niiA(p) and then OE strains of individual genes were constructed by transformation. As shown in Figure 2B, when induced (NO3 [I]), OE of AN1652 and AN3152 resulted in reduced conidiation and enhanced hyphal proliferation. Similar to the multicopy, OE of AN2009 caused restricted colony growth and inhibited conidiation. On the contrary, OE of AN7507, AN5833, and AN9141 did not affect conidiation or growth greatly. Somewhat surprisingly, OE9141 did not cause distinct changes in conidiation, which is very different from results for M-AN9141 (see Figure 1C), suggesting that the fluffy phenotype might be caused by the multicopy presence of the AN9141 cis-acting elements.

Figure 2.

Figure 2

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Overexpression and expression analyses of the six genes. (A) Phenotypes caused by OE of six genes. Colony photographs of WT (TNJ36.1), OE1652 (TNJ98), OE2009 (TNJ104), OE7507 (TNJ122), OE3152 (TNJ110), OE5833 (TNJ116), and OE9141 (TNJ128) strains are shown. Strains were point inoculated on noninducing medium (MMG containing 0.2% ammonium tartrate as a nitrogen source) or inducing medium (MMG containing 0.6% sodium nitrate as a nitrogen source) and incubated at 37° for 3 days. (B) Northern blots for AN1652, AN2009, AN7507, AN3152, AN5833, and AN9141 mRNA levels during the life cycle of A. nidulans WT. Conidia (C) and ascospores (AS) are indicated. The numbers indicate the time (hr) of incubation in liquid MMG (vegetative), postshift to solid MMG with air exposure (asexual), or limited air (sexual). Equal loading of total RNA was confirmed by ethidium bromide staining of rRNA. (C) Northern blots for AN1652, AN2009, AN7507, AN3152, AN5833, and AN9141 mRNA levels. WT (TNJ36.1), ∆fluG (TNJ79), and ∆sfgA (TNJ57) strains were grown in stationary culture in MMG with 0.5% YE. WT (TNJ36.1), OEfluG (TNJ208), and OEsfgA (TNJ70) under the alcA(p) were grown in stationary culture in inducing liquid medium (MM with 1.1% threonine plus 0.5% YE) (MMT) at 37° for 3 days. Equal loading of total RNA was confirmed by ethidium bromide staining of rRNA.

We further examined messenger (m)RNA accumulation patterns of the six genes throughout the life cycle of WT by Northern blot (Figure 2B). Both AN1652 and AN2009 appeared to be expressed at relatively constant levels, whereas AN7507 is expressed at a high level during sexual development (sexual 72–96 hr) and in sexual spores (ascospores, AS). AN3152 (nsdD) appeared to encode two transcripts where the lower one specifically accumulated in conidia, and the upper one accumulated at somewhat constant levels throughout the life cycle. Both AN5833 and AN9141 are expressed at low levels during vegetative growth and longer transcripts appeared to accumulate during development. These results suggest that mRNA accumulation of the six genes is not specifically coupled with brlA expression and/or conidiation.

Finally, we asked whether expression of any of the six genes is under the regulatory control of SfgA and/or FluG and checked mRNA accumulation patterns of each gene in vegetative cells of WT, ∆fluG, ∆sfgA, OEfluG, and OEsfgA strains (Figure 2C). We envisioned that if a given negative regulator is positively controlled by SfgA and repressed by FluG, ∆fluG and ∆sfgA would lead to enhanced and lowered expression of it, respectively. Conversely, OEfluG and OEsfgA would lead to inhibited and elevated mRNA levels of the tested gene. Quantitative analyses of each mRNA band calculated based on the loading control [ribosomal (r)RNA] indicate that mRNA levels the six genes were not distinctly elevated or diminished by the deletion of fluG or sfgA. Moreover, while mRNA levels of AN1652, AN7507, AN3152, and AN9141 appeared to be enhanced by OEsfgA, these were not affected by OEfluG. Interestingly, expression of AN5833 was greatly induced by threonine, but this was abolished by OEfluG. As vegetatively grown cells (liquid submerged culture) were used, mRNA accumulation of AN7507 was almost undetectable. These results indicate that none of the six genes are likely subject to direct regulatory control by SfgA and, if positioned in the FluG pathway, the repressor(s) does not likely act between SfgA and FLB genes.

Deletion analyses and suppression of ΔfluG by ΔnsdD

As we specifically looked for negative regulators of conidiation in the FluG-initiated developmental pathway, we then asked whether the deletion of each gene could suppress the conidiation defects caused by the lack of fluG. To do this, we first generated individual knockout mutants of AN7507, AN2009, AN1652, AN5833, AN9141, and AN3152, using A. fumigatus pyrG+. Then, to generate double mutants, individual deletion strains were used to further knock out the fluG gene, using A. nidulans pyroA+ as the marker (see Table 1). Sporulation and growth phenotypes of individual single- and double-deletion mutants were then compared. As shown in Figure 3A, as AN1652 (msnA) was speculated to be associated with vegetative growth, the deletion of msnA caused extremely restricted colony growth and could not suppress the conidiation defects caused by ΔfluG. The lack of AN2009 or AN5833 did not alter the ΔfluG phenotype either. Furthermore, the deletion of AN7507 or AN9141 even further enhanced the conidiation defects of the ΔfluG mutant. However, the deletion of AN3152 (NsdD) was able to restore conidiation in the ΔfluG mutant, and vegetative growth of the double mutant was similar to that of the ΔnsdD single mutant, suggesting that nsdD is epistatic to fluG. We also tested whether each double-deletion mutant regained the ability to produce ST and found that only ΔnsdD partially restored ST production in the ΔfluG mutant (data not shown, but see Figure 5A for ΔnsdD). In summary, the presence of nsdD in 10 independent multicopy transformants and the suppression of ΔfluG by ΔnsdD strongly suggest that NsdD is a key negative regulator of conidiation. These findings led us to focus on further characterizing NsdD’s role in conidiation.

Figure 3.

Figure 3

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Deletion analyses and the role of nsdD in the FluG pathway. (A) Phenotypes of double mutants. WT (TNJ36.1), ∆AN1652 (TNJ96), ∆fluG ∆AN1652 (TNJ97), ∆AN2009 (TNJ102), ∆fluG ∆AN2009 (TNJ103), ∆AN7507 (TNJ120), ∆fluG ∆AN7507 (TNJ121), ∆AN3152 (TNJ108), ∆fluG ∆AN3152 (TNJ109), ∆AN5833 (TNJ114), ∆fluG ∆AN5833 (TNJ115), ∆AN9141 (TNJ126), and ∆fluG ∆AN9141 (TNJ127) strains were point inoculated on MMG and incubated at 37° for 3 days. (B) Colonies of WT (veA+: TNJ36.1 and veA1: TNJ36.4), ∆sfgA (veA+: TNJ57 and veA1: TNJ135), M-nsdD (veA+: TNJ173 and veA1: TNJ174), and ∆sfgA M-nsdD (veA+: TNJ160 and veA1: TM3152) strains grown on solid MMG at 37° for 3 days. (C) Colony photographs of WT (veA+: TNJ36.1 and veA1: TNJ36.4), ∆fluG (veA+: TNJ79 and veA1: TNJ133), ∆nsdD (veA+: TNJ108 and veA1: TNJ111), and ∆fluG ∆nsdD (veA+: TNJ109 and veA1: TNJ112) strains grown on solid MMG at 37° for 3 days. (D) Quantitative analysis of conidiation of strains shown in C. Conidia in 1-cm2 area of the colonies were collected and counted (***P < 0.005). (E) Plates of WT (TNJ36.1), ∆fluG (TNJ79), ∆nsdD (TNJ108), and ∆fluG ∆nsdD (TNJ109) strains. Spores were spread on solid MMG and incubated at 37° for 3 days. (F) Northern blot for brlA, abaA, wetA, and vosA mRNA levels in WT (TNJ36.1) and ∆nsdD (TNJ108) strains during the life cycle. C, conidia. The numbers indicate the time (hr) of incubation in liquid MMG (vegetative) or on solid MMG under conditions inducing asexual development (postasexual induction). Transcript levels of γ-actin are shown as a control.

Figure 5.

Figure 5

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Multiple roles of nsdD. (A) ST levels (top panels) and Northern blots for aflR and stcU mRNA levels (bottom panels). WT (TNJ36.1), ∆fluG (TNJ79), ∆nsdD (TNJ108), ∆fluG ∆nsdD (TNJ109), ∆flbB (TNJ45), ∆nsdD ∆flbB (TNJ175), ∆flbA (TNJ182), ∆flbA ∆nsdD (TNJ183), ∆rgsA (TNJ63), and ∆nsdD ∆rgsA (TNJ185) strains were stationary cultured in liquid MMG at 37° for 3 days. ST standard was loaded as a positive control. For Northern blot analyses, WT (TNJ36.1) and ∆nsdD (TNJ108) strains were cultured throughout the life cycle. C, conidia. Numbers indicate time (hr) of liquid-submerged culture (vegetative) and developmental induction conditions (postasexual induction). Equal loading of total RNA was confirmed by ethidium bromide staining of rRNA. (B) Cell death of WT (TNJ36.1), ∆nsdD (TNJ108), ∆flbA (TNJ182), and ∆flbA ∆nsdD (TNJ183) strains determined by AB reduction rates for 6 days. (C) Dry weight of WT (TNJ36.1), ∆nsdD (TNJ108), ∆flbA (TNJ182) and ∆flbA ∆nsdD (TNJ183) strains through liquid-submerged culture in MMG were quantified for 6 days. (D) Colony diameter of WT (veA+: TNJ36.1 and veA1: TNJ36.4), ∆nsdD (veA+: TNJ108 and veA1: TNJ111), ∆fluG (veA+: TNJ79 and veA1: TNJ133), and ∆fluG ∆nsdD (veA+: TNJ109 and veA1: TNJ112) strains was measured on MMG grown at 37° for ∼1–4 days. Note veA+ (left) and veA1 alleles (right) in A. nidulans. Error bars indicate standard deviations calculated from biological triplicates. ***P < 0.005; **P < 0.05.

NsdD functions downstream of FluG and represses brlA

VeA is a founding member of the velvet regulators, and it plays a key role in activating sexual development, while repressing conidiation (Kim et al. 2002). The VeA1 mutant protein lacks the N-terminal Nuclear Localization Signal and the ability to interact with another sex-activating velvet regulator VelB, and veA1 mutant strains show highly restricted sexual fruiting with elevated conidiation (Mooney et al. 1990; Stinnett et al. 2007; Bayram et al. 2008). Thus, comparison of the phenotypes resulting from the multicopy or the deletion of NsdD in combination with veA+ and veA1 along with sfgA+ and ΔsfgA was carried out to better understand the role of NsdD in growth and developmental control. As shown in Figure 3B, M-nsdD was sufficient to inhibit conidiation with the veA+ or veA1 allele, yet the greatest reduction of conidiation was observable with the veA+ and sfgA+ alleles (M-nsdD in Figure 3B, left). These results suggest that repression by M-nsdD is maximized when other negative regulators of asexual development, e.g., VeA and SfgA, are functional.

We then checked whether suppression of ΔfluG by deletion of nsdD is affected by the veA allele and found that, when point inoculated, the ΔfluG ΔnsdD double-mutant colonies exhibited high levels of conidiation regardless of veA+ or veA1 (Figure 3C). We then quantified the levels of conidiation by measuring the number of conidia from the WT, ΔfluG, ΔnsdD, and ΔfluG ΔnsdD colonies (all with veA+; Figure 3D). The ΔnsdD mutant formed more conidia than WT with veA+ (P < 0.005). The ΔfluG ΔnsdD double mutant formed ∼10-fold more conidia than the ΔfluG mutant (P < 0.005), but not to WT levels. We then further quantified the levels of conidiation by spreading conidia (∼105 per plate) of WT, ΔfluG, ΔnsdD, and ΔfluG ΔnsdD strains onto solid medium and measuring the number of conidia produced upon 2 and 3 days of incubation (Figure 3E). The ΔnsdD mutant formed ∼1.7- to 1.5-fold more conidia than WT with veA+ and veA1 (P < 0.001). The ΔfluG ΔnsdD double mutant formed ∼2- to 3-fold more conidia than the ΔfluG mutant under these experimental conditions (P < 0.001). However, ΔfluG ΔnsdD strains could not produce conidia to WT levels. These results indicate that NsdD plays an important role in inhibiting conidiation downstream of FluG, yet the removal of nsdD alone is not sufficient to cause full activation of conidiation.

We then checked whether the absence of NsdD altered the patterns of brlA, abaA, wetA, and vosA mRNA accumulation during vegetative growth and asexual development. As shown in Figure 3F, the deletion of nsdD caused accumulation of brlA mRNA at 24, 36, and 48 hr in submerged shake cultures (vegetative), whereas no brlA accumulation was observable in WT vegetative cells. Overall mRNA levels of brlA, abaA, and wetA in the ΔnsdD mutant during asexual developmental induction were much higher than those of WT. Moreover, elevated brlA mRNA accumulation in the ΔnsdD mutant at 10, 12, and 18 hr postdevelopmental induction led to precocious and enhanced accumulation of abaA and wetA mRNAs compared to WT. Levels of vosA mRNA were not much different between the ΔnsdD mutant and WT.

Genetic position of NsdD in the FluG-mediated conidiation pathway

The above genetic and expression data suggest that NsdD functions downstream of fluG but upstream of brlA. This is consistent with the finding that expression of nsdD is not directly regulated by SfgA or FluG, and NsdD does not likely act between SfgA and FLBs. To determine the genetic position of nsdD in the FluG-initiated conidiation control cascade, a series of double mutants were generated. As shown in Figure 4, the deletion of nsdD could restore conidiation in the null mutants of flbE, flbB, flbD, and flbC. In contrast, ΔnsdD could not suppress ΔbrlA or ΔabaA. These results indicate that NsdD functions downstream of FlbE/B/D/C and upstream of brlA. Moreover, nullifying nsdD in the ΔflbA mutant partially rescued the conidiation defects and suppressed autolysis caused by ΔflbA, suggesting that NsdD might also be associated with vegetative growth signaling mediated by FadA (Gα) → PkaA (Shimizu and Keller 2001). In the ΔrgsA mutant, the deletion of nsdD restored conidiation and enhanced growth restriction, suggesting that NsdD and RgsA play an additive role in colony growth.

Figure 4.

Figure 4

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Genetic position of nsdD. Phenotypes of various single- and double-deletion mutants are shown. WT (TNJ36.1), ∆nsdD (TNJ108), ∆flbE (TNJ32), ∆nsdD ∆flbE (TNJ179), ∆flbB (TNJ45), ∆nsdD ∆flbB (TNJ175), ∆flbD (TNJ177), ∆nsdD ∆flbD (TNJ178), ∆flbC (TNJ31), ∆nsdD ∆flbC (TNJ176), ∆flbA (TNJ182), ∆flbA ∆nsdD (TNJ183), ∆brlA (TNJ38), ∆nsdD ∆brlA(TNJ186), ∆abaA (TNJ37), ∆nsdD ∆abaA (TNJ187), ∆rgsA (TNJ63), and ∆nsdD ∆rgsA (TNJ185) strains were point inoculated on solid MMG and incubated at 37° for 3 days. Entire colonies and close-up views of the center of individual colonies are shown. Bar, 100 µm.

Roles of NsdD in ST production, autolysis, and colony growth

Our previous studies demonstrated that the absence of fluG or flbA resulted in the lack of ST production and that biosynthesis of ST required the removal of repressive effects imposed by the heterotrimeric G protein composed of FadA and SfaD::GpgA (Hicks et al. 1997). We also showed that the ΔfluG ΔsfgA mutant regained the ability to produce ST and restored the expression of ST-specific genes to WT levels (Seo et al. 2006). We envisioned that if NsdD functions downstream of most of the developmental activators, the deletion of nsdD might also restore ST biosynthesis in the defective mutants. As shown in Figure 5A, the removal of nsdD partially restored ST production in the ΔfluG, ΔflbB, ΔflbA, and ΔrgsA mutants. However, mRNA accumulation of aflR (encoding an activating TF) and stcU was much reduced in the ΔnsdD mutant compared to WT. These results indicate that NsdD negatively affects ST production acting downstream of FluG, FlbB, FlbA, and RgsA, likely at a post-transcriptional level.

The deletion of flbA causes accelerated and enhanced cell death and autolysis (Lee and Adams 1994b; Shin et al. 2009). As ΔnsdD partially restored conidiation and suppressed autolysis of the ΔflbA mutant in solid culture, we further quantified its effects on suppressing cell death and hyphal disintegration in the ΔflbA mutant in liquid culture. As shown in Figure 5, B and C, WT and ΔnsdD strains maintain cell viability and hyphal integrity at approximately days 3–6, whereas the ΔflbA mutant began to show cell death at day 3 and autolysis at day 4. The deletion of nsdD suppressed both cell death and autolysis caused by ΔflbA. When WT and ΔnsdD strains were compared at approximately days 1–3, the ΔnsdD mutant exhibited reduced AB reduction rates and delayed vegetative proliferation measured by dry weight in liquid submerged culture (P < 0.005; Figure 5, B and C).

We also observed that the deletion of nsdD was epistatic to other developmental mutations in colony radial growth, resulting in restricted colony growth of double mutants similar to that of the single nsdD null mutant (see Figure 4). To quantify the radial growth progression, WT, ΔnsdD, ΔfluG, and ΔfluG ΔnsdD strains were point inoculated and their radial growth was measured at approximately days 1–4. As shown in Figure, 5, D and E, ΔnsdD, and ΔfluG ΔnsdD strains showed a statically significant growth reduction (P < 0.005) at days 3 and 4 in both the veA+ and the veA1 genetic background. These results collectively suggest that NsdD might function downstream of the FadA-PkaA-controlled vegetative growth regulatory network, which is attenuated by FlbA (Shimizu and Keller 2001).

Additive role of NsdD and VosA in repressing conidiation

The above results indicate that NsdD plays its repressive role likely by repressing expression of brlA. We previously showed that VosA is a feedback negative regulator of brlA, and its velvet domain directly binds to the brlAβ promoter (Ahmed et al. 2013). The deletion of either vosA or nsdD caused elevated expression of brlA and formation of conidiophores in liquid submerged culture, where WT strains do not develop (Ni and Yu 2007) (Figure 6B). Based on these observations, we hypothesized that the deletion of two key repressors would have additive effects on expression of brlA, leading to even more enhanced hyperactive conidiation. To test this, we generated the ΔnsdD ΔvosA double mutant in veA+ and examined its developmental phenotypes. The double mutant showed both ΔnsdD and ΔvosA phenotypes, i.e., restricted colony growth and light-green conidia with rapid loss of viability on solid medium (Figure 6A). In liquid submerged culture, while both ΔnsdD and ΔnsdD ΔvosA mutant strains produced conidiophores, the double mutant elaborated conidiophores much more abundantly than the ΔnsdD single mutant (Figure 6B). Moreover, the double mutant exhibited extremely high levels of accumulation of brlA mRNA even at 16 hr of vegetative growth in liquid submerged culture (Figure 6C). These results indicate NsdD and VosA play a negative regulatory role on brlA expression in an additive manner, and both are required for proper control of brlA expression and conidiation during vegetative growth of A. nidulans.

Figure 6.

Figure 6

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An additive role of nsdD and vosA in repressing conidiation. (A) Phenotypes of WT (TNJ36.1), ∆nsdD (TNJ108), ∆vosA (THS15.1), and ∆nsdD ∆vosA (TNJ181). Strains were point inoculated on solid MMG and incubated at 37° for 3 days. Entire colonies (top) and close-up views of the middle of individual colonies (bottom) are shown. Bar, 100 µm. (B) Conidiophore formation in liquid submerged culture. Strains were grown in liquid-submerged MMG culture at 37° for 48 hr (vegetative), and the mycelial aggregates were observed under a microscope. Bar, 50 µm. (C) Northern blot for brlA mRNA levels in strains grown in liquid-submerged MMG culture (vegetative) at 37° for 16 hr. Equal loading of total RNA was confirmed by ethidium bromide staining of rRNA.

Discussion

In this study, to further investigate the regulatory mechanisms of asexual sporulation in Aspergillus, we designed and carried out a unique high-copy repressor screen employing the sfgA deletion mutant strains. Among the six genes verified to inhibit conidiation when present in multiple copies and/or OE, only NsdD, previously identified as an activator of sexual development, has been shown to function in the FluG-mediated conidiation regulatory pathway. Analyses of nsdD function and genetic position reveal that NsdD negatively controls expression of brlA, conidiophore development, and ST production.

The homothallic ascomycete A. nidulans can also reproduce sexually by forming sexual spores (ascospores) in the fruiting bodies called cleistothecia. Sexual development is a complex multistep process that requires special environmental conditions and activities of various regulators, including the mating-type genes matA (HMG-box) and matB (α-box) that transcriptionally coordinate expression of sex-specific genes (Kronstad and Staben 1997; Fraser and Heitman 2005). Sexual fruiting begins with the formation of primordia from ascogenous hyphae in the nest-like structure made of a number of specialized Hülle cells that support fruiting body formation. The primordia mature to cleistothecia in which many mycelia grow and develop into croziers, where a transient nuclear fusion and subsequent meiosis occur (Sohn and Yoon 2002; Han et al. 2007), leading to the formation of macroscopic fruiting bodies (cleistothecia) containing a number of ascospores in asci. The nsdD gene was identified as one of the never in sexual development (NSD) loci (nsdA–nsdD) (Han et al. 1990). Later studies also identified the nsdC gene encoding a putative TF with a novel zinc-finger DNA-binding domain consisting of two C2H2 and one C2HC motifs (Kim et al. 2009). Loss-of-function nsdD or nsdC results in the complete blockage in sexual development under all conditions favoring sexual development. Conversely, overexpression of nsdD or nsdC not only enhances the formation of fruiting bodies but also partially overcomes the inhibitory effects of certain stresses on sexual fruiting (Han et al. 2001; Kim et al. 2009). These results lead to the conclusion that NsdD and NsdC play an essential role in sexual development of A. nidulans.

However, previous studies also have suggested that NsdD might be a potential repressor of conidiation (Han et al. 2001; Cary et al. 2012). For instance, overexpression of nsdD resulted in the near total absence of conidiation with formation of elongated aerial hyphae. Moreover, the conidial yield was reduced by ∼1000-fold by overexpression of nsdD. Furthermore, forced expression of nsdD inhibited conidiation and caused a coiled-hyphal structure in A. fumigatus (Grosse and Krappmann 2008). Importantly, most NSD mutants developed conidiophores earlier than WT by several hours, regardless of environmental conditions (Han et al. 1998). For instance, when WT was cultured on solid medium with limited air and in the dark for >30 hr, the mycelia did not produce any differentiated cells, but were irreversibly determined to undergo sexual development with the formation of a few asexual spores after air was introduced (Han et al. 1990). The NSD mutants, however, began to produce conidiophores instantaneously after air exposure, indicating that the NSD mutants have not been genetically determined to develop sexually. However, as enhanced expression of nsdD also resulted in elevated sexual development even under unfavorable conditions for sexual fruiting, it has been hypothesized that NsdD primarily functions in positively regulating sexual development rather than repressing conidiation. Additional studies in A. flavus demonstrated that the removal of nsdD resulted in elevated expression of brlA (Cary et al. 2012). They found that both NsdC [another sexual activator (Kim et al. 2009)] and NsdD are required for production of asexual sclerotia, normal aflatoxin biosynthesis, and conidiophore development. Along with these important observations, our forward genetic screen clearly supports the idea that NsdD plays an equally important role in repressing conidiation by acting at the brlA level.

The deletion of nsdD suppressed all upstream developmental mutations, but not ΔbrlA. A previous study identified and characterized two suppressors of FlbD (sfdA and sfdB) (Kellner and Adams 2002) that phenocopy nsdD. These sfd mutant alleles restored developmental timing and brlA expression to strains with flbD deletion. Importantly, sfd mutations suppressed the developmental defects of the fluG, flbA, flbB, flbC, and flbE null mutants. All isolated alleles of sfdA and sfdB were recessive to their WT alleles in diploids, suggesting that the mutations were likely loss-of-function ones. Moreover, sfd mutant strains with WT upstream activators exhibited normal conidiation with restricted colony growth. Additionally, they developed conidiophores in liquid submerged culture. These data indicated that sfdA and sfdB activities are required for proper control of conidiation downstream of FLBs and that the absence of sfdA or sfdB can cause hyperactive conidiation. While no sfd mutant alleles were cloned and identified, these phenotypic and genetic characteristics suggested that one might be an allele of nsdD.

NsdD appears to affect other biological processes including ST production, cell death, autolysis, and colony growth. The deletion of nsdD suppressed the enhanced cell death/autolysis of the ΔflbA mutant. Moreover, the absence of nsdD could restore ST production in fluG, flbA, and flbB null mutants, not through enhanced transcription of the ST genes. These observations led us to position NsdD in a multiple-control point that activates sexual development and stimulates vegetative growth, but inhibits conidiation and ST production (Figure 7). Previously, we demonstrated that two antagonistic signaling pathways control A. nidulans growth, conidiation, and ST production. Growth signaling is primarily mediated by FadA and SfaD::GpgA (heterodimer), the α and βγ subunits for a heterotrimeric G protein, respectively. When FadA (Gα) is active (GTP bound), FadA and SfaD:GpgA signal to enhance vegetative growth and repress both asexual sporulation and ST production (Yu et al. 1996; Hicks et al. 1997; Adams et al. 1998; Rosen et al. 1999; Seo et al. 2005). This FadA-dependent growth-signaling pathway is in part transduced through PkaA [a catalytic subunit of cAMP-dependent protein kinase A (Shimizu and Keller 2001)]. FlbA is an RGS domain protein that negatively regulates FadA-mediated growth signaling (Berman et al. 1996; Koelle and Horvitz 1996; Yu et al. 1996). Integrating the previous and present findings, we propose to place NsdD under the control of FadA-PkaA, downstream of FLBs, and upstream of BrlA (Figure 7; see below).

Figure 7.

Figure 7

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Genetic model for growth and developmental control in A. nidulans.

Our recent studies demonstrated that VosA and VelB control expression of various genes in conidia via directly binding to the promoters of their target genes (Ahmed et al. 2013). The removal of vosA causes abnormal accumulation of brlA in conidia and vegetative cells (Ni and Yu 2007; Ahmed et al. 2013). The nsdD gene was one of the target genes that are directly controlled by the VosA-VelB heterodimer in conidia, and the lower transcript of nsdD was not detectable in the ΔvosA or ΔvelB mutant conidia (Ahmed et al. 2013). These results suggest that the VosA-VelB heterodimer activates expression of NsdD in conidia, which confers proper downregulation of brlA during the initial period of vegetative growth. It has been shown that conidiation does not typically occur in A. nidulans until cells have gone through a defined period (∼18 hr) of vegetative growth (Axelrod et al. 1973; Champe et al. 1981); i.e., A. nidulans cells require ∼18 hr of growth before they are competent to conidiate. It is important to note that the deletion of both nsdD and vosA might have reduced this developmental competence period to 16 hr or earlier (Figure 6, B and C). These results imply that developmental competence might be determined by the time and physiological status required to remove the repressive effects imposed by multiple negative regulators, e.g., SfgA, NsdD, and VosA.

Taken together, we propose a working model depicting regulation of conidiation in A. nidulans (Figure 7). In this model, NsdD and VosA are expressed in conidia, preoccupying the brlA promoter in spores, which allows proper vegetative growth to occur after germination of conidia. Upon a certain period of growth (∼18 hr), the FluG factor accumulates in the cells, which then removes the upstream negative controller SfgA (Seo et al. 2006). Such derepression triggers the transition from vegetative growth to development, involving the sequential activation of FlbE-B→D and FlbC. Activated FlbB–FlbD (Garzia et al. 2010) and FlbC then displace NsdD from the brlA promoter and further activates brlA. VosA is likely removed from the brlA promoter by VelB (Park et al. 2012, not indicated in the model). Furthermore, FLBs and VelB activate brlA expression, which collectively leads to (full) activation of conidiation-specific genes and the development of conidiophores. Expression of AbaA and WetA also leads to high-level accumulation of vosA in phialides and conidia, which in turn shuts off expression of brlA at the later phase of conidiation and turns on expression of trehalose biogenesis genes and nsdD in conidia. Stimulation of vegetative proliferation and inhibition of ST biosynthesis mediated by FadA→PkaA might occur in part through NsdD, possibly involving phosphorylation of NsdD by PkaA (indicated by a dashed arrow with a ? in Figure 7). There are six predicted PKA-mediated serine phosphorylation sites in NsdD (http://kinasephos2.mbc.nctu.edu.tw/).

The nsdD gene is predicted to encode a 461-aa polypeptide (49,250 kDa), rich in proline (11.3%) and serine (13.4%), which contains the conserved amino acid sequence of type IVb C-X2-C-X18-C-X2-C zinc-finger domain typically found in the GATA-type TFs (Teakle and Gilmartin 1998; Han et al. 2001). The NsdD polypeptide(s) is highly conserved in most Aspergillus species and many other important fungi including Penicillium, Coccidioides, Ajellomyces, Fusarium, and more. GATA TFs (GATA-1 to -6) bind to a DNA sequence called a GATA motif [(A/T)GATA(A/G)] in the regulatory regions of their target genes through two zinc-finger domains (Urnov 2002), and many can bind to DNA as homodimers or heterodimers. Typically, homodimers recognize inverted repeats within the target nucleotide sequence, whereas heterodimers bind to direct repeats (Laity et al. 2001; MacPherson et al. 2006). There are multiple (at least four) potential GATA TF binding sites in the promoter region (−1000 nt) of brlA. Identification and verification of NsdD binding sites in the brlA promoter and its target genes in the genome and investigation of NsdD interacting partners are in progress to further comprehend the molecular mechanisms of NsdD-mediated developmental regulation in A. nidulans.

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Acknowledgments

The authors thank Dr. Ellin Doyle for critically reviewing this manuscript and Dr. Hee-Soo Park for his technical assistance. This work was primarily supported by a National Research Foundation of Korea grant funded by the Korean Government (NRF-2011-619-E0002) for the Konkuk University–University of Wisconsin collaborative research and by the National Science Foundation (IOS-0950850) (to J.-H.Y.).

Footnotes

Communicating editor: J. Heitman

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