<i>mtfA</i> is necessary for normal expression of terrequinone genes.

<p>A) Wild type (WT) <i>veA</i>+ control (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mftA</i>) and Δ<i>mtfA</i>-com complementation strain (TRVΔ<i>mtfA</i>-com) were inoculated in liquid GMM. Mycelia were collected at 48 h and 72 h after inoculation for RNA extraction. Cultures were grown in a shaker incubator at 37°C at 250 rpm. Expression of <i>tdiA</i> and <i>tdiB</i> was analyzed by Northern blot. 18S rRNA serves as loading control. B) Isogenic wild type (WT) <i>veA</i>+ control (TRV50.1) and over-expression (OE) <i>mtfA</i> strain (TRV60) were inoculated in liquid GMM and grown for 16 h. After that, equal amounts of mycelium were transferred to TMM and further incubated for 24 h and 48 h. <i>tdiA</i> and <i>tdiB</i> expression was analyzed as in (B). Densitometries were carried out with the Scion Image Beta 4.03 software. Asterisk indicates not detected.</p

RM7 mutant presents a single gene mutation at locus AN8741.2.

<p>A) Diagram showing the genomic insert present in the complementation vector from library pRG3-AMA1-NOT1. The insert contains two ORFs corresponding to AN8741.2 and adjacent AN8740.2. The coding region at AN8741.2 locus encodes a putative C₂H₂ zinc finger domain transcription factor. The revertant mutation in RM7 occurred at AN8741.2, designated as <i>mtfA</i> gene. B) Amino acid alignment of <i>A. nidulans</i> MtfA (Ani) and putative orthologs in <i>A. terreus</i> (Ate), <i>A. flavus</i> (Afl), <i>A. clavatus</i> (Acl) and <i>A. fumigatus</i> (Afu). ClustalW (<a href="http://www.ebi.ac.uk/Tools/clustalw2/index.htm" target="_blank">http://www.ebi.ac.uk/Tools/clustalw2/index.htm</a>) land boxshade (<a href="http://www.ch.embnet.org/software/BOX_form.html" target="_blank">http://www.ch.embnet.org/software/BOX_form.html</a>) multiple sequence alignment software programs were utilized in this analysis. The mutation occurred at the codon corresponding to the first methionine. The two conserved zinc finger domains are indicated in A) and B).</p

Effects of <i>mtfA</i> deletion on ST production in <i>A</i>. <i>nidulans</i> strains with a <i>veA+</i> allele.

<p>A) TLC analysis showing ST production in GMM cultures. Wild type (WT) <i>veA</i>+ control (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mtfA</i>) and Δ<i>mtfA</i>-com complementation strain (TRVΔ<i>mtfA</i>-com) were spread-inoculated with 5 mL of top agar containing 10⁶ conidia mL⁻¹ and incubated at 37°C in the dark or in the light for 48 h and 72 h. ST was extracted and analyzed by TLC as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074122#s2" target="_blank">Materials and Methods</a> section. White arrows indicate unknown compounds whose synthesis is also affected by the presence or absence of <i>mtfA.</i> B) Effect of the <i>mftA</i> deletion on <i>aflR</i> and <i>stcU</i> expression. Wild type (WT) <i>veA</i>+ control (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mftA</i>) and Δ<i>mtfA</i>-com complementation strain (TRVΔ<i>mtfA</i>-com) were inoculated in liquid GMM. Mycelia were collected 24 h and 48 h after inoculation. Cultures were grown in a shaker incubator at 37°C at 250 rpm. Expression of <i>aflR</i> and <i>stcU</i> was analyzed by Northern blot. 18S rRNA serves as loading control. Asterisk indicates not detected. C) TLC showing accumulation of ST in the cultures described in (B). Densitometries were carried out with the Scion Image Beta 4.03 software.</p

Fungal strains used in the study.

<p>FGSC, Fungal Genetics Stock Center.</p

The Putative C₂H₂ Transcription Factor MtfA Is a Novel Regulator of Secondary Metabolism and Morphogenesis in <i>Aspergillus nidulans</i>

<div><p>Secondary metabolism in the model fungus <i>Aspergillus nidulans</i> is controlled by the conserved global regulator VeA, which also governs morphological differentiation. Among the secondary metabolites regulated by VeA is the mycotoxin sterigmatocystin (ST). The presence of VeA is necessary for the biosynthesis of this carcinogenic compound. We identified a revertant mutant able to synthesize ST intermediates in the absence of VeA. The point mutation occurred at the coding region of a gene encoding a novel putative C₂H₂ zinc finger domain transcription factor that we denominated <i>mtfA</i>. The <i>A. nidulans mtfA</i> gene product localizes at nuclei independently of the illumination regime. Deletion of the <i>mtfA</i> gene restores mycotoxin biosynthesis in the absence of <i>veA,</i> but drastically reduced mycotoxin production when <i>mtfA</i> gene expression was altered, by deletion or overexpression, in <i>A. nidulans</i> strains with a <i>veA</i> wild-type allele. Our study revealed that <i>mtfA</i> regulates ST production by affecting the expression of the specific ST gene cluster activator <i>aflR</i>. Importantly, <i>mtfA</i> is also a regulator of other secondary metabolism gene clusters, such as genes responsible for the synthesis of terrequinone and penicillin. As in the case of ST, deletion or overexpression of <i>mtfA</i> was also detrimental for the expression of terrequinone genes. Deletion of <i>mtfA</i> also decreased the expression of the genes in the penicillin gene cluster, reducing penicillin production. However, in this case, over-expression of <i>mtfA</i> enhanced the transcription of penicillin genes, increasing penicillin production more than 5 fold with respect to the control. Importantly, in addition to its effect on secondary metabolism, <i>mtfA</i> also affects asexual and sexual development in <i>A. nidulans.</i> Deletion of <i>mtfA</i> results in a reduction of conidiation and sexual stage. We found <i>mtfA</i> putative orthologs conserved in other fungal species.</p></div

Deletion of <i>mtfA</i> affects fungal growth and colony pigmentation.

<p>A) Wild type (WT) <i>veA+</i> (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mtfA</i>) and Δ<i>mtfA</i>-com complementation (TRVΔ<i>mtfA</i>-com) were point inoculated on GMM plates and incubated at 37°C in either dark or light for 6 days. B) Fungal growth was measured as colony diameter. Values are means of four replicates. Standard error is shown.</p

Over-expression of <i>mtfA</i> suppresses <i>aflR</i> and <i>stcU</i> expression and ST production.

<p>A) Northern blot analysis of <i>aflR</i> and <i>stcU</i> expression. Wild-type isogenic control (WT) <i>veA</i>+ (TRV50.1) and over-expression (OE) <i>mtfA</i> strain (TRV60) were inoculated in GMM liquid medium (10⁶ conidia mL⁻¹) and grown for 16 hrs in a shaker incubator at 37°C and 250 rpm. Then, equal amounts of mycelium were transferred and spread onto TMM agar medium. The cultures were further grown for 48 h and 72 h. Mycelial samples were collected at 0 hr (shift time), and 24 and 48 hrs of incubation after shift onto TMM. 18S rRNA serves as loading control. B) qRT-PCR expression analysis of <i>mtfA</i> from mycelial samples collected after 24 h and 48 h of incubation after transfer onto TMM agar medium. C) TLC analysis of ST production from cultures described in (A-B).</p

Revertant mutant 7 (RM7) produces NOR.

<p>TLC analysis of NOR production. Fungal strains were top-agar inoculated on OMM and incubated for six days. Then, mycelial cores were collected and NOR was extracted and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074122#s2" target="_blank">Materials and Methods</a> section. Δ<i>stcE</i> Δ<i>veA</i> (RDAEp206), Δ<i>stcE veA1</i> (RAV1p), Δ<i>stcE</i> Δ<i>veA mtfA-</i> (RM7p), Δ<i>stcE veA1 mtfA</i>- (RM7p-R2), Δ<i>stcE veA1 mtfA</i>- <i>mtfA</i>+ (RM7p-R2-com), Δ<i>stcE</i> Δ<i>veA</i> Δ<i>mtfA</i> (TDAEpΔ<i>mtfA</i>). On the right, densitometry carried out with the Scion Image Beta 4.03 software.</p

Deletion of <i>mtfA</i> results in a reduction of penicillin biosynthesis.

<p>A) Extracts from wild-type (WT) <i>veA</i>+ control (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mftA</i>) and Δ<i>mtfA</i>-com complementation strain (TRVΔ<i>mtfA</i>-com) were analyzed for penicillin content as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074122#s2" target="_blank">Materials and Methods</a> section. B) qRT-PCR expression analysis of <i>acvA</i> from mycelial samples collected after 24 h and 48 h of incubation in PN inducing medium. C) Northern blot analysis of <i>ipnA</i> and <i>aatA</i> from samples collected after 24 h and 48 h of incubation in PN inducing medium. Densitometries were carried out with the Scion Image Beta 4.03 software.</p

MftA localizes in nuclei.

<p>A) Diagram of the strategy utilized to fuse GFP to MtfA. The tagged construct was introduced at the <i>mtfA</i> locus by a double-over event. B) Micrographs showing the subcellular localization of the MtfA::GFP in <i>A. nidulans</i> growing in the light or in the dark. Scale bar represents 10 micrometers.</p