Velvet activated McrA plays a key role in cellular and metabolic development in Aspergillus nidulans

McrA is a key transcription factor that functions as a global repressor of fungal secondary metabolism in Aspergillus species. Here, we report that mcrA is one of the VosA-VelB target genes and McrA governs the cellular and metabolic development in Aspergillus nidulans. The deletion of mcrA resulted in a reduced number of conidia and decreased mRNA levels of brlA, the key asexual developmental activator. In addition, the absence of mcrA led to a loss of long-term viability of asexual spores (conidia), which is likely associated with the lack of conidial trehalose and increased β-(1,3)-glucan levels in conidia. In supporting its repressive role, the mcrA deletion mutant conidia contain more amounts of sterigmatocystin and an unknown metabolite than the wild type conidia. While overexpression of mcrA caused the fluffy-autolytic phenotype coupled with accelerated cell death, deletion of mcrA did not fully suppress the developmental defects caused by the lack of the regulator of G-protein signaling protein FlbA. On the contrary to the cellular development, sterigmatocystin production was restored in the ΔflbA ΔmcrA double mutant, and overexpression of mcrA completely blocked the production of sterigmatocystin. Overall, McrA plays a multiple role in governing growth, development, spore viability, and secondary metabolism in A. nidulans.


Results expression and the role of McrA in asexual development of A. nidulans. Previous study described
that mRNA levels of mcrA in conidia were drastically low by the lack of VelB or VosA, and a promoter region of mcrA contains a putative VosA response element (VRE), suggesting that McrA is a potential target of the VosA-VelB complex in A. nidulans 30 . The mcrA ORF composed of 1,453 bp with four exons predicted to encode a 399 aa-length protein that contains a GAL4-like Zn(II) 2 Cys 6 domain at the C-terminus. To begin to investigate its function, the levels of mcrA mRNA in the life cycle were investigated. As shown in Figs. 1A and S1A, mcrA mRNA was detectable throughout the life cycle and was high at 48 h after asexual developmental induction.
To investigate the roles of mcrA, we generated the mcrA deletion (ΔmcrA) mutant and the ΔmcrA complemented (C'mcrA) strains, which reintroduced a wild-type copy of the mcrA gene back into the other locus. Wild type (WT), ΔmcrA, and complemented strains were point-inoculated onto a minimal medium (MM) with 1% glucose (MMG) agar and their phenotype was checked. In comparison to WT and complemented strains, the ΔmcrA mutant strain produces abnormal conidiophores and brown colony (Fig. 1B). The deletion of mcrA resulted in a reduced number of conidia and reduced levels of brlA compared to WT and complemented strains (Figs. 1C,D and S1B). These results suggest that McrA plays a key role in asexual development in A. nidulans.
McrA is required for conidial integrity. As the expression of mcrA was activated by VosA-VelB, which controls the conidial viability and integrity, we hypothesized that McrA may play a role in spore survival. To test this hypothesis, WT, ΔmcrA, and C' strain conidia were collected from 2, 5, 8, 10 and 20 days grown colonies and checked for the viability. As shown in Fig. 2A, the ΔmcrA mutant conidia rapidly lost viability starting from day 5. As trehalose is a key component conferring the long-term spore viability, conidial trehalose amount was tested in the ΔmcrA mutant conidia. The amount of trehalose in ΔmcrA conidia was about twofold less than that of WT or C' strains ( Fig. 2B). Since the absence of vosA increased the levels of β-(1,3)-glucan in conidia, we investigated the β-(1,3)-glucan levels in the ΔmcrA mutant conidia, and found that β-(1,3)-glucan levels in the ΔmcrA conidia were about twofold higher than those of WT and C' conidia ( Fig. 2C). This was corroborated by the finding that ΔmcrA mutant conidia exhibited elevated mRNA levels of fksA, a gene encoding a β-1,3-glucan synthase (Fig. 2D). These results indicate that VosA-VelB-activated McrA is necessary for the governing the integrity of conidia. the absence of mcrA leads to elevated secondary metabolism. Previously, Oakley and colleagues have shown that the McrA is a negative regulator of the production of secondary metabolites in A. nidulans 34 . We confirmed that the absence of mcrA alters the patterns of secondary metabolites and increases ST production in stationary cultures (Fig. 3A,B). We then tested the secondary metabolite patterns in conidia using the high-performance liquid chromatography (HPLC) and found that the ΔmcrA conidia showed about fourfold enhanced production of ST and an unknown metabolite compared to those of WT and complemented strains ( Fig. 3C-E). We then tested mRNA levels of aflR encoding an essential Zn(II) 2 Cys 6 TF for the activation of the ST gene cluster and found that the ΔmcrA conidia exhibited higher levels of aflR mRNA than those of WT and C' conidia ( Fig. 3F). Overall, these results imply that McrA is a key negative regulator of secondary metabolite production in both hyphae and conidia. However, when cultured in MMT, OEmcrA caused the total blockage of ST and other metabolites' production (Fig. 4A). The previous study described that OEmcrA leads to a reduction of fungal growth that might be related to a reduction of secondary metabolite production 34 . We then cultured WT and OEmcrA strains onto solid MMG and solid MMT and found that OEmcrA led to the fluffy-autolytic phenotype with about tenfold reduction in conidia formation, whereas growth and development of WT and OEmcrA strains were similar in MMG (Fig. 4B,C). We then examined whether OEmcrA-caused fluffy-autolytic phenotypes were coupled with accelerated cell death using the alamarBlue reduction assay and found that, OEmcrA led to dramatically reduced cell viability starting at day 3 compared to WT (Fig. 4D). Taken together, these results imply that McrA plays  These are highly conserved regulators that control the expression of spore-specific genes in Aspergillus species 25,27 . Genome-wide expression and protein-DNA analyses identified certain target genes for these TFs 25,30 . In addition, follow-up studies identified the functions of some target genes and these results provide some clues to elaborate on how these TFs can control spore formation and maturation 29 . For example, the VosA-VelB complex controls the expression of fksA, mtfA, sclB, and vadA, thereby fine-tuning β-glucan synthesis, secondary metabolism, oxidative response, and conidial pigmentation 29   McrA is a multifunctional regulator controlling certain gene expression involved in biosynthesis of secondary metabolites 34 . We revealed that, in conidia, the loss of mcrA increased the mRNA level of aflR, proposing the role of McrA in repressing conidial secondary metabolites. Furthermore, McrA is needed for proper biogenesis of conidial trehalose and downregulation of fksA in conidia, thereby governing the integrity of spores.
Importantly, we have shown McrA's new role in vegetative growth, autolysis, and cell death in A. nidulans. Autolysis is a naturally occurring phenomenon that is reported as enzymatic self-degradation of the cells, affected by nutrient limitation, aging, and other factors 42 . Previous studies have noted that FlbA, an RGS protein, negatively regulates vegetative growth by turning off the FadA-mediated G protein to cAMP-dependent protein kinase (PKA) signaling cascade 37 . The absence of flbA leads to prolonged the activation of FadA-mediated G protein signaling, resulting in the autolytic phenotypes 36  Methods fungal strains and culture conditions. A. nidulans strains used in this study are listed in Table 1. Fungal strains were grown on solid or liquid minimal medium (MM) with 1% glucose (MMG) with supplements as described previously 43 . To determine the numbers of conidia in WT (FGSC4) and mutant strains, 10 5 spores For Northern blot analysis, samples were collected as described previously 26 . Briefly, 10 6 conidia/mL were inoculated in 100 mL liquid MMG in 250 mL flask and cultured at 37 °C and 220 rpm. Samples from liquid submerged culture were collected at designated time points, squeeze-dried and stored at -80 °C until the isolation of RNA. For sexual and asexual developmental induction, 18 h vegetative grown mycelia were filtered and then transferred to solid MMG. The plates were air exposed for asexual developmental induction, or tightly sealed and blocked from light for sexual developmental induction 46 . Table 2. The double joint PCR (DJ-PCR) method 47 was used to generate the ΔmcrA and ΔflbA mutants. Both 5′ and 3′ flanking regions of each gene were amplified from genomic DNA of A. nidulans FGSC4 using OHS767;OHS769 and OHS768;OHS770 (for mcrA), and OMK607;OMK613 and OMK614;OMK610 (for flbA). The A. fumigatus pyrG + marker was amplified from A. fumigatus AF293 genomic DNA with the primer pair OMK589;OMK590. The final mcrA or flbA deletion construct was amplified with OHS771;OHS772 or OMK611;OMK612, respectively. To generate the ΔflbA ΔmcrA double mutant, 5′ and 3′ flanking regions of flbA (OMK607;OMK608 and OMK609;OMK610) were amplified. The A. nidulans pyroA + marker was amplified from FGSC4 genomic DNA with the primer pair ONK395;ONK396. The flbA deletion cassette was introduced into TMK19. Protoplasts were generated using the Vinoflow FCE lysing enzyme (Novozymes) 48 . At least three independent deletion mutant strains were isolated and confirmed by PCR analysis.

Generation of A. nidulans strains. Oligonucleotides used in this study are listed in
To complement the deletion of mcrA, the mcrA locus such as its 2 kb 5′ UTR and coding region was amplified with the primer pair OMK657;OHS878, digested with EcoRI and NotI, and cloned into pHS13 26 , which contains 3/4 pyroA, a 3xFLAG tag, and the trpC terminator. The resulting plasmid pMK23 was then introduced into the recipient ΔmcrA mutant TMK19, in which a single copy mcrA + is confirmed to be inserted into the pyroA4 locus, to give rise to TMK20.
To generate the alcA(p)::mcrA fusion construct, the mcrA ORF derived from FGSC4 genomic DNA was amplified using the primer pair OHS875;OHS878. The amplicon was double digested with EcoRI and NotI and cloned into pHS3, which has the alcA promoter and the trpC terminator 49 . The resulting plasmids pHSN74 was then introduced into TNJ36. The mcrA overexpression (OEmcrA) mutant, THS41, was screened by Western blot analysis using monoclonal anti-Flag antibody (M2 clone, Sigma-Aldrich). nucleic acid manipulation. Genomic DNA isolation was performed as previously described 48 . Total RNA for Northern blot was isolated from each sample using Trizol reagent (Thermo Fisher Scientific) following the protocol provided by the manufacturer's instructions. For Northern blot analysis, DNA probes were prepared by PCR amplification of the coding region of individual genes with suitable oligonucleotide pairs using WT genomic DNA as a template. Probes were labeled with 32 P-dCTP (PerkinElmer) using the Random Primer DNA Labeling Kit (Takara Bio) and purified by Illustra MicroSpin G-25 columns (GE Healthcare).
For quantitative real-time PCR, complementary DNA was synthesized using the GoScript Reverse Transcription system (Promega) using the total RNA was isolated from each sample using Trizol reagent. qRT-PCR was performed with each gene-specific primer set and iTaq universal SYBR Green supermix (Bio-Rad) and using a CFX96 Touch Real-Time PCR system (Bio-Rad).
Determination of cell viability. For spore viability, WT and mutant strains were inoculated onto MMG and cultured for 2, 5, 8, 10, and 20 days 15 . Conidia were collected from the cultured plates. After then, about 100 conidia were spread onto MMG plates and the plates were then incubated for 48 h. Survival rates were calculated as the ratio of the number of colony forming unit to the number of spores inoculated. www.nature.com/scientificreports/ Fungal cell viability was determined by the percent reduction of alamarBlue (Bio-Rad). The alamarBlue assay reagent was placed into each well of a 24-well plate, which has 1 mL of fresh liquid MMG with 0.5% YE and 0.5 mL of individual cultures with an equal amount of the mycelium, at a final concentration of 10% of the reaction volume. After the plate was incubated at 37 °C for 6 h in the dark 38 , the absorbance of each well was detected by A570 and A600 nm wavelength. The reduction percent of alamarBlue was calculated as described previously 50,51 . The values are designated as the mean standard deviation for triplicates of individual cultures.

Sterigmatocystin extraction and thin-layer chromatography (tLc) analysis. Sterigmatocystin
(ST) was extracted from fresh conidia and examined as described 31 . Briefly, 10 5 conidia were inoculated into 2 mL liquid complete medium (CM) and slant cultured at 37 °C for 3 days. ST was extracted by adding 2 mL of CHCl 3 , and the organic phase was transferred into 1.5 mL tubes and then centrifuged at 10,000 rpm for 2 min. The CHCl 3 layer was collected, dried, and then resuspended in 100 μL of CHCl 3 . Approximately 10 μL of each sample was applied onto a TLC silica plate including a fluorescence indicator (Kiesel gel 60, 0.25 mm thick, Merck). ST standard (Sigma-Aldrich) was loaded onto the TLC plate. The TLC 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 onto the TLC plate and the plate was baked at 70 °C for 5 min to enhance the detection of ST. The TLC plate was exposed to UV of A320 nm, and ST levels were measured. This experiment was performed in triplicate.
High-performance liquid chromatography (HpLc) analysis. The HPLC analysis was performed as previous described 31 . Asexual spores (2 × 10 8 ) fungal strains were extracted by adding chloroform into the vials. The samples were vigorously mixed using a vortex mixer. The organic phase was then separated by centrifugation and transferred to new vials. Each sample was evaporated and resuspended with 0.5 mL of HPLC grade acetonitrile:methanol (50:50, v/v). For the control, ST was dissolved using the same solvent and then serially diluted. Samples and the ST standard were filtered using a 0.45 μm pore filter. A linear calibration curve (R 2 = 0.998) was constructed with a ST dilution series, 10 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL, and 0.005 μg/mL. HPLC-diode array detection (DAD) analysis was carried out using a Series 1,100 binary pump with an auto sampler and Nova-Pak C-18 column (Agilent Technologies). The mobile phase was consisted of Table 2. Oligonucleotides used in this study. a Tail sequence is in italic. b Restriction enzyme site is in bold.