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bZIP transcription factors PcYap1 and PcRsmA link oxidative stress response to secondary metabolism and development in Penicillium chrysogenum
Microbial Cell Factories volume 21, Article number: 50 (2022)
Reactive oxygen species (ROS) trigger different morphogenic processes in filamentous fungi and have been shown to play a role in the regulation of the biosynthesis of some secondary metabolites. Some bZIP transcription factors, such as Yap1, AtfA and AtfB, mediate resistance to oxidative stress and have a role in secondary metabolism regulation. In this work we aimed to get insight into the molecular basis of this regulation in the industrially important fungus Penicillium chrysogenum through the characterization of the role played by two effectors that mediate the oxidative stress response in development and secondary metabolism.
In P. chrysogenum, penicillin biosynthesis and conidiation are stimulated by the addition of H2O2 to the culture medium, and this effect is mediated by the bZIP transcription factors PcYap1 and PcRsmA. Silencing of expression of both proteins by RNAi resulted in similar phenotypes, characterized by increased levels of ROS in the cell, reduced conidiation, higher sensitivity of conidia to H2O2 and a decrease in penicillin production. Both PcYap1 and PcRsmA are able to sense H2O2-generated ROS in vitro and change its conformation in response to this stimulus. PcYap1 and PcRsmA positively regulate the expression of brlA, the first gene of the conidiation central regulatory pathway. PcYap1 binds in vitro to a previously identified regulatory sequence in the promoter of the penicillin gene pcbAB: TTAGTAA, and to a TTACTAA sequence in the promoter of the brlA gene, whereas PcRsmA binds to the sequences TGAGACA and TTACGTAA (CRE motif) in the promoters of the pcbAB and penDE genes, respectively.
bZIP transcription factors PcYap1 and PcRsmA respond to the presence of H2O2-generated ROS and regulate oxidative stress response in the cell. Both proteins mediate ROS regulation of penicillin biosynthesis and conidiation by binding to specific regulatory elements in the promoters of key genes. PcYap1 is identified as the previously proposed transcription factor PTA1 (Penicillin Transcriptional Activator 1), which binds to the regulatory sequence TTAGTAA in the pcbAB gene promoter. This is the first report of a Yap1 protein directly regulating transcription of a secondary metabolism gene. A model describing the regulatory network mediated by PcYap1 and PcRsmA is proposed.
In filamentous fungi, reactive oxygen species (ROS) have been shown to trigger and/or modulate different morphogenic processes [1,2,3,4] as well as fungus-plant interactions and biocontrol of plant pathogens [5,6,7]. A role of ROS in fungal secondary metabolism is currently well established too [8, 9]. The biosynthesis of aflatoxins by several Aspergillus species is the best-known case of induction of secondary metabolite (SM) biosynthesis by oxidative stress ( and references therein). Similarly, the production of trichothecene and expression of Tri genes in Fusarium graminearum is stimulated by treatment with H2O2 , and the production of lovastatin by Aspergillus terreus is modulated by ROS levels, which also regulate the expression of lovE, the lovastatin cluster regulatory gene . It has been proposed that the biosynthesis of aflatoxin and trichothecene are part of the defense response against oxidative stress [13, 14].
The response to stress conditions in filamentous fungi shares common features with that in yeasts. A multistep phosphorelay system module transduces the signal to the stress-activated protein kinase/mitogen-activated protein kinase (SAPK/MAPK) module, which results in the activation of specific transcription factors that regulate the expression of target genes involved in the cellular response to the stress signals [8, 15,16,17,18,19,20]. Some of these transcription factors, such as AP-1, AtfA, AtfB, and MsnA, have been shown to participate directly or indirectly in the regulation of SM biosynthesis ([8, 21,22,23,24] and references therein).
Penicillium chrysogenum is one of the most important microorganisms in the biotechnological industry as a producer of penicillin and other β-lactam antibiotic precursors [25, 26]. Penicillin is synthesized from three precursor amino acids (α-aminoadipate, cysteine and valine) in three steps catalyzed by the enzymes δ(α-aminoadipyl)-cysteinyl-valine (ACV) synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, encoded by the pcbAB, pcbC and penDE genes, respectively . The three genes form a cluster, with the pcbAB and pcbC genes being expressed in opposite orientation from promoters situated in a common intergenic region. Unlike many other fungal SM gene clusters, no specific transcription factor is present in the cluster to control the simultaneous expression of the three genes. Instead, several wide domain transcription factors regulate the timing and expression levels of the genes in response to a variety of nutritional and physiological cues . Penicillin biosynthesis is a good example of a process subject to complex global regulatory networks and serves as a model to study fungal secondary metabolism regulation [29, 30].
Several wide domain transcription factors and cis-acting regulatory elements that control the expression of the penicillin genes have been identified (reviewed in ). In a previous work, a fragment-deletion screen of the pcbAB gene promoter fused to the lacZ reporter gene identified a region that was important for transcription . Using electrophoretic mobility shift assay (EMSA) and uracil interference assay (UIA) the sequence TTAGTAA, located 766–760 bp upstream of the pcbAB ATG start codon, was shown to strongly bind an as-then-unidentified transcription factor, which was named PTA1 (Penicillin Transcriptional Activator 1). Deletion and mutations of this sequence confirmed its in vivo functionality in the transcriptional regulation of the pcbAB gene . The TTAGTAA regulatory sequence shows a nucleotide change (TTCGTAA) in the promoter of Penicillium nalgiovense, another penicillin producer usually growing on ripened meat products . The level of transcription of the pcbC gene in P. nalgiovense is much lower than that in P. chrysogenum. Partially purified protein extracts from both fungi failed to bind the TTCGTAA sequence, whereas the P. chrysogenum extract, but not the P. nalgiovense extract, strongly bound the TTAGTAA sequence . These findings confirm the role of the TTAGTAA sequence as an important regulatory element for the transcription of the pcbAB gene and, possibly, the pcbC gene in P. chrysogenum.
The PTA1 binding sequence has structural similarity to sequences bound by the bZIP-type family of transcription factors AP-1 . The AP-1-like factor Pap1 from the fission yeast has been shown to specifically recognize the sequences TTACGTAA and TTAGTAA . In Saccharomyces cerevisiae, a family of eight transcription factors, the Yap family (Yap1-8), has been described as related to the AP-1 family . In many respects, the Yap proteins are functionally redundant, activating the transcription of genes involved in stress response. However, they differ in their specific response to stress agents and also show differences in the discrimination of individual base changes in the DNA-binding sequence [36, 37]. The preferred target sequence of YAP1-4 is TTACTAA.
In filamentous fungi, putative homologs of Yap1 and Yap3 have been cloned and studied. Yap1-like proteins have been found in Aspergillus fumigatus (showing 59% sequence similarity to Yap1)  and other fungi (reviewed in  and ), and they have been shown to participate in the response to oxidative stress. In the case of Aspergillus parasiticus , Aspergillus ochraceus  and F. graminearum , Yap1-like transcription factors were also shown to have a role in the production of the secondary metabolites aflatoxin, ochratoxin A and trichothecene, respectively. For their part, using a multicopy-suppressor approach, Shaaban et al.  cloned the rsmA gene, which was able to restore the deficiency in SM production caused by the absence of the Velvet complex in an A. nidulans mutant strain. The rsmA gene encodes a bZIP transcription factor that shares 31% identity with Candida albicans FCR3  and whose highest identity in the genome of S. cerevisiae is with the FCR3 homolog Yap3 . RsmA binds in vitro to two sequences in the promoter of the sterigmatocystin cluster regulatory gene aflR: TTAGTAA (a typical Yap1-binding sequence) and TGACACA . RsmA orthologs have been found in other fungi and shown to activate SM production [45, 46]. RsmA proteins do not exhibit a consistent pattern regarding the stress response, showing varying effects in different fungi concerning stress challenges [44,45,46,47].
Another bZIP fungal transcription factor, AtfB, has been described as mediating both oxidative stress response and secondary metabolism in some Aspergillus species [13, 23, 48]. AtfB was first identified in Aspergillus oryzae as an ATF/CREB family transcription factor involved in oxidative stress response and conidia tolerance to H2O2 . Using a ChIP approach, Roze et al.  found that, in A. parasiticus, AtfB binds to the promoters of seven aflatoxin genes carrying CRE (cAMP Response Element) motifs (TKACGTMA), whereas EMSA revealed that AtfB binds to a probe containing a CRE-like (TGACATAA) and an AP-1 (TGAGTAC) site from the promoter of the aflatoxin gene nor-1. These sequences show also a resemblance to the TTAGTAA regulatory sequence present in the P. chrysogenum pcbAB gene promoter.
Taking together all these data, we hypothesized that penicillin biosynthesis is regulated by ROS and that the PTA1-binding site TTAGTAA plays a role in this process. Here we describe that the Yap1 ortholog of P. chrysogenum, PcYap1, binds to the TTAGTAA regulatory element in the pcbAB promoter and to a TTACTAA sequence in the promoter of the conidiation regulatory gene brlA. PcYap1 regulates penicillin biosynthesis, conidiation and participates in the oxidative stress response. We also show that PcRsmA regulates penicillin biosynthesis, binds to a sequence (TGAGACA) located 68 bp upstream of the PcYap1-binding site in the pcbAB gene promoter and to a CRE site (TTACGTAA) located in the penDE gene promoter, and plays similar roles to PcYap1 in conidiation and response to oxidative stress. The direct transcriptional activation by a Yap1 protein of an SM gene and the conidiation regulatory gene brlA by binding to their promoters had not been previously reported.
Oxidative stress regulates penicillin biosynthesis
Cultures of the P. chrysogenum Wis54-1255 strain were performed in flasks with complex production medium supplemented with H2O2 at concentrations of 25, 50, 100, 150 and 200 mM (Fig. 1). Concentrations up to 150 mM did not have any effect on the growth and biomass of the fungus, whereas 200 mM negatively affected growth. H2O2 concentrations of 25 and 50 mM did not have any significant effect on penicillin production, while concentrations of 100 mM and above produced important changes in the production pattern. Penicillin production started earlier when 100–200 mM H2O2 was added to the culture. With 100 mM H2O2, production was significantly higher until 72 h of cultivation, while 150–200 mM stimulated penicillin production only during the first 48 h. We concluded that a certain amount of H2O2-induced ROS positively regulates penicillin biosynthesis, and chose the concentration showing higher induction, 100 mM H2O2, for subsequent experiments.
Yap1 and RsmA orthologs in the P. chrysogenum genome
A transcription factor homologous to Yap1 was the first candidate to bind to the TTAGTTA regulatory sequence in the pcbAB promoter. In a search of the P. chrysogenum Wis54-1255 (aka P. rubens Wis54-1255, taxid 500,485) genome at NCBI, we found the gene Pc20g15280, whose deduced amino acid sequence showed 28% overall identity with the Yap1 protein from S. cerevisiae (41% and 52% in two conserved regions of 107 and 58 amino acids located at the N- and C-terminal ends, respectively) and 63.6% overall identity with AfYap1 from A. fumigatus. No other protein with high similarity to Yap1 is encoded in the P. chrysogenum genome. Therefore, we considered Pc20g15280 a Yap1 ortholog and named it Pc-yap1.
Another candidate to bind to the TTAGTAA sequence and regulate penicillin biosynthesis was RsmA, a Yap3-like protein that regulates secondary metabolism in A. nidulans, A. fumigatus and Pestalotiopsis fici (see “Background” section). The protein encoded by the Pc12g16510 gene in the P. chrysogenum genome shows 59.4% overall identity with RsmA from A. nidulans at the amino acid level, 43.2% identity with PfZipA (RsmA ortholog) from P. fici, and the highest similarity in the S. cerevisiae genome is with Yap3p (23.4% identity) and in C. albicans with Fcr3p (29.1% identity). Therefore, we named Pc12g16510 as Pc-rsmA. Pc-yap1 and Pc-rsmA encode putative bZIP transcription factors with a deduced sequence of 582 (PcYap1) and 299 (PcRsmA) amino acids, which conserve the basic DNA-binding and dimerization leucine zipper domains typical of these proteins. We decided to characterize PcYap1 and PcRsmA function by two approaches: (1) EMSA analysis of the binding of the proteins to the TTAGTAA sequence and other regions in the penicillin gene promoters, and (2) Knocking down and overexpression of the genes, and characterization of the resulting phenotypes in relation to penicillin production, oxidative stress defense and conidiation.
PcYap1, but not PcRsmA, binds to the pcbAB gene regulatory sequence TTAGTAA
Purified PcYap1 and PcRsmA (see “Materials and methods” section) were incubated with a 28-bp DNA probe from the pcbAB gene promoter containing the TTAGTAA sequence (probe PTA1-WT) and with a mutated probe with two base changes (probe PTA1-M1), and the reactions were run in a native PAGE (Fig. 2). The results showed that PcYap1 binds to the probe with the TTAGTAA sequence but not to the mutated probe, whereas PcRsmA does not bind to any of the probes. Next, we performed a specificity EMSA adding excess amounts of unlabelled PTA1-WT and PTA1-M1 probes to the binding reactions between PcYap1 and the PTA1-WT probe (Fig. 2C). Only the PTA1-WT probe competed with the labelled probe for binding to PcYap1, thus demonstrating that the binding of PcYap1 to the TTAGTAA sequence is specific.
We then searched for other possible PcYap1 binding sites by performing EMSA with six probes covering the entire pcbAB-pcbC intergenic region. As shown in Fig. 2D, the only probe interacting with PcYap1 was probe 5, which contains the TTAGTAA sequence already proven to be bound by the protein. Therefore, PcYap1 has only one binding site in the pcbAB-pcbC intergenic region.
Both PcYap1 and PcRsmA regulate penicillin biosynthesis
To test the possible involvement of PcYap1 and PcRsmA in the regulation of penicillin production, we carried out cultures in flasks with two Pc-yap1-knocked down strains (si-PcYap1-8 and -12) and two Pc-rsmA-knocked down strains (si-PcRsmA-24 and -25). In CP medium without H2O2, the behavior of the strains with knocked down Pc-yap1 and Pc-rsmA expression was very similar, showing a clear reduction in penicillin production with respect to the control strains from 72 h of cultivation onwards, accumulating at the end of the cultures around 33% and 41% of the amount in the control strains, respectively (Fig. 3A). When 100 mM H2O2 was added to the culture, penicillin production started earlier in all strains, and differences in production showed up from 24 h of cultivation (Fig. 3B). In this case, the effect of Pc-rsmA silencing was less marked than that observed in cultures without H2O2, with penicillin titers of 60–87% compared to the values obtained with the control strains. The final amount of penicillin produced by strain Wis54-1255 when was submitted to oxidative stress was 1.38-fold higher than in the absence of H2O2 (purple bars); this H2O2-mediated induction did not take place when expression of Pc-yap1 was knocked down, and in fact, production in PcYap1-knocked down strains submitted to oxidative stress was only 53% of that in strain Wis54-1255 not submitted to stress. For its part, knocking down of Pc-rsmA also abolished the inducing effect of H2O2 in the late hours of the culture, but in this case penicillin production was similar to that in strain Wis54-1255 not submitted to stress.
When the Pc-yap1 and Pc-rsmA genes were overexpressed under the control of the constitutive pki promoter, a 1.8-fold increase in penicillin production was observed at 48 h of cultivation in medium without H2O2 in both cases (Fig. 3C), whereas in cultures supplemented with 100 mM H2O2 differences between OE and control strains were significant only in OE::PcYap1 strains at 48 h (1.22-fold) (Fig. 3D).
From these results, we concluded that both PcYap1 and PcRsmA are important positive regulators of penicillin biosynthesis and that the induction of penicillin production by H2O2-generated oxidative stress is mediated by PcYap1 and, to a lesser extent, by PcRsmA.
PcRsmA binds specifically to a TGAGACA sequence in the pcbAB-pcbC intergenic region
As concluded in the previous sections, PcRsmA is a positive regulator of penicillin biosynthesis but does not bind to the TTAGTAA regulatory sequence (Fig. 2B). In an attempt to identify possible target sites of PcRsmA we performed EMSAs using six probes of around 190 bp each covering the entire pcbAB-pcbC intergenic region (Fig. 4A). The results showed that only probe 5 was bound by PcRsmA, the same probe in which the PcYap1-binding sequence is located (Fig. 4B). Next, we divided probe 5 into two halves (probes upPta1 and dwPta1), and PcRsmA bound only to upPta1, which comprises the DNA region around and upstream of the PcYap1-binding sequence (Fig. 4C). Within this region and 68 bp upstream of TTAGTAA, there is a sequence (TGAGACA) that shows similarity to a TGACACA sequence present in the A. nidulans aflR gene promoter, previously proven to be bound by RsmA, and to putative RsmA-binding sequences found in a MEME analysis of promoters of genes up-regulated in an A. nidulans OE::rsmA strain . Then, we designed several probes containing and excluding the TGAGACA sequence, and found that PcRsmA only bound to those containing this sequence (Fig. 4D). Specificity of the binding of PcRsmA to the TGAGACA sequence was confirmed by mutating the sequence at positions 3 and 6 to TGCGATA (probe RsmA-2C-M1) and by adding excess amounts of non-labelled probes in competition assays (Fig. 4E). Two TGAGACA sequences are present in the pcbAB-pcbC intergenic region: the PcRsmA-bound sequence at position -835 from the pcbAB gene start codon (named TGAGACA-2), and an unbound sequence at position -377 in reverse orientation (named TGAGACA-1), which is present in probe 3 (Fig. 4A). The fact that only one of the two TGAGACA sequences in the pcbAB-pcbC intergenic region is bound by PcRsmA indicates that its binding to this sequence is context-dependent, and additional nucleotides must be required for it.
The proximity of the PcYap1-binding site (TTAGTAA) to the PcRsmA binding site (TGAGACA) prompted us to explore the possibility that both proteins interact when binding to their respective target sites. We designed an EMSA with several probes of this region that contained one or the other binding site, or both, incubating them with PcYap1, PcRsmA, or both proteins together, expecting to get a supershift in the case that the proteins interact (Additional file 7). Only when the probe contained both binding sites (probe upPta1) and was incubated with both proteins together a supershift pattern appeared, probably as a result of the simultaneous binding of each protein to its specific binding site. This indicates that apparently there is no in vitro interaction between the proteins when binding to their respective target sites.
PcRsmA, but not PcYap1 or PcAtf21, binds to a CRE motif (TKACGTMA) in the penDE gene promoter
Another transcription factor involved in both oxidative stress response and secondary metabolism described in some Aspergilli is AtfB/Atf21 [8, 48], which belongs to the ATF/CREB family of transcription factors. Hai and Curran (1991)  reported that these factors bind as homo or heterodimers to the consensus sequence TKACGTMA. This sequence matches the TTAGTAA regulatory sequence in the pcbAB-pcbC intergenic region but for the presence of an additional S base, and one such sequence (TTACGTAA) is present in the promoter region of the penDE gene at position -697 from the ATG start codon. Therefore, we decided to investigate if a P. chrysogenum AtfB homologue was able to bind some of these motifs. A Blast search in the P. chrysogenum genome using the sequence of Aspergillus flavus Atf21 (XP_002381221) as bait identified a protein of 319 amino acids encoded at locus Pc21g08330, which showed 45.8% overall identity to A. flavus Atf21, A. parasiticus AtfB and A. oryzae AtfB. The highest identity of Pc21g08330 was to Aspergillus clavatus Atf21 (58.7%) and A. fumigatus Atf21 (54.9%), and the highest identity in the Schizosaccharomyces pombe genome was to the ATF-CREB family protein Atf21 (NP_595707), 9.1% identity head to tail, similar to that shown by different Aspergillus AtfB/Atf21 proteins, with 37% identity in a 135 amino acid region containing a bZIP-ATF2 motif. Pc21g08330 is different from a putative Atf1/AtfA ortholog (locus Pc13g09580) and other putative ATF/CREB proteins in the P. chrysogenum genome. Therefore, we decided to name the protein encoded at locus Pc21g08330 as PcAtf21.
PcAtf21 was expressed heterologously as a c-myc-6xHis-tagged protein in Pichia pastoris, purified and used for EMSA (see “Materials and methods” section). PcAtf21 did not bind to any of the six probes covering the pcbAB-pcbC intergenic region (Fig. 5A), which ruled out the possibility that it recognizes the regulatory TTAGTAA sequence located in probe 5. This result is in agreement with the absence of CRE motifs in the pcbAB-pcbC intergenic region. PcAtf21 functionality for in vitro binding was demonstrated by its capacity to bind the NorR4 probe (Fig. 5B, lane 10), whose sequence belongs to the A. parasiticus nor-1 gene promoter and contains an AP-1-like (TGAGTAC) and a CRE-like site (TGACATAA) (see “Discussion” section).
Next, we tested if a 57-bp probe (penDE-CRE123), which belongs to the penDE gene promoter and contains the CRE site TTACGTAA plus two additional CRE-like sequences upstream of it, was bound by some of the three bZIP transcription factors under study. No binding could be detected with PcYap1 and PcAtf21, whereas PcRsmA bound to the probe producing a clear gel retardation pattern (Fig. 5B, lane 3). When the CRE site in the probe was mutated to CTACGAAA binding was abolished, which strongly suggests that PcRsmA specifically recognizes the TTACGTAA sequence. From these results, we concluded that PcAtf21 is not directly involved in the regulation of transcription of the penicillin genes, whereas PcRsmA probably regulates the expression of the penDE gene.
Both PcYap1 and PcRsmA participate in oxidative stress defense
To test the possible involvement of PcYap1 and PcRsmA in the defense against ROS, we measured the amount of ROS present in cultures of knocked-down and overexpressing strains in MCFP medium with or without H2O2. The two proteins showed very similar profiles in the effect caused on the intracellular ROS levels (Fig. 6). In strains with RNAi-mediated attenuation of PcYap1 or PcRsmA expression, ROS levels were higher than in the control strains throughout the cultivation time (Fig. 6A). The highest difference occurred at 24 h in the presence of 100 mM H2O2, when the Pc-yap1-knocked down strains presented 2.28-fold higher amounts of ROS than the control strains and the Pc-rsmA-knocked down strains showed a 1.96-fold increase. In this condition, the differences in ROS levels between strains became smaller as the culture progressed, reflecting the impact of the addition of H2O2 at the beginning of the culture. In cultures without H2O2, differences between either Pc-yap1- or Pc-rsmA-knocked down strains and control strains oscillated in the range of 1.28-fold and 1.75-fold, with a tendency to increase as the culture progressed.
When either Pc-yap1 or Pc-rsmA were overexpressed, the ROS levels showed a decrease with respect to those in the control strains (Fig. 6B). In the case of the strains overexpressing Pc-yap1, this effect was more noticeable in the cultures without H2O2 (ROS levels were 61% of those present in the control strains at 48–72 h), whereas in strains overexpressing Pc-rsmA, ROS levels were around 71% of those in the control strains at 48 h in cultures without H2O2 and oscillated between 68 and 72% in cultures with H2O2.
Participation of PcYap1 and PcRsmA in defense against ROS was further confirmed by assaying the viability of conidia at different concentrations of H2O2 and the effect that knocking-down of both genes had on viability. As shown in Fig. 6C, viability is increasingly affected by silencing of Pc-yap1 and Pc-rsmA as conidia are exposed to higher concentrations of H2O2. With 130 mM H2O2, the number of viable conidia in Pc-yap1- and Pc-rsmA-knocked down strains was 17.3% and 37.6%, respectively, of that in control strains. In the Pc-yap1-knocked down strains, no viable conidia were found with 180 mM H2O2, whereas Pc-rsmA-knocked down strains showed differences between them in conidia viability at high H2O2 concentrations, with 0% viability in strain si-PcRsmA-25 at 160 and 180 mM.
PcYap1 and PcRsmA respond to the presence of H2O2-generated ROS in vitro
Once the participation of PcYap1 and PcRsmA in oxidative stress defense was confirmed, we analyzed wether they were affected in vitro by the presence of H2O2 to get insight into their possible role as ROS sensors. The purified PcYap1::c-myc-6xHis and PcRsmA::c-myc-6xHis proteins were incubated with increasing concentrations of H2O2 for 15 min and immediately loaded onto an SDS-PAGE gel under non-reducing conditions (see “Materials and methods” section). As shown in Fig. 7A, H2O2 concentrations of 200 µM and above produced a change in the mobility of both proteins, which run slightly faster in the gel. Then, the proteins were submitted to different incubation times with 400 µM H2O2, observing the same effect after 15 min of incubation. This change in mobility can be interpreted as a conformational change of the proteins, with the formation of disulphide bonds due to the oxidizing conditions produced by H2O2. In a work by Wood et al. , a fragment of S. cerevisiae Yap1 containing the two cysteine-rich domains fused to GFP was used to test the conformational changes the protein underwent in H2O2-treated cells with respect to untreated cells, and the results showed that the protein from H2O2-treated cells presented an oxidized form with disulphide bonds (as determined by NMR spectroscopy) that moved faster in SDS-PAGE gels in non-reducing conditions. The results in Fig. 7 are analogous to those of Wood et al.  and can thus be interpreted in the same way. To confirm that the higher mobility observed when the proteins were incubated with H2O2 was due to disulphide bond formation, we run the H2O2-incubated proteins in SDS-PAGE in reducing and non-reducing conditions (Fig. 7B). As expected, the mobility change observed in non-reducing conditions did not occur in reducing conditions, a result that can be explained by the rupture of the disulphide bonds upon reduction of the cysteine residues and the subsequent conformational change.
In S. cerevisiae, Yap1 moves into and out from the nucleus under normal physiological conditions. In oxidative conditions caused by H2O2, disulphide bonds form, and the bonding between C303 and C598 produces a conformational change that masks the Nuclear Export Signal (NES) located at the C-terminus, thus preventing recognition by the exportin Crm1 and export from the nucleus [51,52,53]. PcYap1 shows high identity with Yap1 and very high identity with filamentous fungi Yap1 orthologs, and contains all the conserved domains found in these organisms: Nuclear Localization Signal (NLS), NES, bZIP, CRD (cysteine rich domains) (Additional file 8). Nuclear accumulation of filamentous fungi Yap1 orthologs upon induction of oxidative stress conditions has been observed in all cases studied . Besides, in a STRING analysis, PcYap1 showed interaction with a P. chrysogenum exportin Crm1 ortholog (Pc16g01720). Therefore, PcYap1 very likely follows an activation/nuclear localization pattern similar to those described for other fungi. Notwithstanding the results of Fig. 7 indicate that PcYap1 can directly sense oxidizing conditions, its activation in the cell may also be achieved through other mechanisms, such as Gpx3-mediated oxidation, as established for S. cerevisiae , or by other peroxiredoxins, like Asp f3 from A. fumigatus which is required for Afyap1 activation and nuclear localization .
PcRsmA shows the same capacity of ROS-sensing and conformational change in vitro as PcYap1. Analysis of the protein sequence reveals the presence of only two cysteine residues for the formation of disulphide bonds, located at the C-terminus (Additional file 9). The one at position 271 is conserved in A. nidulans RsmA and S. cerevisiae Yap3, and the one at position 228 only in RsmA. A putative NES is located between these two cysteine residues, and thus might be affected by the formation of a disulphide bond. Yap3 localizes in the nucleus upon treatment with hydroquinone ; however, whether the localization mechanism is similar to that of Yap1 remains to be elucidated. Therefore, with the currently available data, we cannot propose a specific mechanism for the entrance/location of PcRsmA in the nucleus.
PcRsmA controls expression of the Pc-yap1 gene
The ability of the transcription factor PcRsmA to participate in the response to oxidative stress in the cell may be due to direct regulation of antioxidant enzyme-encoding genes and/or the regulation of wide domain factors involved in oxidative stress defense, such as PcYap1. We explored this latter possibility by measuring the levels of Pc-yap1 transcript in Pc-rsmA-knocked down strains by RT-PCR (with RNA from mycelium grown in submerged cultures) and by Northern blot (with RNA from mycelium grown on solid medium) (Fig. 8). The results showed that in submerged cultures Pc-yap1 expression in the Pc-rsmA-silenced strains was around 35% of that in the control strain, whereas in solid medium it was 29–52%. Therefore, PcRsmA positively regulates transcription of the Pc-yap1 gene. This regulation may be direct, by binding of PcRsmA to the Pc-yap1 gene promoter, or indirect. No putative PcRsmA binding sites with the sequences TGASACA or TKACGTMA are present in the promoter, but a typical AP-1 binding site, TGAGTCA, is present 145 bp upstream of the ATG start codon, although binding of PcRsmA to this sequence was not tested. The regulation of Pc-yap1 gene expression by PcRsmA implies that this protein can regulate different processes in the cell directly by binding to the promoters of the corresponding genes, indirectly through upregulation of PcYap1 expression or in both ways, as happens in the case of penicillin biosynthesis, where both transcription factors have a direct role in the process.
Conidiation is positively regulated by PcYap1 and by PcRsmA to a similar extent
Next, we studied the function of PcYap1 and PcRsmA in the growth and development of the fungus. First, we analyzed the radial growth of the colonies on plates with PDA medium, finding no significant differences between the strains with knocked down expression of the genes, the strains overexpressing the genes, and the control strains (Additional file 10). Biomass produced in submerged cultures was likewise not affected by PcYap1 or PcRsmA activity (data not shown). Therefore, neither of the proteins regulates the vegetative growth of the fungus. This result is in agreement with studies in other filamentous fungi, where inactivation or overexpression of Yap1 proteins usually has little to no effect on vegetative growth , with a few exceptions, such as Alternaria alternata  and Talaromyces marneffei .
We then analyzed how the onset of the conidiation process responded to the presence of different concentrations of H2O2 in the culture medium (Fig. 9A). Conidiation levels were not significantly affected by the addition of 20, 50, or 140 mM H2O2, whereas with 100 mM there was a 133%, 37% and 15% increase in the number of produced conidia at 72, 96, and 120 h, respectively.
Next, we analyzed the participation of PcYap1 and PcRsmA in the conidiation process. In medium without H2O2, knockdown of both Pc-yap1 and Pc-rsmA had a kind of fluctuating effect on conidiation (Fig. 9C). In the control strains, conidiation started at 48 h, while in Pc-yap1- and Pc-rsmA-knocked down strains it started at 72 h. At this time, the conidia counting in these strains was much higher (3.5- to 9-fold) than in the control strains. However, this initial conidiation-stimulating effect subsides as the conidiation proceeds; in Pc-yap1-knocked down strains conidiation nearly stalls at 72 h, whereas in Pc-rsmA-knocked down strains the number of conidia increased up to 96 h (approximately doubling in the interval 72–96 h) and then conidiation stopped. In the control strains, conidiation proceeds normally, increasing the conidia counting by more than one order of magnitude between 72 and 96 h and then doubling between 96 and 120 h. The final number of conidia at the end of the cultures in the Pc-yap1- and Pc-rsmA-knocked down strains was around 23% of the number in the control strains. The addition of 100 mM H2O2 to the culture media produced some changes in the pattern described above. Conidiation started at 48 h in the Pc-yap1- and Pc-rsmA-knocked down strains, although the number of conidia was one order of magnitude lower than in the control strains. The initial stimulating effect of the attenuation of both proteins also takes place in this condition but to a lower extent (around 2.8-fold more conidia than in the control strains), and then there is a steady moderate increase in conidia counting from 72 to 120 h. The final number of conidia at the end of the H2O2-added cultures in the Pc-yap1- and Pc-rsmA-knocked down strains was around 31% of the number in the control strains. In contrast to the important effect that RNAi-silencing of Pc-yap1 and Pc-rsmA had on conidiation, overexpression of both genes did not result in significant changes in the number of conidia produced at any culture time (Additional file 11).
We can conclude that both PcYap1 and PcRsmA are positive regulators of the conidiation process. Their activity is necessary for normal conidiation kinetics and full completion of the process. Normal (wild type) levels of activity of both proteins are sufficient for conidiation to occur with standard kinetics since overexpression had no effect on conidia production. The addition of 100 mM H2O2 did not result in drastic changes in the effect that expression attenuation of the genes encoding both proteins had on conidiation. Naturally generated ROS seem to be sufficient to trigger the conidiation process, while the addition of H2O2 stimulates the process mainly at the onset, but its overall effect was moderate (Fig. 9A). Interpretation of the particular conidiation kinetics of the knockdown strains is difficult, there is an initial stimulation followed by early termination of the process. A possible explanation is that other factors are prevalent at the beginning of the conidiation process in response to cues like nutrient starvation or others; absence or low amounts of PcYap1 or PcRsmA would have a positive effect on these factors, thus resulting in a stimulation of conidiation. Eventually, oxidative stress response factors (PcYap1 and PcRsmA) become the main inducers of conidiation, and then low amounts of these proteins would result in early termination or slowdown of the process.
PcYap1 regulates brlA expression and binds to a TTACTAA sequence in its promoter
Once regulation of conidiation by PcYap1 and PcRsmA was confirmed, we decided to investigate if they regulate the transcription of the first gene in the conidiation central regulatory pathway: brlA. RNA from control, Pc-yap1- and Pc-rsmA-knocked down strains was extracted from mycelium grown for 120 h on Power medium, the time of highest expression of brlA in P. chrysogenum as determined by García-Rico et al. . A strain expressing an activating dominant allele of the gene encoding a Gα subunit of the heterotrimeric G protein (pga1Q204L) was used as a control since constitutive activation of this pathway results in the nearly absence of conidiation and strong repression of brlA expression . Northern blot using a brlA probe was performed, and the results showed a clear reduction of brlA expression (down to 20–35% of the level of expression in the control strain) in both Pc-yap1- and Pc-rsmA-knocked down strains (Fig. 10A).
Analysis of the sequence of the brlA gene promoter revealed the presence of a typical AP-1 binding site (TTACTAA) 71 bp upstream of the ATG start codon. We performed an EMSA with the purified PcYap1 and PcRsmA proteins using a probe containing this site and another probe with a mutated site: TGACTAG. PcYap1 produced a clear mobility shift with the TTACTAA-containing probe but failed to bind the mutated probe, whereas PcRsmA did not show binding to any of the probes (Fig. 10B).
These results demonstrate that regulation of conidiation by PcYap1 is exerted by regulating the brlA gene expression, most likely through binding to the TTACTAA site in its promoter, not excluding the possibility that PcYap1 has additional targets for the regulation of this complex process. In the case of PcRsmA, the regulation of conidiation is very likely mediated by PcYap1 via regulation of its expression (Fig. 8).
PcYap1 is, with a high degree of certainty, the as-then-unidentified transcription factor PTA1 reported by Kosalková et al.  to bind to the TTAGTAA regulatory sequence in the pcbAB gene promoter. Kosalková et al.  proposed that PTA1 may be a complex formed by more than one protein. In this work, we demonstrate that heterologously expressed and purified PcYap1 is able to specifically bind the regulatory sequence TTAGTAA, and that knockdown of Pc-yap1 causes a decrease in penicillin production. PcYap1 is a typical bZIP transcription factor, orthologous to yeast Yap1 and sharing characteristics with the AP-1 family of transcription factors, whose DNA binding sequences are very similar, and identical in some cases, to the PcYap1-binding site in the pcbAB gene promoter. Therefore, we identify PcYap1 with PTA1, and propose that the name PcYap1 be used henceforth since it is more defining and identifying, not excluding the possibility that PcYap1 may interact in vivo with other proteins forming a complex that binds the TTAGTAA sequence, and in such case PTA1 may be used to refer to this, hypothetical yet, complex.
Oxidative stress has gained considerable attention as a triggering agent of different processes in filamentous fungi since it was first proposed that cell differentiation is a response to oxidative stress . More recently, oxidative stress has been linked to the onset of secondary metabolism [12, 60], including β-lactam biosynthesis [61, 62], and some transcription factors mediating the cell response to the presence of ROS have been implicated in the regulation of the biosynthesis of different secondary metabolites [8, 9]. In this work, we have shown that the transcription factors PcYap1 and PcRsmA participate in the oxidative stress-mediated regulation of penicillin biosynthesis, and characterized the function of both proteins in the oxidative stress response of P. chrysogenum and in the conidiation process.
Penicillin production in flask cultures of the P. chrysogenum Wis54-1255 strain is stimulated by the addition of 100 mM H2O2 to the culture medium (Fig. 1). The importance of oxidative stress for the regulation of penicillin biosynthesis is supported by the results obtained by Jami et al. , who, in a proteomics study, observed an increase in the representation of several proteins involved in oxidative stress defense in a high yield penicillin producer strain with respect to lower yield producers. When the expression of Pc-yap1 was knocked down, the stimulating effect of H2O2 did not take place (Fig. 3A). PcYap1 binds to a TTAGTAA sequence that had previously been shown to be an important regulatory element of the pcbAB gene expression . These data indicate that PcYap1 directly regulates penicillin biosynthesis by binding to the pcbAB gene promoter at the TTAGTAA sequence in response to oxidative stress.
In the case of Pc-rsmA-knocked down strains, penicillin production in H2O2-added medium was lower than in the control strains too. The final amount of accumulated penicillin was in the range of that in the parental strain Wis54-1255 grown without added H2O2. Stimulation of penicillin production by H2O2 in the Pc-rsmA-knocked down strains is lower than in the control strains, but not completely abolished as in the case of the Pc-yap1-knocked down strains. PcRsmA binds specifically to the sequence TGAGACA, located 68 bp upstream from TTAGTAA in the pcbAB gene promoter, and to the sequence TTACGTAA in the penDE gene promoter. In vivo functionality of these sequences has not been tested yet. However, in a deletion screen analysis of the pcbAB gene promoter performed by Kosalková et al. , deletion of the region where the TGAGACA sequence is located resulted in an important decrease in promoter activity. These results strongly suggest that PcRsmA also regulates penicillin biosynthesis in response to oxidative stress by binding to the above-mentioned sequences. The role of RsmA in the regulation of secondary metabolism has been well established for several fungal species, including A. nidulans [42, 44], A. fumigatus , A. flavus  and P. fici .
Normal (wild type) levels of both PcYap1 and PcRsmA seem to be sufficient to positively regulate penicillin biosynthesis in response to oxidative stress, since overexpression of either gene had little effect on production, with only a moderate increase at the start of the production time (Fig. 3C and D). Penicillin production started earlier in both the control and the OE strains when 100 mM H2O2 was added to the culture, but overexpression of either protein did not cause an earlier production in the absence of H2O2. This result can be explained if we consider that induction by ROS is probably necessary for PcYap1 and PcRsmA to become active and exert their function on transcription activation of the penicillin genes. Overexpression will not necessarily produce a specular reflection of the effects caused by silencing; i.e. higher amounts of the proteins in the cell would have no effect if they are not activated.
bZIP-type transcription factors may regulate SM production in two ways, by direct binding to promoters of structural or regulatory genes in a cluster, as shown for AtfB [13, 23] and RsmA , or by modifying intracellular ROS levels through activation of the antioxidant defense system, as demonstrated for Yap1 orthologs in A. parasiticus , A. ochraceus , A. nidulans  and F. graminearum . Our results confirm the role of PcRsmA as a transcription factor binding to promoters of SM genes to regulate SM production and establish for the first time a direct role of a Yap1 ortholog in the transcriptional regulation of an SM gene by binding to a specific regulatory sequence in its promoter. Thus, the role of Yap1 in the biosynthesis of the toxins aflatoxin and trichothecene is opposite to that in penicillin biosynthesis. In the latter case, Yap1 directly stimulates transcription of the pcbAB gene by binding to the regulatory sequence TTAGTAA, resulting in increased levels of penicillin, whereas in the case of aflatoxin and trichothecene the effect is indirect by means of controlling the amount of ROS by induction of the antioxidant defense system, which results in downregulation of toxin production, as revealed by the higher toxin accumulation observed in Δyap1 mutants [21, 40, 41] and decreased production in OE::yap1 strains [41, 47]. Interestingly, in a transcriptomics analysis of conidia from an A. nidulans strain with a deletion of the napA gene (Yap1 ortholog), Mendoza-Martínez et al.  found downregulation of the penicillin biosynthetic gene ipnA (= pcbC) (-3.25-fold) and some other secondary metabolism-related genes with respect to the wild type, which suggests a positive regulation of penicillin biosynthesis and other secondary metabolites by NapA. The authors proposed that in Aspergillus, secondary metabolism may be regulated by NapA in opposite ways during growth and conidiation. Penicillin production has not been tested in A. nidulans napA mutants, and there are no canonical Yap1 binding motifs in the ipnA gene promoter. Therefore, it is still unclear whether the effect of the napA gene deletion on the expression of the ipnA gene is direct or indirect through some other regulatory mechanism.
Veiga et al.  reported that the transcript levels of Pc-rsmA were seven-fold higher in P. chrysogenum Wis54-1255 than in the penicillin high-producing strain DS17690 using a microarray approach, which is in apparent conflict with our findings about PcRsmA being a positive regulator of penicillin biosynthesis. Industrial strains are selected for high production in bioreactors, where conditions are different from cultures in flask. In these conditions, the role of PcRsmA may not be very relevant for penicillin biosynthesis, for instance if the bioreactor process-generated oxidative stress is dealt with by industrial strains in a way that makes the PcRsmA role different or irrelevant. In fact, changes in the oxidative stress response have been observed in a high-producing strain with respect to Wis54-1255 in a comparative proteomic analysis . Other possibilities are that the Pc-rsmA gene had been mutated during the strain improvement program rendering it inactive or altered in its functionality in strain DS17690, or that, also as a result of strain improvement, other regulatory networks may have bypassed the need for PcRsmA to induce high expression levels of the penicillin genes in this strain.
The bZIP transcription factor AtfB/Atf21 was another candidate for oxidative stress-mediated regulation of the penicillin genes since it has been reported to mediate both oxidative stress response and secondary metabolism in some Aspergillus species [13, 23, 48, 49]. AtfB belongs to the ATF/CREB family of transcription factors, which bind as homo or heterodimers to the consensus sequence TKACGTMA . AtfB was first identified in A. oryzae as a transcription factor with bZIP and basic domains characteristic of the cyclic AMP-response element-binding protein (CREB) family . Later, an AtfB ortholog was cloned and characterized in A. parasiticus by Roze et al.  based on the A. flavus AFLA_094010 gene, whose deduced amino acid sequence (XP_002381221) shared 96% identity to both A. oryzae and the newly sequenced A. parasiticus AtfB. The AtfB proteins in these three species did not show high identity with other fungal proteins; they show 9–10% identity to Atf21 of the fission yeast, 53% to an A. fumigatus putative transcription factor Atf21, and less than 45% to similar proteins from other fungi. Hence Roze et al.  concluded that AtfB may be unique to these three Aspergillus species. The highest match to AtfB in the genome of P. chrysogenum was the protein encoded at locus Pc21g08330, which shows 45.8% identity head-to-tail to AtfB from these species. We have named this protein PcAtf21. In A. parasiticus, AtfB binds to a region in the promoter of the aflatoxin gene nor-1; this binding requires both an AP-1-like (TGAGTAC) and a CRE-like site, named CRE1 (TGACATAA), which are adjacent and separated by 12 bp . In addition, Roze et al.  found a consensus sequence (AGCCS) located immediately upstream of the CRE1 site in five aflatoxin promoters that demonstrated AtfB binding. Neither AP-1-like sites nor AGCCS-like sequences are present in the penDE gene promoter close to the CRE site (TTACGTAA), which could explain the inability of P. chrysogenum PcAtf21 to bind in vitro to this DNA region while it does bind the probe NorR4 containing the aforementioned motifs (Fig. 5B). More studies will be required to elucidate the binding properties of PcAtf21 and its possible role in oxidative stress response and secondary metabolism regulation.
While PcAtf21 failed to bind a probe containing the TTACGTAA CRE motif, PcRsmA was able to bind the probe, and this binding was suppressed by mutations at positions 1 and 6 (CTACGAAA) of the motif, which indicates that PcRsmA binds specifically to the CRE motif. Therefore, PcRsmA binds two different sequences in the promoters of the penicillin genes: TGAGACA and TTACGTAA. It also binds the sequence TGACACA from a probe belonging to the A. nidulans aflR gene promoter (Fig. 4B). Versatility in the binding capacity of RsmA proteins might be a common feature, considering that A. nidulans RsmA also possesses the capacity to bind two different sequences in the aflR gene promoter: TGACACA and TTAGTAA, in addition to the sequence TTACTAA of another probe from yeast .
The CCAAT-binding protein complex AnCF (CBC) coordinates the response to oxidative stress in A. nidulans . AnCF binds to the promoter and regulates the expression of napA  and the penicillin genes by binding to CCAAT regulatory elements . Multiple CCAAT boxes are present in the pcbAB-pcbC intergenic region and the penDE gene promoter in P. chrysogenum. Therefore, it will be of great interest to test if these CCAAT boxes play a role in regulating the expression of the penicillin genes and if this regulation is connected to oxidative stress. In addition, the redox state of the cell and the thioredoxin system have been proposed to play another role in penicillin biosynthesis in P. chrysogenum, forming and reducing disulfide bonds that lead to the formation of oxidized disulfide bis-ACV (which cannot be used by isopenicillin N synthase) and free ACV (substrate for this enzyme), respectively . Therefore, penicillin biosynthesis is probably affected by oxidative stress in several different ways.
The role of Yap1 orthologs as mediators in the defense against ROS has been firmly established both in yeasts [15, 70] and filamentous fungi (reviewed by [9, 39]). Yap1 mutant strains show altered ROS levels and expression of genes involved in the defense against oxidative stress. Our results with PcYap1 add to this role of Yap1 proteins (Fig. 6). In the case of RsmA orthologs, their role in general and oxidative stress defense is less consistent. PcRsmA shows the highest identity in the genomes of yeasts to S. cerevisiae Yap3 and C. albicans FCR3. Little is known about Yap3 function in the cell, it shows virtually no response in genomic microarray analyses to multiple forms of environmental insults and cellular stresses , and Yap3-dependent transcription responds to aminotriazole but not to H2O2 or cadmium . North et al.  reported that Yap3 plays a specific role in the cellular response to hydroquinone (HQ). For its part, overexpression of FCR3 confers resistance to fluconazole and 4-nitroquinoline 1-oxide . In filamentous fungi, RsmA proteins do not show a consistent pattern with respect to stress response. In A. nidulans, an OE::rsmA strain revealed no altered response to antifungals, oxidative stressors or heavy metals as compared to the wild type [44, 47], while in a ΔrsmA mutant ROS production was similar to that in the control strain . In contrast, in A. fumigatus, strains containing an OE::rsmA allele grew better than their respective controls on menadione-containing medium, thus indicating a role of RsmA in the defense against menadione-induced ROS . In A. flavus, AflrsmA-overexpressing strains showed increased sensitivity to menadione sodium bisulfite (MSB), whereas AflrsmA deletion caused less sensitivity to tert-butyl hydroperoxide (tBOOH) . For its part, in P. fici, deletion of the rsmA ortholog (PfzipA) resulted in differential responses to oxidative stress agents: it caused resistance to tBOOH, diamide, and menadione sodium bisulfite, but increased sensitivity to H2O2 . In this work we demonstrate that PcRsmA is clearly involved in the defense against H2O2-induced oxidative stress, as shown by the results on ROS accumulation in Pc-rsmA-knocked down/overexpressing strains and on conidia viability after exposure to H2O2 (Fig. 6). PcRsmA is able to sense H2O2-generated ROS in vitro changing its conformation as revealed by its mobility in gels (Fig. 7). In A. nidulans, rsmA expression was found to be induced by H2O2 stress , and in a microarray study performed by Emri et al. , aiming to analyze genome-wide expression changes caused by various stresses, both rsmA and napA genes were found to be part of the COSR (Core Oxidative Stress Response) genes. The authors proposed that RsmA and NapA can be a link between the regulation of stress response and secondary metabolite production in A. nidulans. Our results fully support this hypothesis in P. chrysogenum; both proteins play similar roles in defense against H2O2-induced oxidative stress and directly regulate the expression of penicillin genes by binding to specific regulatory sequences in their promoters. Both proteins are ROS sensors linking oxidative stress to secondary metabolite biosynthesis.
Oxidative stress is an important triggering agent for differentiation in filamentous fungi . Effects on conidiation have been observed in mutant strains lacking Yap1: conidiation was decreased to half in an A. nidulans ΔnapA mutant with respect to the wild type , in A. ochraceus a reduced number of morphologically larger conidia were formed in a ΔAoyap1 strain as compared to the wild type , and in T. marneffei, a ΔyapA mutant strain produced a number of conidia three orders of magnitude lower than the wild type . Opposite results were obtained in a ΔApYapA strain of A. parasiticus, in which premature conidiation occurs and a higher number of conidia is produced during the first 96 h of growth . Involvement of NapA in A. nidulans conidiation is supported by the results obtained by Yin et al. , who found that an OE::napA strain produced more conidia than the wild type. Our results indicate that PcYap1 plays an important role in P. chrysogenum conidiation, since attenuation of PcYap1 expression results in a reduction of conidia production to one-fourth the number in the parental strain (Fig. 9C). A clear association between Yap1 function and development/conidiation was established by Guo et al.  in Magnaporthe oryzae. Disruption of the M. oryzae Yap1 ortholog, MoAP1, caused a 30- to 40-fold reduction in conidiation, morphological conidia abnormalities, reduced aerial hyphal growth and loss of pathogenicity, along with excess ROS accumulation. Therefore, MoAP1 was proposed as a stage-specific regulator of development and plant infection. As happens with secondary metabolism, the regulation of conidiation by Yap1 orthologs may be exerted directly by controlling the expression of genes triggering and coordinating development, or indirectly by modifying the amount of ROS in the cell. In the cases when yap1 disruption or knocking-down has a negative effect on conidiation, which is the norm, direct control of conidiation regulatory genes by Yap1 should be expected. In M. oryzae, Guo et al.  found that disruption of MoAP1 caused a severe down-regulation of MoCOS1, a determinant of conidiophore formation, and other genes required for conidiation. Little is known, however, about the regulation of genes involved in conidiation by oxidative stress. In a transcriptomic analysis of A. flavus submitted to H2O2-induced oxidative stress, some development-related genes were found to be up-regulated , most interestingly flbC, which encodes a transcription factor that, in A. nidulans, activates transcription of brlA, the first of the genes in the conidiation central regulatory pathway . brlA and the other two genes in the central regulatory pathway (abaA and wetA) were also found to be regulated by SakA, a member of the stress-activated MAPK (SAPK) pathway in T. marneffei . In a recent work analyzing the phosphoproteome response to H2O2 in A. nidulans, Carrasco-Navarro and Aguirre  found that StuA, a protein required for proper expression of brlA, was specifically phosphorylated in the H2O2-condition, which suggests that phosphorylation plays also a role in the oxidative stress-mediated regulation of conidiation; interestingly, NapA was also phosphorylated in the H2O2-condition. In this work we describe for the first time a direct regulation of a gene in the conidiation central regulatory pathway, the brlA gene, by an oxidative stress defense protein; PcYap1 binds to a TTACTAA sequence in the brlA gene promoter and upregulates expression of this gene, thus triggering conidiation.
Regarding the role of RsmA proteins in the formation of conidia and other asexual resistance structures, little information is available yet. In A. flavus, AflrsmA deletion decreased sclerotia formation . Results in A. nidulans are inconclusive; only an OE::rsmA strain has been tested for conidia production, showing no conidiation. However, the same profile was observed in the wild type strain in the conditions used . In A. fumigatus, no significant differences were observed in spore production between the ΔrsmA and OE::rsmA strains and their respective controls . In contrast, in P. chrysogenum, we have found clear evidence that PcRsmA is a positive regulator of the conidiation process since Pc-rsmA-knocked down strains sporulated less than the control strains and within a similar range to the Pc-yap1-knocked down strains (Fig. 9C).
It is noteworthy that the concentration of H2O2 that causes a higher stimulation of penicillin production, 100 mM, is the same that induces an increase in conidia production (Figs. 1 and 9). This fact points to a coordinated regulation of secondary metabolism and differentiation by the presence of ROS within a certain concentration range. The results obtained in this work indicate that both PcYap1 and PcRsmA play an important role in this coordination, linking oxidative stress to secondary metabolite production and conidiation. Besides, the functions of both proteins in the cell seem to be largely overlapping, at least concerning the processes studied in this work, and both proteins respond similarly to the presence of the same H2O2 concentrations in vitro (Fig. 7). PcYap1 and PcRsmA might have similar targets in the genome; this actually happens in the penicillin gene cluster, where there is one binding site for PcYap1 and two sites for PcRsmA. The fact that PcRsmA regulates the expression of Pc-yap1 must also contribute to this trait. We have elaborated a model for the cellular processes regulated by PcYap1 and PcRsmA that have been described in this work (Fig. 11).
Oxidative stress is an important factor in industrial processes in bioreactors , hence the interest in characterizing the oxidative stress response and its influence on SM production in industrially important fungi, which may help us to develop more stress-tolerant strains and improve production . The findings obtained in this work may also have applications in the development of P. chrysogenum strains as heterologous expression systems , by means of using cis-acting regulatory elements to increase transcription of engineered promoters modulated by oxidative stress. Our results increase the knowledge on the regulation of the penicillin biosynthetic genes, a model for secondary metabolism gene regulation studies.
bZIP transcription factors PcYap1 and PcRsmA respond to the presence of H2O2-generated ROS and regulate oxidative stress response in the cell. Both proteins mediate ROS regulation of penicillin biosynthesis and conidiation by binding to specific regulatory elements in the promoters of key genes.
PcYap1 binds to a previously identified regulatory sequence in the promoter of the penicillin gene pcbAB (TTAGTAA), thus revealing the identity of the binding protein named PTA1 by Kosalková et al. . This is the first report of a Yap1 protein directly regulating transcription of a secondary metabolite gene.
PcYap1 binds to a TTACTAA sequence in the promoter of the first gene of the conidiation central regulatory pathway: brlA, thus regulating its expression and the conidiation process.
PcRsmA regulates penicillin biosynthesis and binds to the sequences TGAGACA and TTACGTAA (CRE motif) in the promoters of the genes pcbAB and penDE, respectively. PcRsmA also regulates transcription of the brlA and Pc-yap1 genes.
Materials and methods
Strains and growth conditions
P. chrysogenum Wisconsin 54-1255 (also named Wis54-1255) (ATCC 28089) was used as a recipient for genetic transformation with plasmids derived from the silencing vector pGpdPki-RNAi, and as a control in the characterization of strains with knocked down expression of Pc-yap1 and Pc-rsmA. P. chrysogenum Wis54-1255 pyrG, auxotroph of uridine, was obtained from strain Wis54-1255 by random mutation with N-methyl-N-nitro-N-nitrosoguanidine ; it was used as a recipient for genetic transformation with plasmids derived from the vector pBKSpyrG and as a control in the characterization of strains overexpressing the genes Pc-yap1 and Pc-rsmA. P. chrysogenum Q204L was obtained by transformation of strain Wis54-1255 with a plasmid carrying a dominant activating allele of the pga1 gene (pga1Q204L), which encodes a Gα subunit of the heterotrimeric G protein of P. chrysogenum (Zúñiga-León et al. unpublished results); it was used as a control in Northern blot for transcriptional repression of the brlA gene. All P. chrysogenum strains were stored at − 20 °C in 40% glycerol, and grown on plates with Power medium  for 7 days at 26 °C to collect spores for inoculation of submerged cultures. For the growth of strain Wis54-1255 pyrG, the media were supplemented with uridine at a final concentration of 100 µg/mL.
P. pastoris X-33 was used for heterologous expression of P. chrysogenum proteins. It was stored at − 20 °C in 40% glycerol and maintained in YPD agar medium. Escherichia coli DH5α was used as a host for plasmid constructions. Micrococcus luteus ATCC-9341 was used in bioassays to quantify penicillin concentration.
Cultures for penicillin production
Conidia collected from cultures on plates with Power medium were inoculated into 100 mL of complex seed (CS) medium  in flasks, at a final concentration of 5 × 106 conidia/mL. The cultures were incubated for 24 h at 250 rpm and 28 °C. Eight millilitres from the seed cultures were then inoculated, in triplicate, into flasks containing 50 mL of complex production (CP) medium (gxL−1: lactose, 55; corn steep solids, 35; CaCO3, 10; KH2PO4, 7; MgSO4-7H2O, 3; potassium phenylacetate, 4; pH 6.1), which were incubated for 120 h at 250 rpm and 28 °C. Every 12 or 24 h, 2 mL samples were taken to determine penicillin G and dry weight as previously described [84, 85]. For cultures submitted to oxidative stress, H2O2 was added to the CP medium at the time of inoculation at a final concentration of 25, 50, 100, 150 and 200 mM.
Quantification of reactive oxygen species (ROS)
Cultures were performed as indicated above for penicillin production. For the determination of ROS in mycelium from control and 100 mM H2O2-added cultures we followed the method described by Miranda et al.  with some modifications. Every 24 h, 2 mL samples were taken from the CP medium. Each sample was divided in two 1-mL microtubes, centrifuged for 10 min at 14,000 rpm, and the pelleted biomass was resuspended in 1 mL of cold PBS. To one tube, 10 µM of H2DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) was added in the dark, and the tube was maintained on ice for 40 min, the other tube was a background control without H2DCF-DA. From each sample, 200 µL were put in a 96-well microplate, in triplicate, and the absorbance reading was adjusted to 485 nm excitation and 530 nm emission in a DTX 880 multimode plate reader (Beckman-Coulter). The obtained signal was corrected by subtracting the background signal and normalized with the biomass (dry weight).
Analysis of conidiation
Fifty microliters of a suspension with 1 × 106 conidia/mL were inoculated on the center of a Petri dish with Power medium, which was incubated at 28 °C for 120 h. Every 24 h conidia were collected by adding 5 mL of NaCl 0.9% and scraping the surface, then they were centrifuged, concentrated to a final volume of 1 mL, and diluted for counting in a Neubauer chamber. The results were expressed as conidia collected per mL of solution. For cultures submitted to oxidative stress, H2O2 was added to the Power medium at a final concentration of 20, 50, 100, 140, and 180 mM at the time of casting the medium onto the plates.
Oxidative stress resistance tests for conidia
One hundred microlitres from a suspension with 1 × 104 conidia/mL was mixed with 900 µL of a solution of NaCl 0.9% containing different concentrations of H2O2: 0, 100, 130, 160, and 180 mM. The mixture was incubated for 20 min at room temperature and a volume of 50 µL was inoculated on the surface of PDA dishes, which were incubated at 28 °C for 24 h, after which grown colonies were counted.
Genes and proteins used in BLAST searches, sequence alignments and primer design
The sequences of bZip transcription factors AP-1/Yap1 from A. fumigatus (GenBank accession no. XP_750882.1; UniProt: Q4WMH0), Fcr3 (gene ANIA_04562), RsmA from A. nidulans (GenBank accession no. XP_662166.1; UniProt: Q5B4G8), and Atf21/AtfB from A. flavus (GenBank accession no. XP_002381221.1; UniProt: B8NLU5) were used as query to perform BLAST searches to find their putative homologs in the P. chrysogenum genome (P. chrysogenum Wis54-1255, aka P. rubens Wis54-1255, taxid 500485). (See text).
Other protein sequences used for BLAST searches and sequence alignments were: AP-1-like transcription factor YAP1 (NP_013707; P19880) and AP-1-like transcription factor YAP3 (NP_011854; P38749) from S. cerevisiae; transcription factor Pap1/Caf3 (NP_593662; Q01663) and Atf-CREB family transcription factor Atf21 (NP_595707; P78962) from S. pombe; fluconazole resistance protein FCR3 (AAL35299; Q8X229) from C. albicans; AP-1-like transcription factor NapA (XP_680782; Q5AW17) and bZIP transcription factor Atf21 (CBF78271; Q5AST7) from A. nidulans; bZIP transcription factor Fcr3 (AfRsmA) (XP_749389; Q4WIA4) and bZIP transcription factor Atf21 (KMK62786; A0A0J5Q1E3) from A. fumigatus; bZIP transcription factor Atf21 (XP_001274576; A1CBN2) from A. clavatus; basic leucine zipper (bZIP) transcription factor AtfB (XP_001824132; Q2U616) from A. oryzae; CRE1 binding protein/basic leucine zipper (bZIP) transcription factor AtfB (ADZ06147; A0A0F0IP79) from A. parasiticus; hypothetical protein FGSG_08800 (Fgap1) (XP_011319920; I1RWW4) from F. graminearum.
For the designing of primers of the act (gamma-actin) and brlA genes of P. chrysogenum the sequences Pc20g11630 and Pc06g00470 in GenBank were used, respectively.
Total DNA from P. chrysogenum was obtained from mycelium grown in MPPY medium . The mycelium was powdered with liquid nitrogen in a mortar and DNA extraction was performed as described by Fierro et al. . Alternatively, for extraction of small amounts of DNA, the mycelium was broken in 2-mL tubes with 0.5-mm glassbeads using a MINIBEADBEATER (Biospec) for 3 min, and DNA extraction was performed with the Wizard Genomic DNA Purification kit (Promega) following the manufacturer’s indications.
Cloning of Pc-yap1, Pc-rsmA and Pc-atf21 from the P. chrysogenum Wis54-1255 genome
Amplification of the Pc-yap1, Pc-rsmA and Pc-atf21 genes containing their respective promoters and terminators was performed by PCR using primers Yap1-F and -R, RsmA-F and -R, AtfB-F and -R (Additional file 12). The amplified fragments were digested with the appropriate restriction enzymes for the restriction sites present at the 5’-ends of the primers and inserted into the previously digested pBluescript KS + vector. (See text for the identity of the genes in the P. chrysogenum genome).
Construction of plasmids for RNAi-mediated silencing and overexpression of the Pc-yap1 and Pc-rsmA genes
RNAi-mediated gene silencing is an efficient method to knock down the expression of genes in P. chrysogenum [83, 87, 88]. We followed a strategy based on the generation of dsRNA as described by Ullán et al.  and Cepeda-Garcia et al. , using the vector pGpdPki-RNAi (Marcial-Quino, Miranda, Fierro, unpublished) (Additional file 1). Primers siYAP1-F and -R, and siRSMA-F and -R, were used for PCR amplification of fragments from the Pc-yap1 and Pc-rsmA genes, respectively (see Additional file 1 for details). These fragments were inserted into the silencing vector pGpdPki-RNAi at the NcoI site located between the two opposite constitutive promoters (A. nidulans gpd and Aspergillus niger pki), to obtain the silencing plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA.
For gene overexpression, the pki constitutive promoter was fused to a DNA fragment containing the ORF plus 300 bp downstream the stop codon (thus comprising the terminator) from each of the two genes. A strategy based on recombinant PCR was used for the fusion at the ATG start codon (Additional file 2). For the construction of plasmid pPyrG-pki::Pc-yap1, the pki promoter was amplified with primers pki-PcYap1-F and -R (using plasmid pGpdPki-RNAi as template), obtaining a fragment of 1041 bp. The Pc-yap1 gene was amplified with primers PcYap1-Ter-F and -R to obtain a fragment of 2181 bp. The product of both PCRs was mixed and submitted to one PCR cycle with the conditions: 5 min at 95 °C, 5 min at 57 °C and 10 min at 72 °C, and then to 30 cycles with the conditions: 30 s at 95 °C, 1 min at 57 °C, 4 min 72 °C. pfu Ultra II Fusion HS DNA Polymerase (Agilent) was used in all reactions. For the construction of plasmid pPyrG-pki::Pc-rsmA the same steps were followed, using primers pki-RsmA-F and -R, and RsmA‐Ter‐F and ‐R (Additional file 12). The final amplified fusion fragments were digested with the appropriate restriction enzymes (see legend to Additional file 2) and inserted into the vector pBKSpyrG (pBluescript-derived containing the pyrG gene of P. chrysogenum).
Transformation of P. chrysogenum
P. chrysogenum Wis54-1255 was the recipient for transformation with plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA for gene silencing. The transformation was performed as described by Cantoral et al.  using phleomycin at a concentration of 30 µg/mL as selection marker when regenerating the protoplasts on Czapek minimal medium supplemented with 1 M sorbitol. Colonies were then submitted to transfers in different media under selective pressure to ensure the stability of transformants (Power to get conidia – Czapek after dilution to single conidia and plating – Power to grow the final homokaryons).
P. chrysogenum Wis54-1255 pyrG was the recipient for transformation with plasmids pPyrG-pki::Pc-yap1 and pPyrG-pki::Pc-rsmA for gene overexpression. The transformation was performed as described by Díez et al.  using auxotrophy of uridine as selection marker when regenerating the protoplasts on Czapek minimal medium supplemented with 0.7 M KCl. Colonies were then submitted to the same transfers described above.
Confirmation and selection of P. chrysogenum transformants with knocked down expression and overexpression of genes Pc-yap1 and Pc-rsmA
Out of a total of 50 transformants with plasmid pGpdPki-RNAi/PcYap1, we chose eight based on similar macroscopic phenotypes between them, which were named Y1, Y3, Y4, Y5, Y8, Y11, Y12, Y18. The same procedure was followed for the transformants obtained with plasmid pGpdPki-RNAi/PcRsmA, which were named R24, R25, R30, R31, R32, R33, R34, R39. The presence of the silencing plasmids in these transformants was confirmed by PCR (Additional file 3). Preliminary assays with these transformants were carried out consisting of analysis of phenotypic features (rate of conidiation, resistance to ROS) and non-quantitative RT-PCR. From the obtained results, we chose two transformants from each gene: Y8, Y12, R24 and R25 (named as strains si-PcYap1-8, si-PcYap1-12, si-PcRsmA-24 and si-PcRsmA-25), which showed typical, average and stable phenotypes. These strains were submitted to semiquantitative RT-PCR analysis to confirm knocked-down expression of the Pc-yap1 and Pc-rsmA genes (Additional file 3E). The results showed undetectable amounts of Pc-rsmA transcript in both si-PcRsmA-24 and si-PcRsmA-25 strains, and around 28% Pc-yap1 transcript with respect to the amount in the parental Wis54-1255 strain in both si-PcYap1-8 and si-PcYap1-12 strains. These strains were thus selected to study the function of the PcYap1 and PcRsmA proteins in the cell.
A similar approach was followed for transformants obtained with the overexpression plasmids pPyrG-pki::Pc-yap1 and pPyrG-pki::Pc-rsmA. The presence of the plasmids in the transformants was confirmed by PCR (Additional file 4). Two transformants with each overexpressed gene, showing average and stable phenotypes, were chosen for further experiments; they were named: OE::PcYap1-A, OE::PcYap1–E, OE::PcRsmA-C and OE::PcRsmA-H.
Mycelium grown in submerged cultures was collected by filtration, washed with 0.9% NaCl, dried in filter paper and powdered with liquid nitrogen in a mortar. RNA was extracted with Trizol™ reagent (Ambión, Life Technologies) following the manufacturer’s specifications. The RNA was stored at − 80 °C until use.
For mycelium grown on solid agar media the following procedure was used. Discs of 3MM paper (Whatman) were placed on top of Petri dishes with Power medium, and a solution with 1 × 106 conidia was spread onto its surface. The plates were incubated at 28 °C in an oven for 120 h. The mycelium was collected by scraping the surface of the 3MM paper with a sterile steel spatula and powdered with liquid nitrogen in a mortar. RNA was extracted as indicated above.
For Northern blot, a protocol was elaborated based on the standard Northern blot technique with radioactive probes  but using chemiluminescence instead. After RNA extraction from mycelium grown on Power medium, the RNA was quantified in an Epoch microplate reader (BioTek Instruments). A volume containing 10 µg of RNA was mixed (1:1) with standard RNA gel loading buffer (1.25X) and loaded into a denaturing 1.2% agarose gel in MAE buffer 1X (MAE 10X: MOPS 0.2 M, EDTA 50 mM, pH 7) with 3% formaldehyde; running buffer was MAE 1X. Blotting to a Hybond-N+ membrane (GE Healthcare) was made by capillarity with SSC 10X buffer for 12–16 h, after which the RNA was fixed to the membrane in a UVC 500 Crosslinker (Amersham Bioscience) adjusted to 1200 (956 J for 15 s). The probes were labelled with the Biotin DecaLabel DNA Labeling kit (Thermo Scientific) and quantified in an Epoch microplate reader to adjust the concentration to values in the order of ng/µL. Prehybridization of the membrane was performed in a glass container with hybridization buffer (SSC 6X, SDS 0.1%, formamide 40%, Denhart 1X) containing 500 µg/mL salmon sperm DNA (Sigma-Aldrich) for 3 h at 42 °C. This solution was removed and new hybridization buffer containing 100 µg/mL salmon sperm DNA and 15 ng/mL of each labelled probe (previously denatured by boiling for 5 min) was added; the membrane was then incubated for 12 h at 42 °C. The hybridization buffer was removed and the membrane was successively washed with washing buffer I (SSC 2X, SDS 0.1%) at room temperature, washing buffer I at 42 °C, washing buffer II (SSC 0.1X, SDS 0.1%) at 42 °C, and washing buffer II at 65 °C, all washing steps for 15 min. The membrane was left to dry and signal detection was carried out with the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) in a ChemiDoc™ MP Imaging System (Bio-Rad) using the Chemiluminescent channel. The relative density of the hybridization signals was determined with the ImageLab 6–0.1 software (Bio-Rad).
Isolation of the Pc-yap1, Pc-rsmA and Pc-atf21 CDS and insertion in expression vectors for P. pastoris
The CDS of the three genes for heterologous expression of the proteins in P. pastoris was obtained following different procedures (Additional file 5). For Pc-rsmA, a recombinant PCR approach was followed for removal of the introns (see legend to Additional file 5). In the case of Pc-yap1, total RNA was isolated from mycelium grown in submerged culture, then an RT-PCR was performed with primers Yap-1 and Yap-6 (Additional file 12). The Pc-atf21 gene contains no introns, so the CDS was directly amplified from DNA, using as template the previously cloned gene in pBluescript KS + , with primers AtfB-1 and AtfB-2 (Additional file 12). The CDS of Pc-yap1 was inserted in the vector pPICZ-A (Invitrogen) by digestion of the amplified fragment and the vector with the enzymes ApaI and KpnI, whereas the CDS of Pc-rsmA and Pc-atf21 was inserted in the vector pPICZ-B with the enzymes EcoRI and XbaI. In all cases, in-frame insertions were obtained which allowed the expression of c-myc-6xHis-tagged proteins from the three genes (Additional file 5); all insertions were confirmed by sequencing. The resulting plasmids were named: pPICZ-A/PcYap1, pPICZ-B/PcRsmA, and pPICZ-B/PcAtf21.
Transformation of P. pastoris and purification of recombinant 6xHis-tagged proteins
Plasmids pPICZ-A/PcYap1, pPICZ-B/PcRsmA and pPICZ-B/PcAtf21 were introduced in P. pastoris X-33 by electroporation in a GenePulser Xcell (Bio-Rad) following the indications in the manual for the vectors (Invitrogen). The plasmids were linearized with the enzyme SacI prior to transformation. The selection of transformants was performed on YPD agar medium with 50 µg/mL phleomycin. After incubation for 72–96 h at 30 °C colonies of transformants appeared. One colony of each transformant was inoculated in flasks with 50 mL YPD medium containing 2% glycerol and 75 µg/mL phleomycin and incubated at 30 °C, 200 rpm for 16–18 h (reaching an OD600 = 2–6). The cultures were centrifuged for 5 min at 5000 rpm; the cells were resuspended in 100 mL of YP medium with 1% methanol and transferred to 1-L flasks for a new incubation at 200 rpm, 30 °C for 72 h. Methanol was added to the cultures at a final concentration of 1% every 24 h to compensate for the evaporation and thus maintain the induction of the AOX1 promoter to express the recombinant proteins. The cultures were centrifuged at 10,000 rpm, 4 °C for 5 min, and the cells resuspended in 15 mL of breaking buffer (NaH2PO4 50 mM, EDTA 1 mM, glycerol 5%, pH 7.4) containing protease inhibitor (Sigma-Aldrich) and glass beads. The cells were disrupted by vortexing (30 s shaking and 1 min on ice for a total of 45 min) and sonication (20 min in an ice bath at 50/60 HZ intensity in a Bransonic Ultrasonic Cleaner). The lysates were centrifuged at 10,000 rpm, 4 °C for 5 min and the supernatants transferred to 50-mL ultrafiltration units (Amicon Ultra 15, 10 KDa pore size, Merck/Millipore), which were centrifuged at 10,000 rpm, 4 °C for 10 min, washed twice with binding buffer (Na2HPO4 50 mM, NaCl 0.5 M, pH 8) and centrifuged until the samples were concentrated to a volume of 1.5 mL.
Purification of the recombinant 6xHis-tagged proteins was performed with the Ni–NTA Spin Kit (Qiagen). A volume of 600 µL Ni–NTA agarose was added to the empty columns; they were centrifuged at 14,000 rpm for 5 min. Then 600 µL of binding buffer was added, the columns were left to equilibrate for 10 min and centrifuged again at 14,000 rpm for 5 min, this step was repeated three times. Finally, 600 µL of the ultrafiltrated protein solutions were added to the columns and left overnight at 13 °C. The proteins were eluted with 600 μL binding buffer containing increasing concentrations of imidazole (10, 20, 50, 200, 300, and 500 mM), centrifuging after each step at 10,000 rpm, 4 °C for 5 min. The eluted solutions were stored at -20 °C. Aliquots were analyzed by SDS-PAGE to confirm purification of the proteins to homogeneity, which was attained with 300–500 mM imidazole (Additional file 6). Elution of PcYap1 with different concentrations of imidazole gave repeatedly two bands of similar size (around 66 kDa), even using extracts from different P. pastoris transformants. Analysis by mass spectrometry of the two bands separately excised from the gel showed that both corresponded to PcYap1, so we interpreted the result as formation of dimers or, alternatively, isomeric forms produced during expression in P. pastoris.
Electrophoretic mobility shift assay (EMSA)
Probes for EMSA were obtained either by PCR amplification when the length exceeded 80 bp or by annealing (after labelling) of two complementary oligos when the length was below 60 bp (Additional file 12); for annealing, the oligos were mixed, denatured for 3 min at 90 °C, the solution was cooled 1 °C per minute down to the oligos Tm, then maintained at this temperature for 30 min and finally cooled again down to room temperature. A 5% native polyacrylamide gel was made as follows: mixing of H2O, 6.25 mL; TBE 5X, 2 mL; acrylamide/BIS 29:1 at 40%, 1.25 mL; sonication for 10 min, and addition of APS 5%, (500 µL) and TEMED, 6 µL. The gel was casted in a Mini-Protean Tetra Cell (Bio-Rad), left to polymerize and pre-run for 60 min at 100 V with cooled TBE 0.5X. The LightShift Chemiluminescent EMSA kit (Thermo Scientific) was used in all steps of the EMSA process. Labelling was performed with Biotin-11-UTP following the protocol of the Biotin 3’ End DNA Labelling kit part. Reactions were prepared by gently mixing the components in this order: MilliQ H2O, 6 µL; binding buffer 10X, 2 µL; glycerol 50%, 4 µL; MgCl2 100 mM, 1 µL; labelled probe (20 fmol), 4 µL; purified protein (10 µg), 3 µL. The reactions were incubated at room temperature for 20 min, then mixed with 5 µL loading buffer 5X and loaded onto the gel. Electrophoresis was carried out at 80 V with cooled TBE 0.5X. Next, the gel was blotted onto a nylon Hybond-N+ membrane (previously equilibrated with TBE 0.5X for 10 min) in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) with cooled TBE 0.5X at constant 380 mA for 60 min. The membrane was then taken out from the assemblage and excess water was removed with filter paper. The DNA was fixed to the membrane in a UVC 500 Crosslinker (Amersham Bioscience) adjusted to 1200 (956 J for 10 min). Signal detection was performed with the Chemiluminescent Nucleic Acid Detection Module kit (Thermo Scientific) following the manufacturer’s indications.
Oxidation assays of purified proteins
Protein oxidation assays were performed adapting methodologies previously described by Delaunay et al.  and Wood et al. . H2O2 was added at different concentrations (see Fig. 7) to solutions containing 15 or 25 µg of purified PcYap1::c-myc-6xHis and PcRsmA::c-myc-6xHis in binding buffer (see above); the mixtures were incubated for 0, 5, 15, or 30 min at room temperature. Samples from these reactions were run in 8–10% SDS-PAGE gels in reducing or non-reducing conditions. For reducing conditions, 20 µL of protein loading buffer (Tris–HCl 0.5 M pH 6.8, 1.25 mL; Glycerol, 2.5 mL; SDS 10%, 2 mL; bromophenol blue 0.5%, 0.2 mL; MilliQ water, 3.55 mL) containing 5% β-mercaptoethanol was added to the samples, which were heated at 90 °C for 5 min and loaded onto the gels. For non-reducing conditions, the samples were mixed with 20 µL of loading buffer without β-mercaptoethanol and they were not heated. Electrophoresis were performed in Mini-Protean Tetra Cell (Bio-Rad) at 150 V for approximately 90 min. The gels were stained with Blue Silver Coomasie  and analyzed in a Molecular imager Gel Doc EZ System using White tray (Bio-Rad).
Mass spectrometry of proteins
From an SDS-PAGE fixed and stained with Blue Silver Coomassie, the bands of interest were cut and deposited in 0.25-mL microtubes previously washed with acetonitrile. The digestion with trypsin was performed with the In-Gel Tryptic Digestion Kit (Thermo Scientific). The samples were concentrated and desalted in Pierce® C18 Spin Columns (Thermo Scientific). A volume of 2 μL of sample was mixed with 2 µL of the matrix (α-cyano-4-hydroxycinnamic acid), and 1 µL of this solution was loaded in duplicate into the plate of a MALDI-TOF mass spectrometer (Bruker). Proteins were identified by peptide fingerprinting.
Basic Local Alignment Search Tool (BLAST) at NCBI was used to retrieve proteins orthologous to the ones studied in this work from the NCBI and EBI databases.
Clustal Omega from the EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used for multiple protein alignments .
For the prediction of NLS in PcYap1 and PcRsmA we used the cNLS Mapper tool (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi), which predicts importin α-dependent signals .
For prediction of NES we used LocNES (http://prodata.swmed.edu/LocNES/LocNES.php), which predicts signals for CRM1-mediated nuclear export .
The STRING tool (STRING Consortium 2021, https://string-db.org/) was used to identify putative interactions of PcYap1 and PcRsmA .
For statistical analysis of quantifiable data, the NCSS-PASS-GESS program (NCSS, PASS, USA) was used. Means were submitted to a Tukey–Kramer multiple comparison test to determine statistical significance (set at P < 0.05).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional files.
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The authors thank INBIOTEC and the Laboratorio de Medicina Experimental, Unidad de Medicina Traslacional, IIB, UNAM/Instituto Nacional de Cardiología Ignacio Chávez for the use of their facilities to carry out some experiments.
Wylma Pérez and Ulises Carrasco received research grants from CONACyT (scholarships 265789 and 328318, respectively). The equipment and some materials for the development of this research were funded in part by CONACyT (Project CB-2013-01 222207).
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Additional file 1
. Strategy for silencing of expression of Pc-yap1 and Pc-rsmA. (A) A DNA fragment of 458 bp from the exon 2 of Pc-yap1 was amplified by PCR with primers siYAP1-F and -R, digested with NcoI and inserted at the NcoI site of plasmid pGpdPki-RNAi to obtain plasmid pGpdPki-RNAi/PcYap1. (B) A DNA fragment of 409 bp from the exon 3 of the Pc-rsmA gene was amplified by PCR with primers siRsmA-F and -R, digested with NcoI and inserted at the NcoI site of plasmid pGpdPki-RNAi to obtain plasmid pGpdPki-RNAi/PcRsmA. (C) Strategy of silencing. Transcription from the opposite-oriented promoters gpd and pki generates complementary RNA strands that form a dsRNA with the sequence of the inserted 458 bp fragment from Pc-yap1. This dsRNA will cause silencing of the expression of Pc-yap1 through the RNAi pathway.
Additional file 2
. Recombinant PCR for overexpression of genes Pc-yap1 and Pc-rsmA. The pki gene promoter from A. niger was fused to fragments from the genes extending from the ATG start codon to around 300 bp downstream the TGA stop codon to ensure the presence of the transcriptional terminator. The position of the primers used for PCR reactions is indicated (see Materials and Methods for details). The final fragments with the genes fused to the pki promoter were digested with the restriction enzymes EcoRV and SpeI (Ppki::Pc-yap1) or KpnI and XhoI (Ppki::Pc-rsmA) and inserted in the vector pBKSpyrG to obtain the plasmids pPyrG-pki::Pc-yap1 and pPyrG-pki::Pc-rsmA, respectively; the restriction sites in the primers are highlighted in color. Introns are indicated in grey color. Added restriction sites at the 5’-end of the primers are highlighted in orange color.
Additional file 3
. Confirmation of the presence of plasmids for RNAi-mediated silencing of Pc-yap1 and Pc-rsmA and analysis of silencing in P. chrysogenum transformants. (A) Close-up of the pGpdPki-RNAi vector region with the opposite-oriented gpd and pki promoters, and the plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA with the inserted DNA fragments from the Pc-yap1 and Pc-rsmA genes at the NcoI site. Primers Gpd1(pki1)R and Pki1(gpd1)F are shown at the position of annealing with sequences in the gpd and pki promoters, respectively. The expected size of amplified DNA fragments in each type of transformant is indicated with double-headed arrows. (B) Agarose gel with the result of PCR amplification with primers Gpd1(pki1)R and Pki1(gpd1)F using as template DNA from the purified pGpdPki-RNAi vector (lane V), DNA from strain C1 containing the pGpdPki-RNAi vector (lane C1) and DNA from strain Wis54-1255 (lane W). (C) Results of the PCR amplification performed with the mentioned primers and DNA from eight transformants with the pGpdPki-RNAi/PcYap1 plasmid (Y1…. Y18). (D) Results of the PCR amplification performed with the mentioned primers and DNA from eight transformants with the pGpdPki-RNAi/PcRsmA plasmid (R24… R39). (E) Silencing of expression of Pc-yap1 (upper panels) and Pc-rsmA (lower panels) in selected strains containing the RNAi-silencing plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA, respectively. RNA was extracted from mycelium grown for 60 h in MPPY medium and used for semiquantitative RT-PCR (as described by Domínguez-Santos et al. ) using primers qPcYap1-F and -R for analysis of Pc-yap1 expression, qRsmA-F and -R for Pc-rsmA, and qactA-F and -R for act. The left panels show the intensity of the bands in an agarose gel loaded with the products of the RT-PCR reactions, and the right panels the densitometry analysis of the bands. The results were normalized with the bands of the constitutively expressed act gene. The parental strain Wis54-1255 (lane W) was used as reference with a value for integrated optical density (IOD) of 100. Y8: strain si-PcYap1-8; Y12: si-PcYap1-12; R24: si-PcRsmA-24; R25: si-PcRsmA-25.
Additional file 4
. Confirmation of the presence of plasmids for overexpression of Pc-yap1 and Pc-rsmA in P. chrysogenum transformants. (A) Scheme of the fusion of the pki promoter with the Pc-yap1 (top) and Pc-rsmA (down) genes at the ATG start codon in the plasmids pPyrG-pki::Pc-yap1 and pPyrG-pki::Pc-rsmA. Primers Pki1(gpd1)F, OE-PcYap1-R, siYAP1-R, OE-RsmA-R and siRSMA-R are shown at the position of annealing with sequences in the pki promoter and the Pc-yap1 and Pc-rsmA genes, respectively. The expected size of amplified DNA fragments in each type of transformant for every primer pair is indicated with double-headed arrows. (B) Agarose gel with the result of PCR amplification with primers Pki1(gpd1)F and OE-PcYap1-R using as template DNA from the purified pPyrG-pki::Pc-yap1 plasmid (lane P), DNA from strain Wis54-1255 (lane W), a transformant with the empty pBKSpyrG vector (lane Y) and transformants OE::PcYap1-A through –F (lanes A through F). Transformants selected for further characterization are indicated with a red circle. (C) Result of PCR amplification with primers Pki1(gpd1)F and OE-RsmA-R using as template DNA from transformants OE::PcRsmA-A through –H (lanes A through H). (D) Result of PCR amplification with primers Pki1(gpd1)F and siRSMA-R using as template DNA from the purified pPyrG-pki::Pc-rsmA plasmid (lane P), total DNA from strain Wis54-1255 (lane W), a transformant with the empty pBKSpyrG vector (lane Y) and transformants OE::Pc-rsmA-C through –H (lanes C through H). Transformants selected for further characterization are indicated with a red circle.
Additional file 5
. Isolation of Pc-yap1, Pc-rsmA and Pc-atf21 CDS and insertion in expression vectors for P. pastoris. (A) An RT-PCR was performed using RNA from a submerged culture and primers Yap-1 and Yap-6, which contain restriction sites for KpnI and ApaI, respectively. The amplified fragment was inserted in the pPICZ-A vector after digestion with these enzymes and ligation. The in-frame insertion is shown, indicating the position of the cloning enzymes, the Pc-yap1 CDS and the c-myc-6xHis tag. (B) For Pc-rsmA, a recombinant PCR approach was followed, designing primers with sequences linking exon 1 to 2 (RsmA-2 and RsmA-3) and exon 2 to 3 (RsmA-4 and RsmA-5), plus primer RsmA-1, annealing at the start of the ORF and containing an EcoRI restriction site at 5’, and primer RsmA-6, annealing at the end of the ORF and containing an XbaI restriction site. PCR reactions were performed to separately amplify exon 1 (primers RsmA-1 and -2), exon 2 (primers RsmA-3 and -4) and exon 3 (primers RsmA-5 and -6). The products of the first two reactions were mixed together and a new PCR was performed with primers RsmA-1 and -4 to fuse exons 1 and 2. Finally, the product of the latter reaction was mixed with the product of the reaction of exon 3 and a PCR was performed with primers RsmA-1 and RsmA-6 to obtain the entire CDS, from the ATG to the last codon (next to the Stop codon but not including it). This fragment was digested with the enzymes EcoRI and XbaI and inserted in the pPICZ-B vector digested with the same enzymes, so that the Pc-rsmA gene ORF was fused in frame with the sequences in the vector encoding the c-myc epitope and the 6xHis tag. (C) For Pc-atf21, the previously cloned gene was used as template for amplification with primers AtfB-1 and AtfB-2, since this gene contains no introns. The cloning procedure in vector pPICZ-B was as described above. (Asterisks indicate additional amino acids in the fusion proteins resulting from the cloning strategies. In the case of Pc-atf21, the cloning strategy resulted in the loss of the last amino acid, Asn).
Additional file 6
. Purification of recombinant c-myc-6xHis-tagged PcYap1, PcRsmA and PcAtf21 proteins after expression in P. pastoris. Protein extracts were obtained as described in Materials and Methods and loaded onto Ni-NTA Spin 50 (Qiagen) columns. The tagged proteins were eluted with increasing concentrations of imidazole. Aliquots of the eluted samples were analyzed by SDS-PAGE, lane M: size marker, lane CE: protein crude extract, imidazole concentrations were 100, 250, 300 and 500 mM for PcYap1::c-myc-6xHis, and 50, 200, 300 and 500 mM for PcRsmA::c-myc-6xHis and PcAtf21::c-myc-6xHis. Bottom right panel, partially purified PcRsmA::c-myc-6xHis and PcAtf21::c-myc-6xHis run on the same gel for size comparison purposes.
Additional file 7
. EMSA to analyze possible interactions between PcYap1 and PcRsmA upon binding to their respective binding sites. Probes containing either the PcYap1-binding site (PTA1-WT), the PcRsmA binding site (RsmA-2C) or both (upPta1) were incubated with their respective binding proteins (lanes 2, 4, 6, 7) or with both proteins (lanes 3, 5, 8). The results show that no apparent interaction between the proteins occurs when one of them recognizes and binds its specific site (lanes 3, 5). In lane 8 a supershift takes place. Probe upPta1 contains both binding sites for each of the proteins, therefore the supershift pattern does not demonstrate interaction between the proteins and can be explained by the simultaneous binding of the proteins to their respective sites; nevertheless, interaction when this simultaneous binding occurs cannot be excluded.
Additional file 8
. Clustal W alignment of PcYap1 with S. cerevisiae Yap1 and S. pombe Pap1, and conservation of functional domains between them. Nuclear import and export sequences (NLS and NES) are highly conserved. Also conserved are the cysteine-rich domains N-CRD and C-CRD, the latter overlapping the NES, and the key cysteine residues forming disulphide bonds described in Yap1 which mask the NES and prevent export form the nucleus, resulting in nuclear location of the protein in oxidative conditions (see text for details).
Additional file 9
. Clustal W alignment of PcRsmA with A. nidulans RsmA, C. albicans FCR3, and S. cerevisiae Yap3. An NLS is well conserved in all proteins at the same position as in the Yap1 proteins (the beginning of the bZIP domain). Three putative NES were detected with LocNES (from Support Vector Machine) in the amino acid sequence of PcRsmA; two of them, NES-2 and NES-3, show some conservation with the equivalent regions in the other three proteins. NES-3 is located at the C-terminus and between the only two cysteine residues present in PcRsmA, which should form a disulphide bond in oxidative conditions that would cause the conformational change detected by SDS-PAGE (Fig. 7). A cysteine rich domain located at the N-terminus in Yap3 is not conserved in any of the other proteins.
Additional file 10
. Radial growth of strains with knocked down expression of Pc-yap1 and Pc-rsmA (A) and strains overexpressing the respective genes (B). Strain C1 contains the empty pGpdPki-RNAi vector used for gene silencing and strain C2 (pyrG) contains the empty pBKSpyrG vector used for gene overexpression. Three microliters of a suspension with 1x104 conidia/mL were inoculated on the center of a Petri dish with PDA medium. The cultures were incubated in the dark at 28 °C for 144 h. Every 24 h the diameter of the colonies was measured.
Additional file 11
. Conidia production of strains overexpressing Pc-yap1 or Pc-rsmA in Power medium without added H2O2. Strain C2 (pyrG) contains the empty pBKSpyrG vector used for gene overexpression. Bar sizes are the result of three biological replicas, error bars correspond to standard deviation. See Materials and Methods for details.
Additional file 12
. Oligonucleotides used in this work. Added restriction enzyme sites at the 5’ end of several primers are highlighted in color.
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Pérez-Pérez, W.D., Carrasco-Navarro, U., García‑Estrada, C. et al. bZIP transcription factors PcYap1 and PcRsmA link oxidative stress response to secondary metabolism and development in Penicillium chrysogenum. Microb Cell Fact 21, 50 (2022). https://doi.org/10.1186/s12934-022-01765-w
- bZIP transcription factor
- DNA-binding proteins
- Transcriptional regulation
- Secondary metabolism
- Fungal morphogenesis
- Reactive oxygen species
- Oxidative stress defense