YPR2 has its major function in darkness
In order to evaluate the genome wide regulatory function of YPR2 we cultivated ∆ypr2 and wildtype on minimal medium with cellulose as carbon source under controlled light conditions for comparative transcriptome analysis. Transcript levels in the mutant strain were compared to wildtype for light and darkness separately in order to assess distinct regulation patterns by YPR2 under both conditions (>2fold regulation, p-value threshold 0.01). We found that the main regulatory function of YPR2 happens in darkness (Fig. 1b, c). This finding is in accordance with earlier results on global regulation of secreted metabolites by high performance thin layer chromatography (HPTLC), showing a clear alteration in ∆ypr2 in darkness [5].
In darkness, we found 539 genes to be downregulated and 861 genes to be up-regulated in ∆ypr2 upon growth on cellulose, while in light only 20 genes were downregulated and 74 genes upregulated (Fig. 1c, Additional file 1). Fifty genes were regulated by YPR2 in light and darkness (Fig. 1d).
Previously, we evaluated which gene set would be regulated under conditions causing cellulase induction (growth on cellulose, lactose and sophorose) compared to conditions repressing cellulase expression (glucose, glycerol), which revealed 1324 genes, we called “induction specific” [13]. We checked for a possible overlap of this gene set with that influenced by YPR2. In darkness 141 of the genes regulated by YPR2 were previously found to show induction specific regulation [13]. Although the photoreceptor proteins BLR1, BLR2 and ENV1 exert their main function in light, they influence gene regulation in darkness as well [18, 22, 33]. Interestingly, 977 genes (70%) targeted by YPR2 in darkness are subject to regulation by one or more photoreceptors [22] (Additional file 1) indicating that many of the genes influenced by YPR2 are relevant for light response as well. Transcript patterns of SOR cluster genes in this transcriptome dataset on YPR2 are in accordance with detailed RT-qPCR data shown previously [5], hence validating the presented results. Additionally, deletion of ypr2 causes decreased transcript levels of ypr1 (TR_104299), a strong regulator of the SOR cluster [24]. We then tested for non-random distribution of genes regulated by YPR2 in light and darkness and considered three or more neighbouring, coregulated genes as a cluster. Thereby, we detected 40 clusters upregulated in ∆ypr2 in darkness and 30 clusters downregulated. In many cases these clusters included CAZyme encoding genes and secondary metabolism associated genes (Additional file 1). Only one such cluster was found in light.
YPR2 impacts regulation of carbon and secondary metabolism
Functional category analysis was performed to evaluate statistically significant enrichment (p-value < 0.05) of gene functions in the respective groups (Fig. 2a and b, Additional file 2 and Additional file 3: Figure S1). Interestingly, although numerous genes associated with metabolic functions were downregulated in darkness in ∆ypr2, significant enrichment was only observed for genes involved in secondary metabolism (p-value 5.87E-09). Specifically, enrichment occurred with metabolism of polyketides and non ribosomal peptide synthesis. Additionally, functions in siderophore-iron transport along with other transport function and correspondingly, homeostasis of metal ions as well as serine/threonine protein kinase functions were enriched.
Genes up-regulated in darkness in ∆ypr2 showed significant enrichment in metabolic functions (p-value 1.29E-05), particularly in amino acid metabolism as well as regulation of nitrogen, sulphur and selenium metabolism. Moreover, genes involved in C-2, C-4 and organic acid metabolism were enriched as well as those functioning in aliphatic hydrocarbon catabolism. Enrichment of the upregulated gene set in secondary metabolism, particularly metabolism of polyketides, alkaloides and secondary products derived from L-tryptophan, L-phenylalanine and L-tyrosine indicates that lack of YPR2 in the genome causes a shift in secondary metabolite production in darkness that may involve amino acid derived compounds. Moreover, this analysis reflects a broad impact of YPR2 on carbon and secondary metabolism (Fig. 2a and b).
Enrichment of glycolysis and gluconeogenesis related genes among those upregulated in darkness indicates increased investment of resources upon lack of YPR2, which might be fueled by enrichment of genes with functions in C-compound and carbohydrate transport. Interestingly, also genes involved in stress response show significant enrichment in upregulated genes in darkness, including catalase functions and particularly strong enrichment in detoxification functions.
The numbers of genes regulated by YPR2 in light are considerable smaller than in darkness. Among genes down regulated in light in ∆ypr2, genes involved in secondary metabolism are enriched as well along with different functions in transport. Upregulated genes in light are enriched in C-compound and carbohydrate metabolism, polysaccharide metabolism as well as transport facilities. Again, as seen in darkness, the enrichment in functions in secondary metabolism in up- and down regulated genes also in light indicates that the functional shift as observed in darkness, occurs.
Genes regulated by YPR2 in darkness
A total of 61 CAZyme encoding genes are upregulated in ∆ypr2, including 15 carbohydrate esterase genes, 38 glycoside hydrolase genes of diverse families and six glycosyl transferase genes (Additional file 1). Among these genes are four chitinases including ech42 and chit36, which are involved in mycoparasitism, extracellular chitin degradation and recycling of cell wall components upon autolysis and starvation [34,35,36]. Moreover, the alpha-galactosidase genes agl1 and agl2 as well as lxr1 encoding a mannitol dehydrogenase [37] are upregulated in ∆ypr2 in darkness. The heterotrimeric G-protein pathway has been shown to function in sexual development [38], regulation of cellulase gene expression [14] and glucose sensing [13] in T. reesei and diverse functions in other fungi [39]. Of the 57 G-protein coupled receptors of T. reesei [9], 11 are up-regulated in ∆ypr2 including the pheromone receptor gene hpr1 and the peptide pheromone transporter gene ste6p. Additionally the meiosis related genes ecm4, pdc1, gtt1 and msc1 were up-regulated. However, no alterations in sexual development were observed for ∆ypr2 (E. Stappler, unpublished).
Concerning secondary metabolism, we found the regulator vel1, which is involved in chemical communication upon sexual development [40] as well as in cellulase regulation [41] to be up-regulated in ∆ypr2 along with 11 genes encoding cytochrome P450 proteins, the NRPS gene tex19, the PKS/NRPS hybrid gene tex11 and the PKS gene pks9g, for which no functional characterization is available.
The strikingly high number of 59 transcription factor genes positively influenced by YPR2 suggests a flat hierarchical regulatory network triggered by YPR2. Unfortunately, none of these transcription factor genes has been studied in detail so far.
Of the seven catalase genes detected in T. reesei [9], 4 are upregulated in darkness in ∆ypr2 up to more than 20 fold indicating a strong antioxidant response balanced by YPR2.
Among the genes downregulated in ∆ypr2 we found 30 CAZyme encoding genes, including numerous carbohydrate esterase genes, glycoside hydrolases and glycosyl transferases (Additional file 1). However, as with up-regulated genes, the classical genes required for plant cell wall degradation, particularly cellulases and hemicellulases are not the targets of YPR2 and neither are the known cellulase transcription factors. Only vib1, which was recently shown to be involved in cellulase regulation in T. reesei [42] and N. crassa [43] is a target of YPR2 with transcript levels decreased by roughly 60% in darkness (Additional file 1).
The downregulated gene set associated with secondary metabolism (14 genes) includes 5 genes encoding cytochrome P450 proteins, the putative alamethicin synthase tex1 and several more pks and terpenoid synthase genes.
Down-regulation of 9 G-protein coupled receptors, while also several GPCRs are up-regulated in the absence of YPR2, indicates a shift in priorities of signal perception triggered by YPR2.
Interestingly, the hydrophobin genes hfb1, hfb2, hfb3 and hfb5 as well as epl1/sm1 were downregulated in ∆ypr2. Known functions of hydrophobins include many morphogenetic events like sexual (fruiting body formation) and asexual development (sporulation) as well as infection structure formation [44]. An antioxidant activity of T. reesei hydrophobins was suggested by a recent study [45]. The ceratoplatanin elicitor Sm1 is important for plant root interaction and elicitation of disease resistance by Trichoderma spp. [46, 47], while its Sclerotinia sclerotiorum homologue is relevant for pathogenicity [48]. These biological roles may be connected to genes regulated by YPR2 targeting sexual development, signaling and secondary metabolism due to an effect on chemical communication and interaction with fungi and plants in the environment.
Not only fold-regulation, but also absolute transcript levels are relevant as they reflect an investment of considerable resources for expression of a given gene. Therefore we checked for striking alterations among the 100 genes with highest detected transcript levels in ∆ypr2 compared to wildtype. The GMC oxidoreductase gene encoding aox1 was among the 10 genes with the strongest signal in the mutant in contrast to wildtype, with 25fold upregulation in ∆ypr2. Interestingly, aox1 is also strongly upregulated in ∆cre1 in darkness [5]. Additionally, a gene encoding an extracellular membrane protein (TR_123475) and a gene encoding a small cystein rich protein (TR_105533), both with potential effector function as well as a transporter with putative putative tetracyclin resistance function (TR_44956) and a gene of unknown function (TR_44967) show high transcript abundance in ∆ypr2, but not wildtype.
Genes regulated by YPR2 in light
Compared to the effect of YPR2 in darkness, only few genes are directly or indirectly regulated by YPR2 in light (Fig. 1c). Interestingly, in contrast to darkness, upregulation was detected for several genes encoding plant cell wall degrading enzymes. However, transcript levels of these genes in QM6a is at very low levels and even hardly detectable in some cases in light on cellulose and the increase (albeit considerable in fold values) in ∆ypr2 does by far not reach darkness levels of these transcripts. Essentially the same applies also to the putative lactose permease TR_3405 [49], which is upregulated in ∆ypr2 in light, but expressed at considerably higher levels in darkness.
TR_121251 encoding a putative effector protein [9] is upregulated in light in ∆ypr2. The encoded protein is related to the Mad1 adhesin of Metarrhizium anisopliae [50], which is relevant for adhesion and germination.
Consistent and contrasting regulation by YPR2 in light and darkness
Of the genes consistently upregulated in light and darkness in ∆ypr2 (Fig. 1d), TR_74282 encoding a QID74 homologue is particularly interesting. While about 3.7 fold upregulated in light, it is more than 28fold upregulated in darkness, thereby being the most highly expressed gene in ∆ypr2 in darkness. In T. harzianum the cell wall protein QID74 is strongly expressed during starvation and was shown to be relevant cell wall protection and adherence to hydrophobic surfaces. Heterologous expression in yeast further suggested a function in mating and sporulation [51]. Additionally, QID74 was shown to impact plant root architecture upon association with T. harzianum [52]. Together with the regulation of hydrophobin genes, GPCRs and secondary metabolism by YPR2 a function in regulation of pathways important for association with plants in nature would be conceivable.
Analyzing the genes misregulated in ∆ypr2 (including direct and indirect targets) in light and darkness we noted that in many cases the effect of YPR2 in light was the opposite of that in darkness (Fig. 1d). Therefore we wanted to check for a functional relevance of such a light dependent effect of YPR2. Besides TR_43701 encoding SOR4, the multidrug transporter of the SOR cluster [5], several other as yet uncharacterized genes showed contrasting regulation in light and darkness by YPR2.
Intriguingly, we found also a coregulated siderophore cluster located on chromosome 5 ([53]; genes 1083–1088)/scaffold 46 (26764–44,919) [8], which is conserved in Aspergillus fumigatus. It comprises the genes encoding homologues of the NRPS SidD (TR_71005), the transacylase SidF (TR_82628), the siderophore biosynthesis lipase/esterase SidJ involved in siderophore hydrolysis (TR_112590), the ABC multidrugtransporter SitT (TR_71010), the hydroxyornithine transacylase SidF (TR_82628), the enoyl-CoA hydratase/isomerase family protein sidH (TR_6085) and the siderophore iron transporter MirB (TR_71008). Fusarinin that is expected to be produced by the proteins encoded in this cluster [54] was found previously to be produced in T. reesei QM6a [55].
This cluster is in the wildtype differentially regulated in light and darkness. It is consistently downregulated in ∆ypr2 in darkness and up-regulated in ∆ypr2 in light suggesting light specific regulation of siderophore production by YPR2. The high affinity iron uptake system employing siderophores is particularly important under iron limited conditions [56]. Therefore we checked if deletion of YPR2 might cause a general misbalance in iron sensing and uptake systems at the transcriptional level.
Reductive iron assimilation (RIA) represents another high affinity iron uptake system [56] and is represented in T. reesei by two Fet3-homologues, the multicopperoxidases TR_102820 (FET3a) and TR_5119 (FET3b), and two Ftr1 homologues, the high affinity iron permeases TR_54962 (FTR1a) and TR_80639 (FTR1b). FET3a and FTR1a (scaffold 1: 1684330–1,690,370) as well as FET3b and FTR1b (scaffold 1:561024–565,836) are located next to each other and appear to share a bidirectional promotor. fet3a and ftr1a are coregulated and show increased transcript levels in light, but no regulation by YPR2. fet3b and ftr1b are downregulated in light, and ftr1b shows a similar regulation as the siderophore cluster being downregulated in ∆ypr2 in darkness and upregulated in ∆ypr2 in light. Consequently, YPR2 impacts regulation of one of two high affinity iron permeases, although we cannot exclude that the altered transcript levels of ftr1b are due to indirect regulation and caused by altered siderophore availability.
TR_4231 encoding a homologue of the Aspergillus fumigatus siderophore biosynthesis repressor SreA [54] is upregulated in darkness in ∆ypr2. The homologue of the negative regulator of SreA, HapX (TR_77191), which is negatively influenced by increasing iron levels, is not a target of YPR2.
Despite the striking regulation patterns in our data, regulation of the iron uptake systems could also be due to different growth rates between wildtype and mutant strain and hence altered iron consumption/availability. In darkness, biomass formation of ∆ypr2 is indeed decreased compared to wildtype (to 16.4% ± 1.9%). However, in light biomass formation of wildtype and ∆ypr2 are not significantly different, but the cluster still becomes upregulated, indicating that regulation by YPR2 and not merely altered biomass formation is the reason for the difference. Upregulation of sreA in ∆ypr2 in darkness would be in accordance with a reaction to higher iron availability because of lower biomass formation. Nevertheless, regulation of the FET3 and FTR1 homologues as well as of the HapX homologue is not consistent with a hypothesis of regulation of the siderophore cluster solely due to altered iron availability and biomass formation.
A decrease in oxidative stress resistance of siderophore mutants is attributed to an iron limitation, which would be required for several oxidative stress detoxifying enzymes like catalases. Upon deletion of ypr2, 4 catalases are upregulated in darkness which would not contradict this hypothesis, although it remains to be confirmed whether the requirement of iron impacts catalase regulation at the transcriptional level or merely at the activity level.
Regulatory overlap with CRE1 targets
The carbon catabolite repressor CRE1 was shown to regulate ypr2 along with the SOR cluster negatively in light and positively in darkness [5]. Consequently we were interested in investigating if CRE1 and YPR2 share regulatory targets, which would then be subject to a double lock mechanism.
Interestingly, among the 1402 genes regulated by YPR2 in darkness, we found 262 gene regulated by CRE1 either in light or darkness (Fig. 3; Additional file 1). In many cases, we observed contrasting regulation by YPR2 and CRE1 (upregulation by YPR2 and downregulation by CRE1 or vice versa). Consistent regulation by YPR2 and CRE1 was detected for 120 genes, with 58 genes positively regulated by CRE1 and YPR2 and 62 genes consistently negatively regulated by both (double lock mechanism). The gene set of up-regulated genes in both mutant strains compared to the wildtype strain comprises several genes involved in carbon and secondary metabolism and showed enrichment in functions in amino acid metabolism (p-value 8.58e-04) and glycolysis and gluconeogenesis (p-value 3.61e-03).
The consistently upregulated genes include the two transcription factors TR_72611 and TR_102920. TR_72611 is related to Fusarium solani CTF1B, the cutinase transcription factor 1beta, which activates cutinase genes [57]. The consistently downregulated genes include the transcription factors PRO1 (TR_76590) and TR_121682. PRO1 acts as a master regulator of signaling genes involved in development and also targets the cell wall integrity MAPkinase pathway [58], which was reported to regulate cellulase gene expression in T. reesei [59].
Hence the overlap of YPR2 targets with those of CRE1 in metabolic functions suggests that these transcription factors act in part in the same cascade. CRE1 regulates transcript levels of ypr2 [5], but YPR2 does not influence cre1 levels. Together with the differential regulation of the SOR cluster genes by YPR2 on glucose and cellulose [5, 24], we conclude that YPR2 acts downstream of carbon catabolite repression.
YPR2 impacts biosynthesis of alamethicin and orsellinic acid
Previous data indicated that the regulatory function of YPR2 is not limited to the SOR cluster, as besides trichodimerol and dihydrotrichotetronine, also paracelsin B levels decreased in a ypr2 mutant strain [5]. Therefore we performed mass spectrometry analysis on cultures grown under the same conditions as for transcriptome analysis (Additional file 4). We found 6 clusters of secondary metabolite profiles obtained for the culture supernatants, which show the light-dependent involvement of YPR2 in the underlying metabolic processes (Fig. 4a). In agreement with transcriptome data, the major differences between wildtype and ∆ypr2 can be seen upon cultivation in darkness (Fig. 4b).
Our transcriptome data clearly confirmed regulation of the SOR cluster genes by YPR2 (Additional file 1) as shown previously [5]. Surprisingly, the predicted paracelsin synthase, the NRPS TR_123786 [60] is not regulated by YPR2 and although paracelsin B levels are strongly decreased in light in ∆ypr2 [5], transcript abundance of TR_123786 increases in light in both the wildtype and in ∆ypr2. As coregulation of genes indicates a regulatory relationship, we checked for coregulated genes with ypr2 under conditions known to be relevant for secondary metabolism (different carbon sources, light/photoreceptors). We chose regulation on cellulose, glucose, lactose, glycerol and sophorose in light and darkness (dataset from [13]) as well as in photoreceptor mutants in light and darkness (dataset from [22]). Comparison showed one consistently coregulated NRPS gene, TR_60751, which is however related to a ferrichrome synthase and supports the relevance of YPR2 for siderophore regulation rather than a function in paracelsin production. We conclude that the regulatory effect of YPR2 on paracelsin levels is indirect and does not occur on the transcriptional level.
Our findings on regulation rather indicate that higher order regulation mechanisms should be considered. One such mechanism would be regulation by upstream open reading frames (uORFs), which could interfere with translation of the downstream target ORF [61]. Several short exons at the start of the predicted ORF of TR_123786 encoding a predicted paracelsin synthase could indeed represent such uORFs. Since no characterized homologues of TR_123786 are available from other fungi, clarification of the regulation mechanism of paracelsin biosynthesis warrants further detailed investigations.
A targeted screening by a mass spectrometry approach using a standardized method and internal standard compounds revealed the regulation of alamethicin biosynthesis by YPR2 in darkness on cellulose (Fig. 4c). Alamethicin was previously reported to be produced by Trichoderma spp. [62], albeit only by those species of the brevicompactum clade [63]. Alamethicin is reported to permeabilize Arabidopsis seedlings, which can be counteracted by prior treatment with cellulase [64]. These findings on a relevance of alamethicin in plant interaction are in agreement with both a carbon source depending function of YPR2: the function of YPR2 on glucose [24] is different to that on cellulose [5]. As cellulase regulation also happens in response to different carbon sources, a reaction to sensing the presence of a plant in terms of cellulase expression with an involvement of YPR2 would not be without precendent. Moreover, ypr2 transcript levels are subject to carbon source dependent regulation [13]. While a functional annotation of an alamethicin synthase is not available, the annotation of Druzhinina et al., 2016 [65] as supported by antismash analysis indicates TR_23171 for this function. In agreement with alamethicin levels (decreased to 23.8% of wildtype, 4.2 fold), our transcriptome data showed decreased transcript levels (4.3fold down in ∆ypr2) for the predicted alamethicin synthase gene tex1/TR_23171 [60] and hence supports the predicted function. Interestingly, alamethicin levels are also decreased in a strain lacking sor5 (TR_73623; Fig. 4c), which is positively regulated by YPR2. It remains to be shown whether this regulation is direct or indirect and if it involves the function of SOR5.
The same screening also showed production of orsellinic acid by T. reesei, but only in constant darkness in QM6a and this metabolite was not detected in the absence of YPR2 or SOR5 (TR_73623). Presence of orsellinic acid in the wildtype was confirmed with three independent, subsequent sample sets. Therefore we aimed to identify the cluster responsible for orsellinic acid production in T. reesei. The closest homologue of the PKS encoding gene of the A. nidulans ors-cluster [66], orsA (ANID_07909), was found to be T. reesei pks4 (TR_82208), which however represents the PKS responsible for pigment biosynthesis [67] and is related to the wA gene with the same function in Aspergilli [68]. Also a blast search with only the PksD domain (COG3321) yielded the same result. Accordingly, the whole ors cluster does not have direct homologues in T. reesei and pks4 is not significantly regulated by YPR2.
Nielsen et al., [69] suggest a function for ANID_07903 in orsellinic acid biosynthesis. The homologue of this gene is TR_73621, which was recently shown to be involved in sorbicillin biosynthesis [5, 24]. However, deletion of TR_73621 has no significant influence on orsellinic acid production (data not shown) that would support such a function in T. reesei. The same study [69] reports detection of traces of orsellinic acid in strains lacking ANID_07903 and ANID_07909/orsA. These traces are attributed to unmethylated byproducts of the PKS ANID_08383 that produces dimethylorsellinic acid, but this PKS has no homologue in T. reesei.
Besides YPR2, also the monooxygenase TR_73623/SOR5 is required for orsellinic acid production in T. reesei (Fig. 4d) and deletion of ypr2 strongly decreases sor5 transcript levels in light and darkness [5]. The homologue of sor5 in A. nidulans, ANID_07902, is located close to the ors cluster in the genome, but a connection to orsellinic acid has not been shown.
Using only the PksD domain of AN07909 (COG3321) for the homology analysis with T. reesei, we found again pks4 (TR_82208) as best homologue, but another pks gene, TR_81694/pks8g with only marginally lower e-value and even higher identity with OrsA than PKS4 within this domain. Using the PksD domain of TR_81694 for a BLAST search against A. nidulans showed best homology to several PKSs other than OrsA, with highest score for PkgA. However, in contrast to pks4, TR_81694 is strongly down regulated in light and positively regulated by YPR2, which is in agreement with the levels detected for orsellinic acid. Additionally, three further genes within the cluster surrounding TR_81694 are coregulated and show light dependent downregulation and decreased transcript levels in ∆ypr2. AN7071/PkgA was found to be involved in production of several metabolites including alternariol [70] and the cluster in T. reesei is similar to that in A. nidulans.
These findings suggest that the biosynthesis of orsellinic acid in T. reesei is altered compared to A. nidulans and may involve the cluster around pks8g, which remains to be proven.
