Introduction

Lignocellulose is the most abundant biomass and cellulase is crucial for its degradation. Trichoderma reesei is an industrially-important filamentous fungus that secretes vast amounts of cellulases and hemicellulases, which have a variety of industrial applications and are currently also exploited for the production of biofuels (Seiboth et al. 2011). However, as the high cost of cellulase production is still a major bottleneck in lignocellulosic biofuel production, a comprehensive understanding of the complex regulatory system that governs cellulase formation in T. reesei is imperative. Such knowledge can be used to increase the yield and efficiency of cellulases produced by T. reesei thereby enabling more economical production of biofuels (Kubicek et al. 2009).

In T. reesei, the expression of cellulase genes is strictly dependent on the presence of inducers (Ilmen et al. 1997). Cellulose, sophorose, lactose, and cellobiose can all induce the expression of cellulase genes in T. reesei. However, the induction strengths and cellulolytic enzyme profiles vary among different inducers (Ilmen et al. 1997), suggesting that the expression of cellulase genes is finely tuned (Tisch et al. 2011). To date, four transcriptional activators, Xyr1(Stricker et al. 2006), HAP2/3/5 complex (Zeilinger et al. 2001), Ace2 (Aro et al. 2001), and Ace3 (Häkkinen et al. 2014), and two repressors, Cre1 (Portnoy et al. 2011a, b) and Ace1 (Saloheimo et al. 2000), have been documented to regulate the expression of the major cellulase genes. Interestingly, the full induction of the two activators, Xyr1 and Ace2, is dependent on the function of the repressor Cre1 (Portnoy et al. 2011a, b). Despite a strong cellulase production capability, only a few cellulase-encoding genes are found in the T. reesei genome (Martinez et al. 2008). It is reasonable to assume that this fungus has developed sophisticated signal transduction networks by which the cells sense and respond rapidly to environmental signals (Schmoll 2008). Research on T. reesei signal transduction has been initiated for a few years and has been mainly focused on elucidating the effects of the cAMP signaling-related pathway on cellulase biosynthesis (Tisch et al. 2011). However, little work has been performed to explore the relationship between the MAPK signaling pathways and T. reesei cellulase biosynthesis (Wang et al. 2013).

Eukaryotic MAPKs are important signal transduction proteins involved in a variety of essential biological processes, including growth, development, pathogenesis, and environmental responses (Li et al. 2005; Maerz et al. 2008). In addition, MAPK homologs belonging to the YERK1 (yeast and fungal extracellular signal-related kinase) class and Hog1 (high-osmolarity glycerol) type have been implicated in governing the expression of cellulase genes in fungi. For example, Saccharamyces cerevisiae Kss1(Madhani et al. 1999), Botrytis cinerea Bmp1(Zheng et al. 2000), Cochliobolus heterostrophus Chk1(Lev et al. 2003), and Trichoderma Virens TmkA (Mukherjee et al. 2003) are essential for the expression of the endopolygalacturonases PGU1, BCPG1, pectatelyase, cellulases, and mycoparasitism-related hydrolytic enzymes.

inducer of meiosis 2 (Ime2) was first identified as a meiosis-specific MAPK indispensable for the production of asci in Saccharomyces cerevisiae (Smith and Mitchell 1989) and then shown to be also involved in pseudohyphal growth (Strudwick et al. 2010). In contrast, Ime2 homologues are not required for meiosis in Neurospora crassa and Aspergillus nidulans, and their inactivation does not result in pronounced defects in sexual development (Bayram et al. 2009; Hutchison and Glass 2010). In addition, increasing evidence has shown that the Ime2 protein kinase family has more cellular functions than just meiosis (Irniger 2011). Ime2–like protein kinases are involved in spore formation in Schizosaccharomyces pombe (Abe and Shimoda 2000), and morphogenesis environmental responses in the plant pathogen, Ustilago maydis (Garrido and Pérez-Martín 2003). These results indicate the functional diversity of this kinase family among fungal species. Since T. reesei is an important workhorse for cellulase production, it would be attractive to investigate the role of Ime2 in T. reesei cellulase biosynthesis.

The T. reesei genome contains a gene named TrIme2 which shows high amino acid sequence identity to Aspergillus nidulans Ime2. Our previous study showed TrIme2 was probably involved in cellulase biosynthesis (Chen et al. 2014). And now we provide evidence that TrIme2 is indeed involved in cellulase expression in T. reesei, an industrial cellulase-producing workhorse.

Materials and methods

Strains and media

Trichoderma reesei QM9414 (ATCC26921) and ku70 (uridine auxotrophic strain) were used. The T. reesei strain ku70 was kindly provided by Prof. Monika Schmoll (Guangtao et al. 2009) which can be easily used for homologous recombination and had no significant different cellulase expression compared with QM9414. T. reesei spores were maintained on potato/dextrose/agar (PDA) plates. Liquid minimal medium (MM) was prepared without peptone as described (Ilmen et al. 1997). When necessary, PDA and MM were supplemented with 10 mM uridine. The pH of MM was adjusted to 5.3 ± 0.2. The Escherichia coli DH5α was used as the host strain for plasmid propagation.

Identification of TrIme2 gene

TrIme2 was predicted by BLAST analysis of N. crassa and A. nidulans Ime2 proteins against the T. reesei genome (http://genome.jgi-psf.org). The conserved residues were analyzed by alignment of amino acid sequences and phylogenetic tree was constructed for protein sequences of nine Ime2-related proteins using MEGA version 4.0 by the neighbor-joining method with 1000 bootstrap replicates. T. reesei Tmk1 (GenBank accession no: XP_006965066.1) was an outgroup.

Construction of the T. reesei ΔTrIme2 mutant and complement strain

The primer pairsTrIme2-5f/TrIme2-5r, pyr4F/pyr4R, andTrIme2-3f/TrIme2-3r were used to amplify the 5′-flanking region of TrIme2, the pyr4 gene, and the 3′-flanking region of TrIme2, respectively. The genomic DNAs of T. reesei strain QM9414 and ku70 were used to amplify the pyr4 gene and homologous arms, respectively. PCR products obtained using the above primers were digested with HindIII/EcoRI, EcoRI/BamHI, and BamHI/NotI, respectively. A four-fragment ligation was performed by mixing the three PCR products with the pBluescriptIISK(+) vector (Stratagene) digested with HindIII/NotI. The resulting plasmid containing the deletion cassette ΔTrIme2::pyr4 was obtained and transformed into T. reesei ku70 according to the method of Penttilä et al. (1987). Positive transformants were selected and further confirmed by PCR using two primer pairs upupF (upstream of the 5′-flanking region of TrIme2)/pyrmR (located in the pyr4 gene expression cassette) and pyrmF (located in the pyr4 gene expression cassette)/dodoR (downstream of the 3′-flanking region of TrIme2) to obtain 1.3 kb and 1.5 kb fragments, respectively. A subsequent Southern hybridization was performed to determine the copy number of the deletion cassette integrated into the T. reesei genome. The probe was generated by PCR using the primer pair TrIme2-5f/TrIme2-5r and hybridized with SalI-digested genomic DNA using the DIG Easy Hyb kit (Roche, Germany). The detection of a single 2.8 kb hybridizing band in the ΔTrIme2mutant and a 4.2 kb hybridizing fragment in the recipient strain suggested that only one copy of the deletion cassette was integrated into the genome.

To construct the complement strain, the entire TrIme2 gene, including the promoter, was amplified using the upF and downR primer pairs and the purified PCR products were directly transformed into protoplasts generated from the ΔTrIme2 mutant with the assay method described above.

Assay of enzyme activity and gene expression analysis

For mycelia biomass determination, 8 × 107 spores were inoculated into 100 ml liquid MM containing one of the following carbon sources including 1 % (w/v) Avicel cellulose, 3 % (w/v) lactose, 2 % (w/v) cellobiose, 2 % (w/v) glucose, 2 % (w/v) fructose, 2 % (w/v) sorbitol, or 2 % (v/v) glycerol. The dry weights of the mycelia harvested were measured according to the method as described (Aro et al. 2003). The biomass for the fungus cultivated in Avicel cellulose medium was represented by the amount of protein, which was extracted as described previously (Schuster et al. 2011).

To analyze the effects of TrIme2 deletion on the expression of cellulases and transcriptional factors, pre-grown mycelia in MM-glucose were transferred into liquid MM supplemented with 1 % (w/v) Avicel cellulose for induction of cellulase expression according to the method as described (Verbeke et al. 2009). Avicel cellulose was chosen to induce cellulase expression, because cellulose induces high levels of cellulases (Ilmen et al. 1997). The pre-grown cultures continued to grow for 2, 4, 8, 12, and 24 h with shaking at 28 °C in cellulose-containing MM, and the mycelia were harvested for RNA isolation. The sampling points were chosen mainly according to the method as described (Portnoy et al. 2011a, b). Parallel experiments were designed for assaying the filter paper activities (FPAs) of cellulases. Supernatant samples were collected at 48, 72, 96, 120, and 144 h, and FPAs were measured according IUPAC (Ghose 1987). One international filter paper unit (FPU) is defined as the amount of cellulase that releases 1 µmol glucose per min. The activity was recorded as FPU/ml.

Nucleic acid isolation

The genomic DNA of T. reesei was isolated using a plant genomic DNA kit (Tiangen, Beijing, China). Plasmid extraction from E. coli was performed according to the TIANprep mini plasmid kit (Tiangen, Beijing, China). Total RNA was isolated using Trizol.

Quantitative real-time PCR (qRT-PCR)

DNase I-treated total RNA was used to synthesize the first-strand cDNA according to the Invitrogen Superscript III first-strand synthesis kit. The qRT-PCR assays were performed in a Bio-Rad CFX96TM real-time system using SYBR Premix Ex TaqTMII (Perfect Real Time) kit (Takara). All PCRs were performed in triplicate. Specificity of the amplification was identified by checking the melting curves. Supplementary Table 1 lists the primer sequences used in this study.

Results and discussion

Identification of an Ime2-like protein kinase from T. reesei

Blastp analysis of N. crassa and S. cerevisiae Ime2 revealed the existence of one Ime2-like protein encoding gene in the T. reesei genome, which was designated TrIme2 (GenBank accession no.XP_006967044). TrIme2 consists of an ORF of 2292 bp encoding 763 amino acids, that shares 55 and 61 % amino acid sequence identity with N. crassa Ime2 and A. nidulans ImeB, respectively. The search for conserved domains in Ime2–related proteins against the NCBI Conserved Domain program revealed that TrIme2, like other Ime2-like proteins from other filamentous fungi, contains the conserved catalytic domain of male germ cell-associated serine/threonine kinases (residues 23–351) and a TTY (residues 211–213) motif which is characteristic for the activation loop of MAPK (Kültz 1998). To determine the evolutionary relationship among Ime2 proteins from nine different fungi, phylogenetic tree was constructed for the full-length protein sequences of nine Ime2 proteins with TrTmk1 which belongs to the YERK1 class MAPK as an outgroup. The results showed that TrIme2 clusters well with N.crassa Ime2 (Fig. 1). Taken together, the TrIme2 gene encodes an Ime2-related MAPK.

Fig. 1
figure 1

Phylogenetic analysis of Ime2-related proteins. AnIme2: Aspergillus niger Ime2 (XP_001399336.1); AnImeB: Aspergillus nidulans ImeB (XP_001399336.1); AoIme2: Aspergillus oryzae Ime2 (XP_001821753); NcIme2: Neurospora crassa Ime2 (CAB99241.2); TrIme2: Trichoderma reesei Ime2 (XP_006967044); ScIme2: Saccharomyces cerevisiae Ime2 (CAA89401.1); Um Crk1:Ustilago maydis Crk1(AAM21640.3); SpPit1:Schizosaccharomyces pombe Pit1(NP_593607.1); Sp Mde3:Schizosaccharomyces pombe Mde3 (NP_595581.1); TrTmk1:Trichoderma reesei Tmk1 (XP_006965066.1)

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The disruption of TrIme2 enhances cellulase expression in T. reesei

As shown in Fig. 2a, deletion of the TrIme2 gene led to a marked increase in cellulase activity against filter paper (up to 90 % during earlier period time of cellulase induction) as well as the extracellular protein production (Fig. 2b). And the re-transformation could also restore the phenotype of the recipient strain (Fig. 2a, b). In order to rule out the possibility that the improvement of extracellular protein production was due to altered growth, we measured the biomass accumulation. No pronounced differences were observed in growth profiles for ∆TrIme2 mutant (Fig. 2c), suggesting that the enhanced extracellular protein production is caused by the disruption of TrIme2. Having observed the role of TrIme2 in cellulase production, we also wondered whether the deletion of the TrIme2 gene could result in increased transcriptional levels of the major cellulase genes, which in turn resulted in enhanced cellulase production and enzyme activity. As shown in Fig. 3a, the ∆TrIme2 mutant showed highly increased levels of transcripts of the three cellulase genes, with the greatest impact being on the transcriptional level of cbh1. These data strongly indicate that TrIme2 is involved in cellulase gene expression in T. reesei.

Fig. 2
figure 2

Deletion of TrIme2 enhances cellulase expression and extracellular protein production. Strains were cultivated in glucose-containing MM for 48 h at 30 °C. Pre-grown mycelia were shifted to MM supplemented with 1 % Avicel cellulose as sole carbon sources for additional cultivation. The cultures were collected at indicated time points to determine filter paper activity and extracellular protein. a Comparison of filter paper activities of ku70, ∆TrIme2 mutant and the complement strain (Comp); b SDS-PAGE analysis of the extracellular protein in the supernatants. Lanes 1, 2, 3 represent the supernatants from the strains ku70, △TrIme2 mutant and complement strain, respectively; c comparison of biomass accumulation in different carbon sources, and there were no pronounced differences when they were cultivated in the same carbon source; DW: dry weight

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Fig. 3
figure 3

a Comparative analysis of steady state mRNA levels of the three major cellulase genes in ku70 and △TrIme2 mutant strain grown in cellulose-containing medium for 8, 12, and 24 h. The culture conditions and the carbon sources used were as described in Fig. 2. The relative transcriptional levels of each target gene in the ΔTrIme2 mutant versus that in ku70 were determined by a previously described method (Foreman et al. 2003). mRNA samples from the ∆TrIme2 mutant was directly compared with samples from ku70 cultures grown under cellulose condition. Quantitative real-time PCR was performed to examine the expression levels of cbh1, cbh2, and egl1 under cellulose cultivation. The relative quantification of gene expression was analyzed by the ddCt method (Arocho et al. 2006). The actin gene was used as an internal control to normalize the expression levels of target genes (Furukawa et al. 2009); b relative mRNA level analysis of cellulase transcriptional regulator genes in ku70 and △TrIme2 mutant strain from 2 to 24 h of cellulase induction by cellulose. The culture conditions and the carbon sources used were as described in Fig. 2. The method used here was the same as described in a

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TrIme2 modulates the expression of the cellulase transcription regulatory factors Cre1 and Xyr1

Since the deletion of TrIme2 enhances expression of cellulase genes in T. reesei, it is relevant to know whether the expression of the transcriptional factors responsible for cellulase production had been altered. We investigated the transcriptional levels of four cellulase transcriptional regulators (xyr1, ace2, cre1, and ace1) between ∆TrIme2 mutant and ku70 grown on cellulose. A real-time PCR analysis indicated that, at all the time points tested, the deletion of TrIme2 markedly decreased the mRNA levels of cre1 and xyr1 on cellulose medium with almost the same fold change (Fig. 3b), suggesting that TrIme2 positively modulates the transcription of cre1 and xyr1. Conversely, TrIme2 appeared to have no influence on the transcriptional levels of ace1 and ace2. Since the full induction of Xyr1 requires the involvement of the Cre1 (Portnoy et al. 2011a, b), we speculate that TrIme2 regulates Xyr1 through Cre1. Cre1 phosphorylation is required for DNA binding and carbon catabolite repression (Cziferszky et al. 2002); however, it remains unknown whether TrIme2 affects Cre1 phosphorylation at the protein level.

Because the disruption of xyr1 eliminated the expression of the major cellulase genes, Xyr1 is thought to be the main transcriptional activator of xylanase and cellulase-encoding genes in T. reesei (Stricker et al. 2006). Interestingly, in the present study, the reduced xyr1 transcriptional level did not reduce but rather increased the expression levels of the major cellulases genes. One explanation for this phenomenon is that post-translational modification of Xyr1 would function in controlling cellulolytic and xylanolytic enzyme formation in T. reesei (Mach-Aigner et al. 2008). On the other hand, as main transcriptional regulator mediating carbon catabolite repression, the decrease of cre1 transcriptional level might be an important treason that why cellulase expression improved.

The present result, that downregulation of cre1 reduced Xyr1 and led to an increased transcript accumulation of cellulase genes, is to some extent similar with the previous study of Portnoy et al. (2011a, b) in which decreased Xyr1caused the increase of cbh1 transcription in the ∆cre1 strain. Since the full induction of Xyr1 requires the involvement of the Cre1, we speculate that TrIme2 regulates Xyr1 through Cre1.

In conclusion, TrIme2 is associated with cellulase gene expression in T. reesei. Thus it is a good candidate for genetically engineering T. reesei to achieve higher cellulase production.