Fungal strains and growth conditions
Throughout this study, the T. reesei strains listed in Table 1 were used. All strains were maintained on malt extract (MEX) agar at 30 °C. If applicable, 5-FOA or uridine was added to final concentrations of 1.5 mg/ml or 5 mM, respectively.
For carbon source replacement experiments, mycelia were pre-cultured in 250 ml of Mandels–Andreotti (MA) medium  supplemented with 1% (w/v) glycerol as the sole carbon source on a rotary shaker (180 rpm) at 30 °C for 24 h. A total of 109 conidia per litre (final concentration) were used as the inoculum. Pre-grown mycelia were washed, and equal amounts were resuspended in 20 ml MA medium without any carbon source (reference condition) or MA medium containing 1% (w/v) d-glucose or 1.5 mM sophorose. Samples were taken after 3 h of incubation from three biological replicates.
For direct cultivations for the β-glucosidase assays, 30 ml MA medium containing 1% (w/v) oat spelt xylan (Sigma-Aldrich, St. Louis, MO, USA) were inoculated with 108 conidia per litre (final concentration) and incubated at 30 °C and 250 rpm for 66 h. For direct cultivations for cellulase assays, the Dhax1 strains were incubated for 48 h and 70 h at 30 °C and 180 rpm in 100 ml MA medium containing 1% (w/v) α-d-lactose inoculated with 109 conidia per litre (final concentration). For time course experiments, the OEhax1 strains were incubated for 72 h and 10 ml samples were taken after 37, 48, and 61 h.
Construction of p5-A-3 used for disruption of the hax1 locus in the T. reesei wild-type strain QM6a was done as follows. The 452 bp 5′- and the 1066 bp 3′-flanks of the targeted site for insertion of the amdS marker gene were amplified using the primers locus 5′for SalI/locus 5′rev HindIII and locus 3′for Acc65I/locus 3′rev XbaI, respectively, and cloned into pGEM-T (Promega, Madison, WI, USA). Subsequently, the 5′-fragment was released with HindIII and SalI and ligated into the vector pAMDS (a derivate of pUC19 containing the 3864 bp amdS gene ) digested with the same enzymes. Finally, the 3′-fragment released with Acc65I and XbaI was introduced into the plasmid p5-A resulting from the first cloning step, downstream of the amdS marker gene. The final construct p5-A-3 contains the hax1 5′- and 3′-flanks interrupted by the amdS marker gene in forward orientation.
For generation of the hax1 overexpression constructs pCD-∆pyr4-Pbgl1-hax1QM6a, pCD-∆pyr4-Pbgl1-hax1QM9414 and pCD-∆pyr4-Pbgl1-hax1Rut-C30, the respective hax1 versions were amplified using chromosomal DNA of T. reesei QM6a as template. The following primers were used: hax1 for_QM6a_BcuI and hax1 rev_3′QM6a for the 262 bp hax1QM6a; hax1 for_QM9414_BcuI and hax1 rev_3′QM6a for the 299 bp hax1QM9414 and hax1 for_Rut-C30_BcuI and hax1 rev_3′QM6a for the 428 bp hax1Rut-C30. The purified PCR products were blunt-end ligated into pJET1.2 (Thermo Scientific, Waltham, MA, USA) and the appropriate orientation was verified by digest with BcuI and XbaI. In a next step, a 997 bp fragment of the bgl1 promoter was PCR-amplified using the primers Pbgl1 for_Kpn2I and Pbgl1 rev-NheI, then digested with Kpn2I and NheI, and subsequently cloned into the respective pJET-hax1 vectors digested with Kpn2I and BcuI. To prevent positioning of the hax1 gene and the foreign 5′-untranslated region of bgl1 next to each other, the terminal point of the promoter fragment was chosen to be equal to the transcriptional start point previously defined for this gene . For construction of the final hax1 overexpression cassettes, the Pbgl1-hax1 fusion products were isolated from the plasmids by digestion with Kpn2I and XbaI, extracted from a gel, and introduced into BcuI/Kpn2I-digested pCD-∆pyr4 (carrying the cbh2 terminator ) in forward orientation.
For cloning of the constructs, Escherichia coli strain Top10 (Invitrogen, Life Technologies, Paisley, UK) was used. It was maintained on LB supplemented with 100 µg/ml ampicillin or spectinomycin and grown at 37 °C. All PCRs were performed applying peqGOLD Pwo DNA polymerase (PEQLAB, Biotechnologie, Erlangen, Germany) according to the manufacturer’s instructions. The primers used are listed in Table 2. Final constructs were verified by sequencing (Microsynth, Balgach, Switzerland).
Accidental disruption of hax1 (Dhax1) in QM9414 occurred by usage of 9 µg of the vector pAMDS (a derivate of pUC19 containing the 3864 bp amdS gene) in an optimized protocol for particle bombardment . Selection for positive transformants carrying the amdS gene, was performed on minimal medium (72.9 mM KH2PO4, 10.2 mM sodium citrate, 1% (w/v) d-glucose, 20 ml/L trace element solution (see MA medium ), and 1.5% agar noble dissolved in ultrapure H2O) containing acetamide. After autoclaving, acetamide, CsCl and MgSO4 were added from sterile-filtered stock solutions to final concentrations of 10, 10, and 24.3 mM, respectively.
Protoplast transformation of T. reesei was performed as described previously . QM6a_Dhax1 strains resulted from transformation of 13 µg hax1 5′-amdS-3′ DNA derived from a HindIII/Acc65I-digest of p5-A-3 and extraction from a gel. The template DNA was resuspended in 15 µl sterile dH2O and used for transformation of 107 protoplasts (in 150 μl) of QM6a_∆tmus53. 100 μl–2 ml of the transformation reaction were added to 20 ml melted, 50 °C warm amdS selection medium supplemented with 1.2 M sorbitol. This mixture was poured into sterile petri dishes.
For overexpression of hax1 (OEhax1), 200 µg of the NotI-digested construct pCD-∆pyr4-Pbgl1-hax1QM6a, pCD-∆pyr4-Pbgl1-hax1QM9414, or pCD-∆pyr4-Pbgl1-hax1Rut-C30 were used for transformation of 107 protoplasts (in 150 μl) of QM9414_Dhax1 or QM6a_∆tmus53. Selection for pyr4 deleted transformants was performed on MEX agar containing 1.2 M sorbitol, 1.5 mg/ml 5-FOA, and 5 mM uridine as described by Derntl and co-workers .
Plates were incubated at 30 °C for 3–7 days until colonies were visible.
For an initial identification of uridine auxotroph OEhax1 strains, candidates were grown on MA medium containing 1% glycerol as a carbon source without peptone or uridine. The OEhax1 candidates that were unable to grow under these conditions were tested by PCR using the primers 5pyr4_fwd(BglII) and hax1 rev_3′QM6a.
For Dhax1 strains, homokaryotic strains were generated by three rounds of vegetative spore propagation on selection medium. In case of QM9414_Dhax1 strains, the locus of integration was identified via inverse PCR and verified by Southern blot analysis. In case of QM6a_Dhax1 strains correct integration of the construct was tested by PCR and by sequencing.
Extraction of chromosomal DNA for candidate screening and Southern blot analysis was performed as described previously [65, 66]. For Southern blot analysis, the chromosomal DNA of QM9414_Dhax1 strains was digested with SacII, resulting in a 2960 bp fragment specific for the wild type and a 5178 bp fragment specific for disruption of hax1. The locus-specific biotinylated probe applied for hybridization was derived from a PCR using the primer pair locus for and locus rev and the Long-PCR Enzyme Mix (Thermo Scientific) for amplification.
PCR analysis for screening for Dhax1 candidates was performed using the same polymerase and primers. For verification of OEhax1 transformants via PCR, GoTaq G2 polymerase (Promega) was applied. DNA sequencing was performed at Microsynth. All primers used for candidate screening are listed in Table 2.
For the identification of the locus that was targeted by integration of the amdS marker in QM9414_Dhax1 strains, an inverse PCR was performed as follows. 20 µg of chromosomal DNA were digested with either Acc65I or NotI (Thermo Scientific) at a final concentration of 1 U/µl in a total reaction volume of 20 µl according to the manufacturer’s instructions. After heat inactivation, 2 µl of T4 Ligase (Promega, 1–3 U/µl) and the corresponding buffer were added and ligation was performed at 18 °C for 90 min. Subsequently, the ligation was stopped by heat inactivation and 1 µl was applied as template in a 25 µl inverse PCR reaction, initially using the primers amdS inv for and amdS inv rev. For further approaches, the primers locus for and locus rev annealing to the identified regions were applied. All amplifications were performed in an iCycler (Bio-Rad, Hercules, CA, USA) using the Long-PCR Enzyme Mix (Thermo Scientific) and the following program: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 59 °C and 3 min at 72 °C, and final elongation at 72 °C for 5 min. The DNA fragments were sequenced by MWG Biotech (Ebersberg, Germany).
RNA stability assay
The strains QM6a, QM9414, and Rut-C30 were cultivated in 25 ml MA medium containing 1% (w/v) α-d-lactose and 109 conidia per litre (final concentration) at 30 °C and 180 rpm for 24 h. The cultivation was carried out in biological triplicates. Then, the transcription was stopped by addition of 16 µg/ml (final concentration) of the inhibitor DRB. The cultures were further incubated and after 30, 60, 90, and 120 min, 500 µl samples were taken. A reference sample was taken from each culture before addition of DRB. The three biological replicates of each strain were pooled to one 1.5 ml sample. Mycelia were harvested by centrifugation and frozen in liquid nitrogen. Finally, the RNA was extracted, cDNA synthesis was performed and the samples were analysed via quantitative PCR as described in “Transcript analysis” section.
Extraction of RNA from fungal mycelia, cDNA synthesis, and quantitative PCRs were performed as described previously . Template cDNAs were diluted 1:100 (QM9414) or 1:20 (QM6a). Analysis was carried out in triplicates. The following PCR protocols were run: 3 min initial denaturation at 95 °C, followed by 50 cycles of 15 s at 95 °C, 15 s at 60 °C and 20 s at 72 °C (for hax1 and act) or 3 min initial denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 120 s at 64 °C (for sar1). Control reactions, data normalization using sar1 and act as reference genes and calculations were performed as published previously .
Qualitative PCRs for estimating the abundance of hax1QM6a, hax1QM9414, and hax1Rut-C30 were performed using the Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. The template cDNAs were diluted 1:20. Amplification was performed running the following program: initial denaturation at 98 °C for 30 s, followed by 35 cycles of 10 s at 98 °C, 10 s at 70 °C and 20 s at 72 °C, and final elongation at 72 °C for 2 min. The resulting fragments (262, 299, and 428 bp) were analysed on a 1.5% agarose gel applying a GeneRuler 50 bp DNA Ladder (Thermo Scientific) for size estimation. Primer sequences for all transcript analyses are provided in Table 2.
RACE and enrichment of HAX1
5′ and 3′ RACE was performed using the 5′/3′ RACE Kit, 2nd generation (Roche, Basel, Switzerland). For PCRs, the GoTaq G2 polymerase (Promega) was applied. Primer sequences for RACE are provided in Table 2.
5′ RACE was carried out according to manufacturer’s instructions, applying 0.9–1 µg of DNase I digested RNA extracts for cDNA synthesis in a total reaction volume of 20 µl. For this initial reverse transcription step, the gene specific primer hax1 rev_1.Intron was used. After RNase A digestion and purification with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) based on the modified protocol for RACE applications recommended by Roche, a poly(A)-tailing was performed. Subsequently, the hax1 fragments were specifically amplified from the total pool with the Oligo dT-Anchor Primer (included in the kit) and hax1 rev_5′RACE_2 in a first PCR. The resulting product was diluted 1:50 and used as a template for a nested PCR with either hax1 rev_up-Intron or hax1 rev_ 5′RACE_4 and the PCR Anchor primer included in the kit.
For 3′ RACE, classical analysis was performed according to manufacturer’s instructions, applying 0.45 µg of DNase I digested RNA from T. reesei QM9414 in a total reaction volume of 20 µl. After reverse transcription with the Oligo dT-Anchor Primer, cDNAs were RNase A digested and used for amplification of hax1 applying the gene specific primers up hax1 for_2 and hax1 for_3′RACE_3 for the initial and nested PCRs, respectively. Further 3′ RACE approaches based on prior enrichment of HAX1 were performed. For this purpose, HAX1 was enriched from 1.25 to 2.1 mg total RNA extract using a biotinylated and HPLC-purified hax1 specific DNA probe (sonde hax_5-Biotin) as well as streptavidin-linked magnetic beads included in the µMACS™ Streptavidin Kit and the corresponding µMACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany) based on manufacturer’s instructions. According to the unusual high calculated melting temperature of the biotinylated probe, initial denaturation was performed at 85 °C for 5 min and annealing to appropriate amounts of streptavidin-linked magnetic beads was done at 70 °C for 15 min. RNase-free TEN buffer (10 mM Tris/HCl, pH 8.0; 1 mM EDTA; 100 mM NaCl2) and TE buffer (10 mM Tris/HCl, pH 8.0; 1 mM EDTA) were used for binding and washing, respectively. Enriched RNA was eluted with 150 µl RNase-free dH2O and digested with DNase I (Thermo Scientific). For poly(A)-tailing followed by RACE analysis, the enriched RNA was precipitated with isopropanol, washed with 70% (w/v) ethanol and dissolved in 20 µl RNase-free dH2O. Poly(A)-tailing was performed using 19 µl of the resulting RNA in a total reaction volume of 25 µl. For proceeding to cDNA synthesis without prior poly(A)-tailing, the RNA was purified using the GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Scientific) as instructed in the protocol for purification of DNase I digested samples. In this case, the RNA was eluted from four columns with 10 µl each and pooled for cDNA synthesis. Reverse transcription and further 3′ RACE analysis steps were performed as described before using the RACE Kit and applying the gene specific primers up hax1 for_2 and hax1 for_3′RACE_3 for the initial and nested PCRs, respectively.
For both, 3′ and 5′ RACE, the final PCR products were extracted from a gel, blunt-end ligated into pJET1.2 (Thermo Scientific) and analysed by sequencing (Microsynth).
The β-glucosidase activity was assayed in 50 mM sodium citrate buffer at pH 5.0 using p-nitrophenyl-β-d-glucopyranoside as a substrate. The enzyme assay was performed at 50 °C for 30 min as previously described  and the activity was calculated from the absorbance at 405 nm based on the Lambert–Beer law.
The cellulase activity was assayed in 25 mM sodium acetate buffer pH 4.5 using Azo-Cellazyme C tablets (Megazyme, Wicklow, Ireland) as substrate, essentially according to the manufacturer’s instructions. The reaction time was increased to 30 min–5 h until detectable values were obtained. The same reaction times were used for all strains cultivated for the same time period within one experiment, and samples with higher cellulase activity were adjusted by dilution to enable comparison. Cellulase activities given in μ were calculated from the absorbance at 590 nm for 10 min reaction time based on the equation μ = 232.6 * Abs + 5 .
One unit is defined as the amount of enzyme required to release 1 µmol of d-glucose reducing-sugar-equivalents per minute under the respective assay conditions. For the final timepoints of cultivation, the enzymatic activities were referred to the biomass dry weight derived from incubation of the harvested mycelia at 80 °C for 24 h.
Structure and sequence analysis tools
Genome analysis and BLAST were performed using the NCBI database  and the sequence of Trichoderma reesei QM6a v2.0 accessible in the JGI database . For structural gene prediction, the web server AUGUSTUS described by Stank and Morgenstern was used . Codon usage was calculated applying the online Codon Usage Calculator of Biologics Corp . In silico prediction of RNA secondary structures of minimized free energy was performed using the RNAfold Web server  included in the ViennaRNA Package 2.0 , provided by the University of Vienna. Displayed structures are based on HAX1 sequences without polyA-tail; however, the addition of a random polyA-tail did not change structures.
For statistical analyses of the data, the program GraphPad Prism 5.00.288 was used to perform ANOVA (P < 0.05) tests and Tukey’s multiple comparison test as posttest.