## 3 Materials and Methods
### Construction of the TaST Knockout Strains  
Using the highly efficient genetic modification strategy of _Trichoderma hypoxylon_[25], a chassis strain of ACCC 33109, \(\Delta\)_Talig4:meo_, was constructed and used for the creation of other _TaTS_ knockout strains by the double homologous gene replacement approach. Hygromycin B (K547; Amresco, Solon, OH, USA) and G418 (FG401-01; TransGen) were used to evaluate the fungal sensitivity. PDA plates supplemented with 100-800 \(\upmu\)g/mL G418 and 10-100 \(\upmu\)g/mL hygromycin B were inoculated with a 6 mm diameter culture plug of ACCC 33109 in the center and incubated for 5 days at 28 \({}^{\circ}\)C. To yield the maximum amount of protoplasts, fresh mycelia of ACCC 33109 cultured for 24 h in PDB were disrupted with sonication (50 Hz and 60 s) and digested by 20 mg/mL lysing enzyme (L1412; Sigma-Aldrich, St. Louis, MO, USA) for 2-10 h. Protoplasts were suspended in STC solution (1.2 M sorbitol, 10 mM Tris-HCl, pH = 7.5, 50 mM CaCl\({}_{2}\)) and enumerated under a microscope. The recovery rate of the protoplasts was determined by serial dilution and inoculation on TB3 agar plates (200 g/L sucrose, 5 g/L yeast extract, 3 g/L casein, and 15 g/L agar). The oligonucleotide primers used for the construction of the _\(\Delta\)Tslig4:meo_ and _\(\Delta\)Tslig4:meo_XTaTS:lph_ strains are given in Supplementary Table S4. In the first round of the PCR, approximately 800 bp fragments upstream and downstream of the target genes, 1300 bp of the neomycin phosphotransferase gene (_neo_), and 800 bp of the hygromycin phosphotransferase gene (_lroph_) were amplified from the genomic DNA of ACCC 33109 and plasmids pKS666 and pKH-KO, respectively. The 50 \(\upmu\)L PCR reaction systems consisted of 25 \(\upmu\)L of 2\(\times\) PCR buffer, 1 \(\upmu\)L each of forward and reverse primers (10 \(\upmu\)M), 1 \(\upmu\)L of genomic DNA or plasmids, 1 \(\upmu\)L of TksGflex DNA Polymerase (TaKaRa, Dalian, China), and 21 \(\upmu\)L of distilled water. The PCR conditions were as follows: 5 min at 95 \({}^{\circ}\)C, 34 cycles of 10 s at 94 \({}^{\circ}\)C, 45 s at 60 \({}^{\circ}\)C, and 30 s at 72 \({}^{\circ}\)C, and 10 min at 72 \({}^{\circ}\)C. The PCR fragments were purified with an EasyPure Quick Gel Extraction Kit (Transgene) and quantified. In the second round of the PCR, the upstream and downstream of the _Tslig4_ gene were ligated with the forward 2/3 and backward 1/3 fragments of _neo_, respectively, as well as the upstream and downstream of the _TaTS_ gene with the forward 2/3 and backward 1/3 fragments of _hph_. The 50 \(\upmu\)L PCR reaction systems consisted of 25 \(\upmu\)L of 2\(\times\) PCR buffer, 1 \(\upmu\)L each of forward and reverse primers (10 \(\upmu\)M), 1 \(\upmu\)L of _Tslig4_ or _TaTS_ PCR products (100 ng/\(\upmu\)L), 1 \(\upmu\)L of _neo_ or _lroph_ PCR products (100 ng/\(\upmu\)L), 1 \(\upmu\)L of TksGflex DNA polymerase, and 20 \(\upmu\)L of distilled water. The PCR conditions were as follows: 5 min at 95 \({}^{\circ}\)C, 34 cycles of 10 s at 94 \({}^{\circ}\)C, 90 s at 60 \({}^{\circ}\)C, and 30 s at 72 \({}^{\circ}\)C, and 10 min at 72 \({}^{\circ}\)C. The fused fragments were purified and quantified as described above. The fused fragments of _Tslig4:meo_ and _TaTS:lph_ were mixed at a molar ratio of 1:1, and 10 \(\upmu\)L of the mixture were added into 250 \(\upmu\)L of ACCC 33109 or _\(\Delta\)Tslig4:meo_ protoplasts (10\({}^{7-8}\) cfu/mL). After 30 min storage on ice, the mixture was added with 1.4 mL of PEG, placed on ice for another 20 min, and transferred into 5 mL of TB3 medium supplemented with 100 \(\upmu\)g/mL ampicillin for overnight incubation at 28 \({}^{\circ}\)C with a constant agitation of 80 rpm. The liquid cultures were mixed with 50 \({}^{\circ}\)C preheated TB3 agar medium containing 200 \(\upmu\)g/mL G418, poured into 9 cm in diameter Petri dishes, and covered with 400 \(\upmu\)g/mL G418.