Generation of epl1 deletion and overexpression mutants of Trichoderma spp.

For constructing the epl1 gene replacement cassette, a 1.2-kb upstream flanking fragment (5′ homologous arm) and a 1.2-kb downstream flanking fragment (3′ homologous arm) of the epl1 gene were amplified from Trichoderma genomic DNA by PCR using the primer pairs e1-upF/e1-upR and e1-dnF/e1-dnR, respectively (all primers used are listed in Table 4). The hygromycin B expression cassette (hph, 2.3 kb) was PCR amplified from the pPcdna1 plasmid using the primer pair e1hph-F/e1hph-R and was inserted between the two flanking arms described above via overlapping PCR with the primers e1-upF/e1-dnR. The amplified fragment (4.7 kb) was then purified and used for the standard polyethylene glycol (PEG)-mediated protoplast transformation for Trichoderma (3). For overexpressing epl1 in Trichoderma spp. under the constitutive promoter P_cdna1, the primer pair OEe1-F/OEe1-R was used to amplify a 1.6-kb fragment, which contains the ORF of epl1 and a 1.2-kb terminator region, from the genomic DNA of the wt strain. The PCR product was purified and fused into the ClaI-digested pPcdna1 plasmid with the In-Fusion HD cloning kit (TaKaRa, Japan), resulting in plasmid pP_cdna1::epl1::T_epl1. Protoplast transformation was performed using the PscI-linearized plasmid. Stable transformants were verified by sequencing and maintained on PDA medium containing 200 μg ml⁻¹ of hygromycin B (Thermo Fisher Scientific, USA).

Primers used in this study