Trichoderma Transformation

To transform the Trichoderma sp. strain T154, a hygromycin B test was performed to determine its sensitivity to different concentrations of hygromycin B (50, 100, 150, and 200 μg mL⁻¹) in PDA medium. It was obtained that 200 μg mL⁻¹ was the optimum concentration for performing the experiment, no growth of Trichoderma was observed ( Supplementary Data S2: Figure S3 ). The transformation of Trichoderma sp. T154 with plasmid pBHt2-tdTom (Caasi et al., 2010) was then carried out from a fresh Trichoderma sp. T154 spores suspension according to Cardoza et al. (2006). The binary vector pBHt2-tdTom contains Td tomato fluorescent protein under the control of the toxA promoter and the hygromycin resistance marker hph. Plates containing PPG [mashed-potato-glucose agar (Sousa, 2004)] medium were inoculated with 1 × 10⁷ spores and incubated at 28°C for 3 days. The spores collected from the plate were then used to inoculate 50 ml of CM (5 g malt extract, 5 g yeast extract and 5 g glucose and distilled water up to 1 L) medium and incubated in an orbital shaker at 250 rpm and 28°C for 24 h. Then, 25 mL of that culture were filtered through Nytal^® (30 μM pore diameter) (Maissa, Barcelona, Spain) and washed twice with 0.7 M NaCl. After that, the mycelium was re-suspended in 20 ml of NaCl 0.7M containing a mix of lytic enzymes (Lysing enzymes L-1412, Driselase D-8037, Chitinase C-6137, Sigma, USA) at concentrations of 5, 15, and 0.05 mg mL⁻¹, respectively. The mycelium was then incubated at 30°C on an orbital shaker at 80 rpm for 20 h. Protoplast formation was analyzed under the microscope at the end of the incubation period to verify the hydrolysis of the mycelium cell-walls. Once the protoplasts were released, they were collected by filtration through Nytal^® filters (30 μm pore diameter) and centrifuged for 15 min at 4,000 rpm. The pellet was re-suspended in 0.5 ml STC buffer (10 mM Tris HCl; pH 7.5, 1.2 M sorbitol, and 50 mM CaCl₂), counted with a Thoma cells, and diluted with STC solution to a concentration of 1 × 10⁸ protoplasts per ml. Finally, the protoplasts were mixed with solution 1 (v:v), to obtain a 5 × 10⁷ protoplast mL⁻¹ (solution 1 was prepared by mixing five volumes of STC with 1 volume of PEG [10 mM Tris HCl; pH 7.5, 50 mM CaCl₂, 30% polyethylene glycol 8000]). One-hundred microliters of this protoplast suspension containing 5 × 10⁷ protoplasts mL⁻¹, were then mixed with 10 μg of linearized plasmid, pBHt2-tdTom, resuspended in 100 µl of STC, and 50 µl of PEG. This plasmid was previously linearized with the enzyme HindIII to facilitate the integration of the vector into the fungal genome. The plasmid and the protoplast suspension were then mixed and maintained at room temperature for 15 min, followed by another 15 min at 42°C. Then, 2 ml of PEG were added, and the mixture was incubated at room temperature for another 5 min. Finally, the mixture was diluted with 2 ml of STC and poured as an overlay on regeneration medium plates (27.4% sucrose; 0.1% yeast extract, 0.1% NZ-amine, and 1.2% Bacto-agar). The plates were maintained at room temperature for 5 to 10 min until the medium has solidified, and subsequently incubated at 28°C for 24 h to allow the regeneration of the protoplasts. Finally, a 1% agar overlay containing hygromycin B at a concentration of 200 µg mL⁻¹ was added to the plates, and they were left in incubation, at 28°C, until the appearance of the transformants, for 3 to 5 days ( Supplementary Data S2: Figure S3 ).

The transformants were analyzed and confirmed by PCR following the TERRA method (PCR Direct polymerase mix. Clontech, Mountain View, CA).