4.3. ATMT Transformation

A. tumefaciens strain EHA105 transformants carrying pDHt/ZafA::hph or pDHt/ZafA-bar were grown on solid YEB medium supplemented with 50 μg/mL of rifampicin, 25 μg/mL of chloramphenicol, and 50 μg/mL of kanamycin at 28 °C for 48 h, collected in 10 mL of sterile 0.9% sodium chloride solution, centrifuged at 3500 rpm/min, collected in 10 mL of the liquid YEB medium (50 μg/mL kanamycin, 50 μg/mL rifamycin, and 25 μg/mL chloramphenicol), and cultured for 48 h at 28 °C. After centrifuging at 3500 rpm/min, the A. tumefaciens cells were suspended in liquid AIM to an optical density of 0.7 at 660 nm. After adding 200 μM of acetosyringone, the bacterial suspensions were incubated on a shaker at 150 rpm/min at 28 °C for 6 h.

The wild-type T. mentagrophytes strain 28,185 was cultured on solid SDA medium at 28 °C for 14 days, then gently washed with sterile 0.9% sodium chloride solution, and the conidial concentration was adjusted to 1 × 10⁷ colony-forming units (CFU)/mL. A mixture of 100 μL of A. tumefaciens suspension and 100 μL of T. mentagrophytes conidial suspension was spread onto sterilized nylon membranes (Sigma, USA), placed on solid AIM medium (supplemented with 200 μM of acetosyringone) and incubated at 28 °C in the dark for 60 h. The nylon membranes were transferred onto a solid SDA medium plate supplemented with 600 μg/mL of hygromycin B and 50 μg/mL of cephalexin, and incubated at 28 °C for two to three days [28,40]. After two to three days, fungal colonies were produced on the nylon membranes, and 10 samples were randomly chosen on one solid SDA medium plate and cultivated on solid SDA medium supplemented with 600 μg/mL of hygromycin B at 28 °C for 14 days. The T. mentagrophytes ZafA mutant was named ZafA-hph. The T. mentagrophytes ZafA complemented strain was obtained by the same methods as above, then selected by 2 mg/mL of glufosinate-ammonium and named ZafA+bar.