Reagents
Nourseothricin and G418 were purchased from Jena Bioscience (Dortmund, Germany) and Enzo Life Science, Inc. (Farmingdale, NY, USA), respectively.
Culture of T. asahii
The T. asahii strain (MPU129 ku70 gene-deficient mutant) used in this study was generated as previously reported (Matsumoto et al. 2021). The strain have been deposited in MPU culture collection. The T. asahii MPU129 ku70 gene-deficient mutant was grown on Sabouraud dextrose agar (SDA) containing G418 (50 μg/ml) and incubated at 27°C for 2 days.
Preparing competent T. asahii cells
Competent T. asahii cells were prepared according to a previous report with slight modification (Matsumoto et al. 2021). The T. asahii MPU129 ku70 gene-deficient mutant was spread on SDA and incubated at 27°C for 1, 2, or 5 days. T. asahii cells on the agar were suspended in physiologic saline solution (2 ml), and the suspension was transferred to a 1.5-ml tube. The fungal cells were collected by centrifugation at 8,000 rpm for 3 min (TOMY-MX100, TOMY Digital Biology Co. Ltd, Tokyo, Japan) and suspended by adding 1 ml of ice-cold water and centrifuged at 8,000 rpm for 3 min. This washing process was repeated four times. The washed cells were suspended by adding 1 ml of 1.2 M sorbitol solution and centrifuged at 8,000 rpm for 3 min. The obtained fungal cells were suspended with 0.2 ml of 1.2 M sorbitol solution as competent cells.
Electroporation
Electroporation was performed according to a previous report with slight modification (Matsumoto et al. 2021). The PCR-amplified 5'-UTR (cnb1) -NAT1-3'-UTR (cnb1) fragment (180 ng/2 µl) was added to the competent T. asahii cells (40 µl) and placed on ice for 15 min. The suspension was added to a 0.2-cm gap cuvette (Bio-Rad Laboratories, Inc.) and electroporated (time constant protocol: 1.8 kV, 5 ms) using a Gene Pulser Xcell (Bio-Rad Laboratories, Inc.). The cells were suspended by adding 500 µl YPD containing 0.6 M sorbitol and incubated at 27°C for 3 h. After incubation, the cells were collected by centrifugation at 10,000 rpm for 5 min, suspended in 100 µl of physiologic saline solution, and applied to SDA containing nourseothricin (300 µg/ml). The cells were incubated at 27°C for 3 days, and the growing colonies were isolated as cnb1 gene-deficient mutant candidates.
Genotyping PCR
Genotyping PCR was performed according to a previous report with slight modification (Matsumoto et al. 2021). The transformants were grown on SDA containing nourseothricin (300 µg/ml). Colony PCR was performed using Primers-2 for cnb1 genotyping (Table 1). The genome of transformants selected by the colony PCR was extracted using a Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA). The mutation of the genome of the transformants was confirmed by PCR using the primers shown in Table 1.
Amplification of DNA fragments by joint PCR
Joint PCR was performed according to a previous report with slight modification (Lin et al. 2015). The genome of T. asahii MPU129 strain was used as a template to amplify the 5′-UTR (cnb1) and 3′-UTR (cnb1), and the previously generated 5'-UTR (cnb1)-NAT1-3'-UTR (cnb1) was used as a template to amplify the NAT1 gene (1st PCR). Double-joint PCR was performed using the PCR products obtained by the 1st PCR to amplify 5'-UTR(cnb1)-NAT1 and NAT1-3'-UTR(cnb1) (2nd PCR). The 5'-UTR (cnb1)-NAT1-3'-UTR (cnb1) was amplified by double-joint PCR using the PCR product obtained by 2nd PCR (3rd PCR).