A joint PCR-based gene-targeting method using electroporation in the pathogenic fungus Trichosporon asahii

Abstract

Trichosporon asahii is a pathogenic fungus that causes deep-seated fungal infections in immunocompromised patients. Established methods for generating gene-deficient T. asahii mutants exist, but the frequency of obtaining transformants by electroporation remains low. In the present study, we optimized the conditions for gene transfer by electroporation using a ku70 gene-deficient mutant with high recombination efficiency. Introducing a DNA fragment by electroporation into T. asahii cells on Sabouraud dextrose agar to generate a cnb1 gene-deficient mutant and incubating for 1 day led to the growth of approximately 100 transformants. When the incubation period was extended to 2 days or 5 days, however, only 2 or no transformants, respectively, were grown. The highest number of transformants was grown by electroporation when a square wave at 1.8 kV (9 kV/cm) was applied for 5 ms. In addition, the number of transformants increased with an increase in the length of the homologous region, and transformants did not grow when the homologous region was less than 500 base pairs. A DNA fragment was produced for deletion of the cnb1 gene by joint PCR, and the cnb1 gene-deficient mutant was obtained by introducing the DNA fragment by electroporation. These results indicate that DNA fragments produced by joint PCR can be used to generate gene-deficient mutants of T. asahii through gene transfer by electroporation.

Keywords: Electroporation; Gene targeting; Joint PCR; Mutant; Trichosporon asahii.

Fig. 1

Optimization of the conditions for gene transfer by electroporation in T. asahii. A Structure of the DNA fragment for constructing the cnb1 gene-deficient mutant and the predicted genome of the cnb1 gene-deficient mutant. B Scheme for obtaining drug-resistant strains by gene transfer via electroporation. C Effect of the number of incubation days for preparing competent T. asahii cells. The T. asahii MPU129 ku70 gene-deficient mutant was spread on SDA and incubated at 27 °C for 1, 2, or 5 days. 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 electroporated (time constant protocol: 1.8 kV, 5 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted. D Effect of voltage on gene transfer by electroporation. The PCR-amplified 5'-UTR (cnb1) -NAT1-3'-UTR (cnb1) fragment (180 ng/2 µl) was added to competent T. asahii cells (40 µl) prepared by culture for 1 day and electroporated (time constant protocol: 1.2–2.1 kV, 5 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted. E Effect of time constant on gene transfer by electroporation. The PCR-amplified 5'-UTR (cnb1) -NAT1-3'-UTR (cnb1) fragment (180 ng/2 µl) was added to competent T. asahii cells (40 µl) prepared by culture for 1 day and electroporated (time constant protocol: 1.8 kV, 3–10 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted

Fig. 2

Effect of the length of homologous regions for gene transfer by electroporation in T. asahii. A Illustration of DNA fragments used in this study. B Electrophoresis of DNA fragments amplified by PCR. C The PCR-amplified 5ʹ-UTR (cnb1) -NAT1-3ʹ-UTR (cnb1) fragments were added to the competent T. asahii cells prepared by culture for 1 day and electroporated (time constant protocol: 1.8 kV, 5 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted

Fig. 3

Effect of the DNA concentration for gene transfer by electroporation in T. asahii. The DNA fragment 2 (0–13.5 nM) was added to competent T. asahii cells prepared by culture for 1 day and electroporated (time constant protocol: 1.8 kV, 5 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted

Fig. 4

Construction of DNA fragment for gene targeting by joint PCR. Scheme of constructing DNA fragments for gene targeting by joint PCR to generate cnb1 gene-deficient mutants and electrophoresis of DNA fragments amplified by PCR or joint PCR

Fig. 5

Generation of cnb1 gene-deficient mutant using DNA fragment produced by joint PCR. A DNA fragment 2 produced by joint PCR was added to competent T. asahii cells prepared by culture for 1 day and electroporated (time constant protocol: 1.8 kV, 5 ms). Colony PCR was performed on colonies (samples 1–15) grown on SDA containing nourseothricin (300 µg/ml). B Location of the primers for confirming the genome structure of the cnb1 gene-deficient candidate by PCR. C Confirmation of the cnb1 gene-deficiency of the cnb1 gene-deficient candidate by PCR using extracted genome DNA