Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii

Figures

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  Figure 1. Comparison of pathogenicity of T. asahii strains against silkworms. (a–f) The number of surviving silkworms under a rearing condition at 37 °C was determined 48 h after administration of the fungal cells (1 × 102 to 2 × 107 cells/larva) into the hemolymph of silkworms. Survived and dead silkworms are indicated as 1 and 0, respectively. n = 4/group. The curves were drawn from the combined data of 2–6 independent experiments by a simple logistic regression model. (g) LD50 values of T. asahii strains. Data for JCMM2466 are cited from Matsumoto et al.20.

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  Figure 2. Construction of the ku70 gene-deficient mutant in the T. asahii MPU129 strain by the ATMT system. (a) Structure of the plasmid used to construct the ku70 gene-deficient mutant and the predicted genome of the ku70 gene-deficient mutant. (b) The wild-type (MPU129) and ku70 gene-deficient candidate were spread on Sabouraud agar medium with or without G418 (50 µg/ml) and incubated at 27 °C for 2 days. (c) Location of the primers for confirming the genome structure of the ku70 gene-deficient candidate by PCR. (d) Confirmation of the ku70 gene-deficiency of the ku70 gene-deficient candidate by PCR using extracted genome DNA. Cropped blots were used. Full-length blots are presented in Supplementary Fig. 1.

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  Figure 3. Effects of ku70 gene-deficiency in the T. asahii MPU129 mutant on growth, morphology, and pathogenicity against silkworms. (a–d) The wild-type (MPU129) and ku70 gene-deficient mutant were inoculated on RPMI-1640 medium and Sabouraud medium and incubated at 27 °C or 37 °C. Absorbance 630 nm of the culture was monitored. Data are shown as means ± standard error of the mean (SEM). (e) The wild-type (MPU129) and ku70 gene-deficient mutant were incubated at 2 days after inoculation and observed with a microscope. (f–h) Saline, T. asahii MPU129 (WT), or the ku70 gene-deficient mutant (∆ku70) [1.1 × 104 cells/larva (f), 2.2 × 103 cells/larva (g), 4.4 × 102 cells/larva (h)] were injected into the silkworm hemolymph and incubated at 37 °C. The survival of the silkworms was monitored for 74 h. The significance of differences between groups in silkworm infection experiments was calculated by the log-rank test based on the curves by the Kaplan–Meier method. NS Not significant (P > 0.05). n = 10/group.

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  Figure 4. Construction of cnb1 gene-deficient mutant in the ku70 gene-deficient mutant of T. asahii MPU129 strain by electroporation. (a) Structure of the DNA fragment for construction of the cnb1 gene-deficient mutant and the predicted genome of the cnb1 gene-deficient mutant. (b) The parent strain (MPU129 ∆ku70) and cnb1 gene-deficient candidates (∆cnb1 #1, #2, and #3) were spread on Sabouraud agar medium with or without nourseothricin (100 µg/ml) and incubated at 27 °C for 2 days. (c) Location of the primers for confirming the genome structure of the cnb1 gene-deficient candidate by PCR. (d) Confirmation of the cnb1 gene-deficiency of the cnb1 gene-deficient candidate by PCR using extracted genome DNA. Cropped blots were used. Full-length blots are presented in Supplementary Fig. 1.

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  Figure 5. Attenuated pathogenicity of T. asahii against silkworms by cnb1 gene-deficiency. (a) Saline, T. asahii MPU129 ∆ku70 (parent strain) (5.4 × 104 cells/larva), or the cnb1 gene-deficient mutants [∆cnb1 #1 (7.1 × 104 cells/larva), #2 (5.2 × 104 cells/larva), and #3 (2.5 × 104 cells/larva)] were injected into the silkworm hemolymph and the silkworms were incubated at 37 °C. The survival of the silkworms was monitored for 72 h. The significance of differences between parent strain group and the cnb1 gene-deficient mutant groups was calculated by the log-rank test based on the curves by the Kaplan–Meier method. n = 10/group. (b) The number of surviving silkworms under a rearing condition at 37˚C was determined at 48 h after administration of the fungal cells (3.3 × 102 to 6.2 × 106 cells/larva) into the hemolymph of silkworms. Survived and dead silkworms were indicated as 1 and 0, respectively. n = 4/group. The curves were drawn from combined data of 2–3 independent experiments by simple logistic regression model.