The Tetracycline-Inducible/CRISPR-Cas9 System is an Efficient Tool for Studying Gene Function in Phytophthora sojae

Abstract

The present study presents a novel approach combining a tetracycline-inducible system (Tet-On) and CRISPR-Cas9 techniques to investigate the function of two essential genes in Phytophthora sojae. We constructed a donor vector in which the reverse tetracycline transactivator (rtTA) is driven by an oomycete promoter. Additionally, it contains a fused TetR binding site and the minimum oomycete promoter, as well as 1000-bp homologous arms of the promoter upstream and downstream sequences. The promoter of the target gene was replaced with a tetracycline-responsive promoter (P_tet) using a CRISPR-Cas9 system. In the native transformants, the target gene was induced by the administration of tetracycline and repressed in its absence. Using the Tet-On/CRISPR-Cas9 system, we obtained inducible transformants of PsAF5 and PsCesA3. The phenotype of PsAF5 inducible transformants without doxycycline was consistent with that of ΔPsAF5 transformants, specifically characterised by an increase in oospore production and heightened sensitivity to H₂O₂. PsCesA3 inducible transformants could not grow in the absence of doxycycline, which means PsCesA3 is an essential protein for P. sojae. In conclusion, the Tet-On/CRISPR-Cas9 system represents an effective approach for investigating crucial genes in P. sojae.

Keywords: Phytophthora sojae; CRISPR‐Cas9; inducible transformants; tetracycline‐inducible system.

FIGURE 1

Sensitivity of Phytophthora sojae to doxycycline. The concentration of doxycycline in the red‐framed box is the concentration used in subsequent experiments. All experiments were repeated three times with consistent results. The bar graphs indicate the mean values of triplicates and error bars indicate the standard error. Tukey's honestly significant difference analysis of variance was used for statistical analysis. Different letters above the bars indicate significant differences (p < 0.05).

FIGURE 2

The Tet‐On/CRISPR‐Cas9 mediated system. P _tet: Tetracycline‐responsive promoter, ham34 _P: Ham34 promoter, ham34 _T: Ham34 terminator, P _min: the smallest promoter, rTetR: the reverse Tet repressor protein, AD: the activation domain used in Toxoplasma gondii.

FIGURE 3

Regulatory expression of PsAF5 and mycelial growth. (A) The expression levels of PsAF5 in hyphae were quantitatively evaluated using reverse transcription‐quantitative PCR (RT‐qPCR). The wild type (WT), control transformants (PsAF5‐CK), and two PsAF5 inducible transformants (PsAF5‐T1, PsAF5‐T2) were tested at doxycycline concentrations of 0 and 0.5 μg/mL. (B) Growth characteristics after 6 days on V8 medium. (C) Measurement of colony diameter after 6 days on V8 medium. Scale bar: 1 cm. All experiments were repeated three times with consistent results. The bar graphs indicate the mean values of triplicates and error bars indicate the standard error. Tukey's honestly significant difference analysis of variance was used for statistical analysis. Different letters above the bars indicate significant differences (p < 0.05).

FIGURE 4

Sensitivity of PsAF5 inducible transformants to 4 mM H₂O₂ and oospore production. The sensitivity of the wild type, control transformants (PsAF5‐CK), PsAF5 inducible transformants (PsAF5‐T1, PsAF5‐T2), PsAF5 knockout (ΔPsAF5‐2) and PsAF5 complementary transformants (C‐PsAF5) to H₂O₂ was detected in the absence (A) or presence (B) of doxycycline. (C) Quantification of inhibition by 4 mM H₂O₂. (D) Oospore number of the wild type (WT), control transformants (PsAF5‐CK), PsAF5 inducible transformants (PsAF5‐T1, PsAF5‐T2), PsAF5 knockout (ΔPsAF5‐2), and PsAF5 complementary transformants (C‐PsAF5) at doxycycline concentrations of 0 and 0.5 μg/mL. C‐PsAF5: Transformed full‐length PsAF5 into the knockout mutant ΔPsAF5‐2 to generate a complementary transformant C‐PsAF5. Scale bar: 1 cm. All experiments were repeated three times with consistent results. The bar graphs indicate the mean values of triplicates and error bars indicate the standard error. Tukey's honestly significant difference analysis of variance was used for statistical analysis. Different letters above the bars indicate significant differences (p < 0.05).

FIGURE 5

Regulatory expression of PsCesA3 and mycelial growth. (A) Reverse transcription‐quantitative PCR (RT‐qPCR) detection of the expression levels of PsCesA3 in hyphae of the wild type (WT), control transformants (PsCesA3‐CK), and two PsCesA3 inducible transformants (PsCesA3‐T1, PsCesA3‐T2) was measured at doxycycline concentrations of 0 and 0.5 μg/mL. (B) Growth characteristics after 6 days on V8 medium. (C) Measurement of colony diameter after 6 days on V8 medium. Scale bar: 1 cm. (D) Determination of the EC₅₀ values of the wild type (WT), control transformants (PsCesA3‐CK) and PsCesA3 inducible transformants (PsCesA3‐T1, PsCesA3‐T2) to dimethomorph at 0.5 μg/mL doxycycline. All experiments were repeated three times with consistent results. The bar graphs indicate the mean values of triplicates and error bars indicate the standard error. Tukey's honestly significant difference analysis of variance was used for statistical analysis. Different letters above the bars indicate significant differences (p < 0.05).