DclB and PpdA are required for IRT-mediated silencing in A. fumigatus. (A) Droplet growth assays for wild-type (WT), ΔdclA/B, ΔppdA/B, and ΔrrpA/B strains grown on AMM or Malt agar plates for 48 h. Image shown is representative of three independent experiments. (B) Wild-type and RNAi knockout strains transformed with IRT constructs were spot inoculated on AMM with and without inducer (10 µg/mL doxycycline [dox]) for 2 d. Image shown is representative of three independent experiments. (C) Wild-type and IRT-transformed strains were spot inoculated on AMM agar plates with inducer (10 µg/mL doxycycline) for 2 d. Spores were collected and the experiment was repeated without IRT induction. (D) The wild-type (CEA17ΔakuBKU80) strain was transformed with a plasmid (pSilent-pabA) expressing a dox-inducible IRT targeting pabA, a para-aminobenzoic acid synthetase-encoding gene. Wild-type and pabA-IRT-expressing strains were spot inoculated on AMM agar plates supplemented with different concentrations of dox. Images were taken after 2 d of incubation at 37°C. (E) Wild-type and pabA-IRT-expressing strains, as well as a control pksP-IRT-expressing strains were grown with and without dox induction for 24 h. Mycelia were collected and dried for 3 d at 60°C before measuring weight. Plot displays data from four biological replicates, with each replicate having three technical repeats. Significance was determined by two-way ANOVA with Bonferroni-corrected post hoc t-test comparing all columns. (F) Validation of silencing of pabA gene after induction of pabA-IRT using qRT-PCR. Plot shows standard error of the mean of three biological replicates performed in technical triplicates using the 2−ΔΔCT method (Livak and Schmittgen 2001). Significance was determined by one-way ANOVA followed by Bonferroni corrected post hoc t-test comparing all columns and assuming Gaussian distributions.
