dsRNAs of ~300 bp in size that were complementary to MaATG8F and MaATG4B were generated with the T7 RNAi Transcription Kit following the manufacturer’s protocol (Vazyme Biotech, Nanjing, China). The PCR technique was used to add the T7 promoter sequence to both ends of the RNA interference (RNAi) target fragments. The primers used for PCR are listed in Table S2. Sequences including the T7 promoters were purified and used as the templates for dsRNA amplification. Infection assays were then performed to establish the effects of knocking out target genes performed as in a previous study with some modifications [32 (link)]. Specifically, banana leaves of the same age were detached and pressed with the tip of a pipette head to make even wounds, then treated with 10 μL dsRNA at a concentration of 500 ng/μL with 0.02% Silwet L-77 (Solarbio Science & Technology, Beijing, China). The sites in the same leaves treated with 10 μL water with 0.02% Silwet L-77 were used as a control. The Silwet L-77 was a surfactant used for facilitating the adsorption and spread of dsRNA or water on the banana leaves. The dsRNA solution and water were allowed to dry for about 1 h, then mycelial plugs of 5 mm in diameter were taken from plates containing 6-day-old Foc TR4 strain II5. Plugs were taken only from the growing edges and were placed onto the portion of banana leaves treated with dsRNA. The mycelium was facing down, touching the banana leaves. A total of 10 banana leaves were used for each treatment. Each experiment was repeated three times independently. All leaves were then moved to plastic trays, each of which was lined with two layers of moistened paper towel. To maintain sufficiently high humidity, each tray was covered with plastic film. Trays containing inoculated leaves were incubated at 28 °C in the dark for 10 days. Leaves were then photographed, and the fungal lesion size was quantified in ImageJ V1.8.0 software (https://imagej.nih.gov/ij/, access on 20 November 2023). Leaves were also harvested for qRT-PCR to verify gene silencing.
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