Transient Silencing of Fungal Genes

Small interference RNAs (siRNA) design primers, preparation of fungal protoplast and siRNA delivery were performed according to Hanano et al. (2018a) (link). In brief, delivery of siRNA to protoplasts was done in sterile 1.5 mL tubes. A total of 10 μL of siRNA primer (100 nM) was mixed with an equal volume of Lipofectin reagent (Invitrogen Life Technologies, United Kingdom) and kept for 15 min at 20°C. A volume of 50 μL of protoplasts was added, gently mixed and incubated at 20°C for 24 h to allow transfection to proceed. The transfected protoplasts were therefore inoculated in 10 mL of PD medium with 1.2 M of sorbitol for 7 days at 28°C in the dark. All experiments were performed using three biological replicates.