Na c apochromat oil immersion objective

To examine endocytosis, strains were cultured in liquid CM medium. After growing at 28°C for 24 h, the hyphae were stained with N‐(3‐triethylammoniumpropyl)‐4‐(p‐diethylamino‐phenyl‐hexatrienyl) pyridinium dibromide (FM4‐64) (Molecular Probes, Eugene, OR, USA). This dye is used for staining the Spitzenkorper, plasma membrane, septum, early endosomes, late endosomes and vacuolar membranes, as well as for the examination of endocytosis, as described previously (Fischer‐Parton et al., 2000). Photographs were taken using a Zeiss LSM 710 confocal microscope with a 63/1.2 NA C‐Apochromat oil immersion objective (Zeiss, Oberkochen, Germany). The relative fluorescent density was analysed using Image‐pro Plus (Media Cybernetics, Shanghai, China). 7‐amino‐4‐chloromethylcoumarin (Sigma‐Aldrich) (10 mM stock solution in dimethyl sulfoxide) was used for vacuole staining, as previously described (Ohneda et al., 2002; Shoji et al., 2006). Photographs were taken using the confocal laser scanning microscope, as described earlier.

Wild type F. oxysporum f.sp. lycopersici (Fol) strain 4287 and mutant strains used in this study are listed in Supplementary Table S1. Vegetative growth rate was determined using potato dextrose agar (PDA) and complete medium (CM) at 28°C for 5 days (Li et al., 2019b (link)). Conidiation was assayed as previously reported (Li et al., 2019b (link)). The conidial morphology of wild type and mutants was examined using a Zeiss LSM 710 confocal microscope with a 63/1.2 NA C-Apochromat oil immersion objective (Zeiss, Oberkochen, Germany). Liquid CM was used to grow cultures for extraction of genomic DNA and total RNA.

To track the fluorescein-labeled FolRDR1/GFP-dsRNA, plant tissues and fungal materials were collected after dsRNA treatment with the concentration of 200 ng/μL and 150 ng/mL, respectively. Images were taken using a Zeiss LSM 710 confocal microscope with a 63/1.2 NA C-Apochromat oil immersion objective (Zeiss, Oberkochen, Germany). The relative fluorescent density was analyzed usingImage-pro Plus (Media Cybernetics Inc., Shanghai, China).