Jem 1400 series 120 kv transmission electron microscope

Germination of conidia was examined with a Nikon Optiphot 2 Microscope (Tokyo, Japan). Lipid bodies within fungal cells were stained with 2.5 μg/mL Nile red (Sigma-Aldrich) solution for 20 min at room temperature and examined by a ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA, USA) microscope equipped with the 530-nm excitation and 635-nm emission wavelength filters. Transmission electron microscopy (TEM) was performed using a JEOL JEM-1400 series 120 kV Transmission Electron Microscope (Jeol, Tokyo, Japan). Samples were treated with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and 1% osmium tetraoxide, and immersed in LR White Resin after being dehydrated with an ethanol series. Samples were cut to thin slices, stained with uranyl acetate and lead citrate, and examined. Photographs were taken with an Gatan Orius SC 1000B bottom mounted CCD-camera (Gatan Inc., Pleasanton, CA, USA). For penetration studies, leaf samples inoculated with fungal strains were sectioned to thin slices by hand using a sharp razor blade, stained with lactophenol cotton blue (Sigma-Aldrich), and examined by light microscopy.

Transmission electron microscopy (TEM) was performed to observe MDV formation. Brain tissues were fixed in 2.5% glutaraldehyde (Sigma) in phosphate buffer overnight at 4 °C. Next, the samples were fixed in 1% osmium tetroxide for 2 h, dehydrated with a graded series of acetone washes, infiltrated with propylene epoxide, and embedded in Epon 618. After sample preparation, 90–100-nm-thick sections were mounted onto a 200-mesh copper grid and imaged with a JEM-1400 series 120 kV transmission electron microscope (JEOL, Japan) with an integrated high-sensitivity complementary metal oxide semiconductor (CMOS) camera.

Fluorescence was examined by a ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA, United States) microscope equipped with specific wavelength filters. Red fluorescence from mCherry was visualized using 543 nm excitation and 560–615 nm emission. Red fluorescence after staining with 1 μg/ml MM 4–64 (Enzo, New York, NY, United States) for 30 min was observed using 588 nm excitation and 734 nm emission to track endocytosis. Green fluorescence in hyphae after staining with 40 μM 2'-7'-dichlorofluorescin diacetate (DCFHDA, Sigma-Aldrich) was examined using 504 nm excitation and 529 nm emission. Blue fluorescence in hyphae after staining with 100 μM monodansylcadaverine (MDC, Sigma-Aldrich) was examined using 335 nm excitation and 518 nm emission to identify autophagosomes, amphisomes, and autolysosomes. Fluorescence after staining with 20μM CellTracker^™ Blue CMAC Dye (Molecular Probes C2110, Thermo Fisher Scientific, Waltham, MA, United States) was visualized using 353 nm excitation and 466 nm emission to detect the vacuoles. Transmission electron microscopy (TEM) using a JEOL JEM-1400 series 120 kV Transmission Electron Microscope (JEOL, Tokyo, Japan) was conducted to examine hyphae/conidia, which were soaked in 2.5% glutaraldehyde and 1% osmium tetraoxide, embedded in LR White Resin, sliced, and stained with uranyl acetate and lead citrate.