Zen system

For immunofluorescence, cells were seeded at approximately 50% confluence in 24-well chamber slides (Cellvis, USA) and subsequently were transfected with 100 nM siRNA or treated with 50 μM FDW028. After removal of culture medium, the chamber slides were washed twice with PBS. Then, cells were fixed with 4% formaldehyde for 20 min and washed three times with PBS for 5 min each. Cells were then permeabilized with 0.2% Triton X-100 and blocked in 3% BSA in PBS for 1 h at room temperature. The primary antibodies were diluted in the blocking buffer (1:200) and incubated in chamber slides for 50 min at room temperature. The cells were rinsed by PBS three times for 5 min each. Secondary antibodies were diluted in PBS (1:800) and incubated for 20 min at room temperature, followed by washing three times with PBS for 5 min each. Cells were examined with Zeiss LSM710 confocal microscope (Carl Zeiss, Germany) fitted with a 63 × oil immersion objective or AIR HD25 confocal microscope (Nikon, USA) fitted with a 100 × oil immersion objective. Micrographs were captured by means of confocal software ZEN system (Carl Zeiss) or NIS-Elements Viewer (Nikon). Colocalization analysis was performed using the ImageJ software.

The confocal laser scanning microscope (CLSM) LSM880 (Carl Zeiss, Jena, Germany) equipped with a 63×/0.9 numerical aperture Plan-Apochromat objective was used to acquire confocal microscopic images. We visualize fungal and rice cells by CLSM and confocal reflection microscopy. Fungal nuclei and rice cells were visualized with 488 and 400-nm lasers, respectively. Acquired confocal images were analyzed using ZEN Software (Version 3.5, Carl Zeiss) and ImageJ software. Another CLSM TCS SP5 (Leica, Mannheim, Germany) equipped with HC PL APO 20x/0.75 IMM CORR CS2 objective lens was used to acquire confocal dual-color microscopic images. For epi-fluorescent inverted microscopy, cells were observed using an Axio Observer Z1 (Carl Zeiss) microscope equipped with a Plan-Apochromat 63  ×  1.4 Oil or 10 or 20 times objective lens, an AxioCam 506 mono camera and Colibri.2 LED light (Carl Zeiss). Temperature of the stage was kept at 30 °C by a thermo-plate (TOKAI HIT, Japan). For zoom microscopy, plates were observed by AXIO Zoom V16 and HXP 200C illuminator (Carl Zeiss). Images were collected and analyzed using the Zen system (Carl Zeiss) and ImageJ software.

Cells grown on coverslips were fixed with 3.7% formaldehyde in PBS, 10 min at room temperature, followed by permeabilisation in PBS containing 0.1% TritonX-100, 5 min at room temperature. Blocking and incubations with secondary antibodies were performed at room temperature in PBS containing 0.05% Tween and 3% BSA. Primary antibodies used were: mouse anti-TOMM20 (Santa Cruz Biotechnology, sc-17764), rabbit anti-LC3A/B (Cell Signalling Technologies, 12741S). Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 0.1 μg/ml; Sigma-Aldrich) and mounted using Flouromount Mounting Media (Sigma-Aldrich, F4680). Samples were analysed using a Zeiss LSM 800 microscope equipped with 63X or 100X (oil immersion) objectives. Images were acquired using ZEN system (ZEISS, Germany). ImageJ software was used for image analysis. Calculation of the mitochondrial content as percentage of cell area occupied by mitochondria, was performed using the “Mitophagy” macro [40 (link)].

Live-cell imaging of germlings and young hyphae: cells were grown on cover slips in 0.5 ml MMGly (de-repression of the alcA promoter) were used. Although GFP-ChsB was expressed under the native promoter, to compare the data of GFP-CsmA, which was expressed under the alcA promoter, MMGly was selected. Cells were incubated at 30°C overnight or 1 day. The cover slips were mounted on a microscope slide. Tempcontrol mini (Pepcon) was used as needed to control the temperature of the specimens during microscopy. Images were captured using an Axiophot microscope using a Planapochromatic 63 times oil immersion objective lens, the Zeiss AxioCam MRM camera (Zeiss, Jena, Germany), and the HBO103 mercury arc lamp (Osram) or HXP 120 (Zeiss, Jena, Germany) possessing faster speed wavelength switching. Images were collected and analyzed using the AxioVision and Zen system (Zeiss). Kymographs were made using ImageJ software (http://rsb.info.nih.gov/ij/) and Zen system (Zeiss).

Cells were grown either in Fluorodishes FD35-100 (World Precision Instruments) or on cover slips (Carl Roth) with minimal medium + 2% carbon source at 28°C overnight. Images were captured using an Axiophot microscope using a planapochromatic 63x/1.4 N.A. oil immersion objective lens, the ZEISS AxioCam MRM camera (ZEISS, Jena, Germany), and the HBO103 mercury arc lamp (Osram) or HXP 120 (ZEISS) possessing faster speed wavelength switching. Images were collected and analyzed using the Zen system (ZEISS).