Spe dm2500

For visualization of DNA content and cellular morphology, cells were stained with DAPI and observed by differential interference contrast (DIC) and fluorescence microscopy using a DeltaVision microscopy system comprising an Olympus IX71 microscope (Olympus, Shinjuku, Tokyo, Japan) and CoolSnap HQ camera (Photometrics, Tucson, AZ, USA).
For in plant quantification of filament and appressoria formation in co-infection experiments with U. maydis CFP and RFP labelled strains, 20 hpi leaf samples were stained with calcofluor white (Sigma-Aldrich, St. Louis, MO, USA) to visualize fungal material and then checked for CFP or RFP fluorescence in the DeltaVision microscopy system.
To analyze the U. maydis progression inside the maize plant, leaf samples stained with PI and WGA-AF488 (described above) were examined using a Leica SPE (DM2500, Leica, Wetzlar, Germany) confocal microscope.
Image processing was carried out using Adobe Photoshop CS5 (Adobe, San Jose, CA, USA) and ImageJ (public domain).

To analyze the filamentation capability of U. maydis in PD-charcoal plates, single colonies where visualized using the Leica M205 Stereoscope equipped with an ORCA-Flash4.0 LT Hamamatsu digital camera. The area of the colonies was measured by selecting the perimeter of each colony using the plugging convex hull of ImageJ software. To determine sirtuins’ localization, sir2:eGFP, hst2:eGFP, hst4:eGFP, hst5:eGFP and hst6:eGFP cells were visualized using a DeltaVision microscopy system comprising an Olympus IX71 microscope and CoolSnap HQ camera (Applied Precision, Issaquah WA, United States). To visualize mitochondria, 0.5 mM Mito-Tracker CM-H2Xros (Molecular Probes, Eugene, OR) was added to the U. maydis YEPSL cell culture and cells were incubated for 15 min at 25°C (Bortfeld et al., 2004 (link)). To analyze the U. maydis progression inside the maize plant, leaves samples from 3, 4 and 6 dpi infected plants were distained with ethanol, treated for 4 h at 60°C with 10% KOH, washed in phosphate buffer and then stained with propidium iodide (PI) to visualize plant tissues in red and wheat germ agglutinin (WGA)/AF488 to visualize the fungus in green. At least four leaves from two independent experiments were stained and visualized by fluorescence microscopy (Leica SPE DM2500, Leica, WZ, Germany). Image processing was carried out using the ImageJ software.

For on plant filamentation and appressoria quantification, wild-type cells harboring cytoplasmic CFP and mutant cells harboring cytoplasmic RFP were grown in liquid YEPSL until exponential phase, washed twice with bi-distilled water, and concentrated to an OD₆₀₀ of 3. Cells from wild-type and mutant strains were mixed in equal proportions, centrifuged, and concentrated to an OD₆₀₀ of 3. Maize seedlings were infected, and leaves were recovered 18–20 h post-infection. Infected leaves were stained with calcofluor white (Sigma-Aldrich, St. Louis, MO, USA) to visualize U. maydis filaments and appressoria in a fluorescence microscope.
To analyze maize plant colonization capability, U. maydis cultures were grown at 28 °C to exponential phase in liquid YEPSL and concentrated to an OD₆₀₀ of 3, washed twice in water, and injected into seven-day-old maize seedlings. Three days post-infection, leaves were de-stained with ethanol for at least 24 h, treated at 60 °C with 10% KOH for 4 h, washed four times in phosphate buffer, and then stained with propidium iodide (PI) to visualize plant tissues in red and wheat germ agglutinin (WGA)-AF488 to visualize the fungus in green for 30 min in a vacuum pump with 5 min vacuum and 5 min rest cycles. At least four leaves from two independent experiments were stained and visualized by fluorescence microscopy (Leica SPE DM2500, Leica, Wetzlar, Germany).