Lsm780 axio observer confocal laser scanning microscope

To visualize fungal proliferation in infected tissue, the area 1–3 cm below the injection site was excised at 2–7 dpi. Fungal hyphae, i.e. chitin, were stained with wheatgerm agglutinin (WGA) coupled to AlexaFluor488 (Invitrogen). Plant cell walls were stained with propidium iodide (Sigma‐Aldrich, St. Louis, MO, USA). Leaf samples were incubated in staining solution (1 µg/mL propidium iodide, 10 µg/mL WGA‐AF488) and observed with an LSM780 Axio Observer confocal laser scanning microscope (Zeiss, Jena, Germany) under the following conditions: WGA‐AF488, excitation at 488 nm and detection at 500–540 nm; propidium iodide, excitation at 561 nm and detection at 580–660 nm. For fluorescent protein detection, leaf samples were directly observed by confocal microscopy using the following conditions for mCherry: excitation at 561 nm and detection at 580–630 nm. For plasmolysis experiments, a 1 m mannitol solution was infiltrated into the leaves and observed 30 min later. All experiments were carried out at least three times. To measure appressoria formation and penetration efficiency, infected maize leaves were harvested 18–20 h post‐infection and stained with calcofluor white (10 ng/mL). Calcofluor white stains fungal hyphae only on the leaf surface as it cannot stain penetrated hyphae. The appressorial marker used has been described previously (Mendoza‐Mendoza et al., 2009).

Cell walls of fungal hyphae were stained with the chitin specific Wheat Germ Agglutinin - Alexa Fluor 488 conjugate dye (WGA-AF 488; Thermo Fisher Scientific, Waltham, MA). Plant membranes were stained with FM4-64 (Thermo Fisher Scientific, Waltham, MA). Staining and confocal laser scanning microscopy was performed as previously described by Doehlemann et al. (2008) (link). For DAPI and WGA staining of germinating spores and sporidia, germinating spores/sporidia were pelleted and fixed by incubating them with acetone for 15 min. Fixation was followed by 10 min incubation with WGA-AF 488 to visualize fungal cell walls and 15 min incubation with 1 µg ml⁻¹ DAPI solution (Sigma-Aldrich, Taufkirchen, Germany) to stain nucleic acid. Cell/spore pellets were resuspended in PBS and subjected to confocal laser scanning microscopy. Images were acquired with a LSM780 Axio Observer confocal laser scanning microscope (Zeiss, Jena, Germany). WGA-AF excitation was at 488 nm and detection at 517–552 nm, DAPI excitation at 405 nm and detection at 421–508 nm.

For colocalization experiments U. maydis cells were grown in YNB‐N medium with 10 mM sodium salicylate for 5 h (OD_600nm = 0.8). Cells were harvested by centrifugation (2400 g, 5 min) and fixed by addition of 2% formaldehyde in 1x PBS. Samples were incubated for 5 min, centrifuged at 2400 g for 5 min, and washed once with 1x PBS. Afterwards the pellet was resuspended in DAPI (4′,6‐Diamidine‐2′‐phenylindole dihydrochloride) solution (0.5 µg ml⁻¹) and incubated for 10 min. After an additional washing step, samples were subjected to confocal microscopy. Colocalization of mCherry‐HA‐Rss1 and DAPI stained nuclei was microscopically assessed by employing an LSM780 Axio Observer confocal laser‐scanning microscope (Zeiss, Jena, Germany) with the following settings: mCherry: Laser DPSS 15 mW, excitation 561 nm, detection 578‐648 nm; DAPI: Laser Diode 25 mW, excitation 405 nm, detection 415–510 nm.