Microscopy

For microscopic observation of E. festucae strains, cultures were grown on slide plates. Approximately 20 mL of PD agar was poured into Petri dishes and allowed to set.

Sterile slides were then placed on top of the PD agar and overlayed with 8-10 mL of PD agarose (2% w/v). Cultures were then inoculated adjacent to the slide and incubated at 22°C for ~7 days or until the colonies started to grow across the slide. Fluorescence and DIC microscopy was performed using an Olympus IX71 microscope with a ×63 or a ×100 oil immersion objective, NA = 1.4. Photographs were taken with a Hamamatsu ORCA- ER C4742-80 digital CCD camera (Hamamatsu Corporation) and analysed using Metamorph software (Molecular Devices Corporation). Confocal microscopy was performed using a Leica TCS SP5 confocal microscope. Bright field microscopy was performed using a Zeiss Axiophot light microscope and images recorded using a Leica DCF320 digital camera.

2.11.1. Light microscopy

To quantify changes in S. pombe cell length, cells were observed by DIC (differential interference contrast) microscopy 19, 22, 25 and 27 h following release of thiamine repression at a density of 2-4 x 106 _{cells/mL. At least 100 cells were measured for each}

sample and average lengths calculated. Results are representative of three independent experiments. Growth of E. festucae strains in culture was examined using DIC microscopy. Superoxide production by E. festucae strains was visualised by bright field microscopy of NBT stained samples. In planta samples stained with Alcian blue/safranin O, aniline blue, toluidine blue, or Lactophenol trypan blue were also observed by light microscopy.

2.11.2. Fluorescence microscopy

Samples for fluorescence microscopy were kept in the dark after staining to prevent photobleaching.

2.11.2.1. GFP

GFP localisation in S. pombe was performed in an imaging chamber (CoverWell, 20 mm diameter, 0.5 mm deep) (Molecular Probes) filled with 800 µL of 2% agarose in EMM

and sealed with a 22x22 mm glass coverslip. Cells were observed under UV light, and for osmotic stress induction, the EMM agarose was supplemented with 0.6 M KCl.

2.11.2.2. FM4-64

For visualisation of vacuoles in E. festucae strains, samples were stained with FM4-64. 4-5 x 1 µL aliquots of FM4-64 (stock concentration 1.64 mM in DMSO, diluted to 16.4 µM

with milli-Q prior to use; Invitrogen) were spotted around the edge of the colony. A sterile coverslip was then mounted on the colony and incubated at 22°C for ~28 h. Fluorescence was then observed under UV light.

2.11.2.3. Calcofluor white (CFW)

For visualisation of chitin deposits, samples were stained with calcofluor white by spotting 4-5 x 1 µL aliquots of 3 µg/mL CFW (Fluorescent brightener 28; Sigma)

around the edge of the colony then mounting a coverslip. Samples were incubated at RT for 2-5 min then fluorescence observed under UV light. Cell compartment lengths were then measured as the distance between septa using Metamorph software. The width of cell compartments was measured at the septa.

2.11.3. Confocal microscopy

Calcofluor white stained fungal hyphae and EGFP-expressing hyphae growing in planta were examined using a Leica SP5 DM6000B Confocal microscope. For examination of fungal growth in planta, whole leaf sheathes were mounted in water and optical sections taken at 2 µm intervals through 50 µm depth.

2.11.4. Transmission electron microscopy

For examination of hyphal structure, small pieces (0.5-1 mm thick) of pseudostem tissue from endophyte-infected plants were fixed in 3% glutaraldehyde and 2% formaldehyde

in 0.1 M phosphate buffer, pH7.2 for 1 h and then transverse sections prepared for light microscopy and TEM as described by Spiers and Hopcroft (1993). For light microscopy, the sections were stained with toluidine blue as described by Christensen et al. (2002). A Philips CM10 transmission electron microscope was used to examine hyphal structure and images were recorded using a SIS Morada digital camera.

2.11.4.1. Cerium chloride staining for ROS

To detect hydrogen peroxide in planta, a modified cytochemical method was used (Briggs et al., 1975; Bestwick et al., 1997). Approximately 3-4 mm sections from the meristematic zone of endophyte-infected plants were vacuum-infiltrated with 5 mM CeCl3 solution buffered with 50 mM 3-(N-morpholino)-propanesulfonic acid (MOPS),

pH 7.2 or MOPS only. Samples were then fixed in 3% glutaraldehyde buffered with 0.1 M cacodylate buffer, pH 7.2 for 1 h. Samples were then incubated in 1% osmium tetroxide at RT for 30 min – 1 h, followed by dehydration through an acetone series and infiltration and embedding in Procure 812 epoxy resin. Samples were prepared for TEM as described by Shinogi et al. (2001), observed with a Philips CM10 transmission electron microscope and images recorded using a SIS Morada digital camera.

2.11.5. Scanning electron microscopy

For examination of epiphyllous hyphae by scanning electron microscopy (SEM), plants were inoculated with the E. festucae strains then grown under sterile conditions on MSO- phytoagar to prevent surface contamination by other fungi. Blade and sheath samples were incubated in fixative (3% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.2) for a minimum of 24 h at room temperature. Samples were then washed three times with 0.1 M phosphate buffer (pH 7.2) before being dehydrated by passage through a graded ethanol series. Samples were then critical point dried using liquid CO2. Dried samples were mounted onto aluminium specimen support stubs,