, 2003). In contrast, autophagic PCD is induced by the heterokaryon incompatibility system of Podospora anserina (Pinan-Lucarréet al., 2003). Thus, the mechanism for PCD appears to vary among organisms rather than being consistent within a taxon. In previous research, we collected H. mompa isolates belonging to numerous kinds of mycelial compatibility groups (Ikeda et al., 2004). In this study, we performed cytological

analysis of mycelial incompatibility in H. mompa using light and transmission electron microscopy (TEM). The H. mompa isolates used were V18 (MAFF No. 305915), V670 (MAFF No. 328063). Cultures were maintained in Petri dishes on oatmeal agar (26 g L−1 oatmeal, 5 g L−1 sucrose, 15 g L−1 agar) at 4 °C until use. We paired H. mompa isolates (V18 vs. V670) on rectangular cellulose membranes (1 × 1.5 cm size) laid on water agar plates (15 g L−1 agar) or on 1/10-strength oatmeal selleck compound agar plates (2.6 g L−1 oatmeal, 5 g L−1 sucrose, 15 g L−1 agar) with or without 0.2% w/v activated charcoal (Wako, Osaka, Japan) in 50-mm-diameter Petri dishes (AS One, Osaka, Japan). We placed one mycelial plug on one of the short sides of the rectangle and the confronting mycelial plug at the opposite LY2109761 side, separated by 7.5 mm. After incubation of the cellulose membranes for 3 days at 25 °C, we transferred the plugs to 50-mm-diameter glass-bottomed culture dishes (MatTek,

Ashland, MA) that contained no growing medium and observed the hyphal contact zones with a fluorescence microscope (BIOREVO BZ-9000, Keyence, oxyclozanide Osaka, Japan). To evaluate the nature of the hyphal contact, we searched for hyphae that were in contact and for which both tips were visible in the field of view at of × 40 objective lens. We evaluated hyphal fusion on the basis

of whether the cell walls had merged. We tracked hyphae backwards from the zone of contact to clarify the origin of each hypha (i.e. which isolates produced it) to confirm that the hyphae represented reciprocal pairs rather than self-pairs. As some hyphae also extended vertically, we excluded the hyphal crossing in which one hypha passed over another hypha without hyphal contact; we judged this to occur when it was not possible to view both hyphae simultaneously in the same focal plane. We paired the H. mompa isolates (V18 vs. V18 or V18 vs. V670) on Aclar film (Nisshin EM, Tokyo, Japan) laid on a glass slide coated with 1/10-strength oatmeal agar medium and incubated at 25 °C for 4 days in 90-mm-diameter Petri dishes that included agar medium (15 g L−1 agar) to maintain moisture. The mycelia on the glass slide were first fixed with 2.5% glutaraldehyde (Nisshin EM) in 0.1 M phosphate-buffered saline (PBS), pH 7.4 at 4 °C overnight. The pieces were rinsed with PBS three times at the intervals of 10 min, and postfixed with 1% osmium tetroxide (Nisshin EM) in PBS at room temperature for 1 h.