The aqueous phase was added to a 0.7 volume of dPEG solution (20% polyethylene glycol 6000, 2.5 M NaCl) on

ice for 10 min. Genomic DNA was finally recovered by centrifugation at 10 000 g at 4 °C for 10 min, washed in 70% ethanol, and resuspended in 50 μL of TE buffer. The concentrations of each genomic DNA were measured by NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE). Each 1.5 μg of genomic DNA was electrophoresed on 1.0% agarose gel in TAE buffer. We observed buy LY294002 hyphal contact in the compatible and incompatible combinations (Fig. 1). In the compatible pairing (V18 vs. V18), almost 70% of hyphal contact zones were anastomosed (Fig. 2). Various types of hyphal anastomosis (Rayner et al., 1994) were observed: tip to tip, tip to side, side to side, and side to side extending hyphal neogenesis (data not shown). In the incompatible pairing, the frequency of hyphal anastomosis was reduced and most of contacting hyphae crossed over each other (Fig. 2). We also evaluated the effects of the growing medium (i.e. water Ipilimumab agar and 1/10-strength oatmeal agar media), with or without activated charcoal. In the compatible pairing, supplementation with activated charcoal significantly decreased the frequency of fusion but significantly increased the frequencies of cross-over. In the incompatible combination, supplementation with activated charcoal increased

the frequency of hyphal fusion and cross-over but the changes were not dramatic (Fig. 2). TEM observation in the compatible combination (V18 vs. V18) revealed that hyphal anastomosis, cell wall fusion and reconstruction of septa had occurred (Fig. 3b). We also recognized a healthy plasma membrane, tonoplast, mitochondria, and nuclear membrane. In the incompatible combination (V18 vs. V670), we observed the disconnection of membranes (plasma membrane, tonoplast, nuclear membrane, and mitochondria membrane) and collapse of cell contents (cytoplasm, nuclei and nucleolus, mitochondria, and vacuole) (Fig. 3c–f). When we noticed that some

cell structures had collapsed at the hyphal contact zones, we rated the parts of the cell Meloxicam collapse by each cell component. We calculated the percentage of collapsed organella from 50 hyphae in which the following were observed: disconnection of tonoplast (10%), disconnection of tonoplast and plasma membrane (22%), disconnection of tonoplast, plasma membrane, and nuclear membrane (36%), collapse of most cell contents except mitochondria (28%), and collapse of all cell contents (4%). We found that the collapse of plasma membrane, nuclear membrane, and mitochondria never preceded the collapse of tonoplast. We estimated that PCD occurred in the following order: (1) collapse of the tonoplast, (2) collapse of the plasma membrane and nuclear membrane, (3) reduction of the electron density of nuclei and nucleolus, and (4) collapse of mitochondria.