MoEnd3 is important for appressorium formation and virulence.

(A) Appressorium formation assay. Conidia were incubated on hydrophobic surfaces and the samples were observed at different time points. Bar = 10 μm. (B) Appressorium formation rates at different time points were calculated and statistically analyzed. The percentage at a given time was recorded by observing at least 200 conidia for each strain and the experiment was repeated three times. Error bars represent SD and asterisks represent significant differences (P < 0.01). (C) Images show appressoria after 24 h incubation on hydrophobic surfaces. Bar = 10 μm. (D) Mean appressorium diameter. The values were recorded by observing at least 100 appressoria for each strain and the experiment was repeated three times. Error bars represent SD and asterisk represents significant difference (P < 0.01). (E) Appressorium turgor was measured by an incipient cytorrhysis (cell collapse) assay. The percentage of collapsed appressoria was recorded by observing at least 100 appressoria and the experiment was repeated three times. Error bars represent SD and asterisks represent significant differences (P< 0.01). (F) Conidial suspensions of strains were sprayed onto 2-week old rice seedlings (CO-39) and 7-day old barley. Diseased rice and barley leaves were photographed after 7 and 5 days of inoculation, respectively. (G) Penetration assay with rice sheath. Excised rice sheath from 4-week-old rice seedlings was inoculated with conidial suspension. Images show invasive growth in rice sheath epidermal cells at 36 hpi. Bar = 10 μm. (H) Pmk1 phosphorylation level analysis with proteins extracted from mycelium, conidia, and conidia or appressoria incubated on hydrophobic surfaces for 3 h, 8 h and 16 h. The phosphorylation levels of Pmk1 (42-kDa) were detected using a phosphor-MAPK antibody (upper panel). The endogenous Pmk1 was detected using a MAPK antibody (lower panel). (I) Appressorium formation assay on the hydrophobic surfaces. (J) Appressorium formation rates were calculated and statistically analyzed. Asterisks represent significant differences (P<0.01). (K) Pathogenicity assay on rice (CO-39). (L) Quantification of the lesion numbers per 5 cm length of rice leaf. Error bars represent SD and asterisk represents significant difference (P<0.01). (M) Penetration assays in rice sheath. IH growth on rice cells was observed at 36 hpi and 4 types of IH were quantified and statistically analyzed. Error bars represent SD. Micrographs show 4 types of IH in rice cells. Bar = 10 μm. MoEnd3 is involved in endocytosis of Pth11 and MoSho1.