Figure 1.

MoHat1 is important for pathogenicity in M. oryzae. (a) Rice spraying assays. Four milliliters of conidial suspension (5 × 10⁴ spores/ml) of each strain were sprayed on two-week old rice seedlings (Oryza sativa cv. CO39). Diseased leaves were photographed after 7 days incubation. (b) Quantification of lesion types (per 1.5 cm²) on susceptible rice spayed with conidia of wild-type Guy11, ∆Mohat1, and ∆Mohat1/MoHAT1 complemented strains. Disease lesions were quantified by a ‘lesion-type’ scoring assay which divided the lesions into 1–5 types according to their severity (type 0, no lesion; type 1, pinhead-sized dark brown specks without visible centers; type 2, small brown lesions that are approximately 1 mm in diameter; type3, 2- to 3-mm gray spots with brown margins; type 4, elliptical gray spots longer than approximately 3–4 mm; type 5, large eyespot lesions that coalesced infecting 50% or more of the leaf area). Error bars represent SD and asterisks represent significant differences (P < 0.01). (c) Diseased leaf area analysis. Data are presented as a bar chart showing percentage of lesion areas analyzed by ImageJ. Error bars represent SD and asterisks represent significant differences (P < 0.01). (d) Severity of blast disease was evaluated by quantifying M. oryzae genomic 28S ribosomal gene (rDNA) relative to rice genomic RUBQ1 DNA (7 days post-inoculation). Mean values of three determinations with standard deviations are shown. The asterisks indicate a significant difference from the wild-type Guy11 (P < 0.01). (e) Conidial suspension (2 × 10⁵ spores/ml) of each strain was injected into rice sheath of 3-week-old seedlings and 30 healthy rice sheaths were used for each strain. The arrow points out the injection site. (f) Detached barley cultivar Four-arris leaves were dropped with different concentrations of conidial suspensions (10⁴, 10³, 10² spores/ml) and diseased leaves were photographed 5 days after inoculation. Experiments were performed more than 3 times. (g) Close observation for 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 24 and 36 hpi. CO, conidium; Ap, appressorium; IH, invasive hyphae. Scale bar: 10 μm. (h) The penetration rate was counted for more than 100 appressoria in (g) and the experiment was repeated 3 times. Error bars represent SD and asterisk represents significant difference (P < 0.01). (i) Detailed observation and statistics for infectious growth in rice sheath cell at 24 and 36 hpi. Appressorium penetration sites (n = 100) were observed and invasive hyphae (IH) were rated from type 1 to 4 (type1, no hyphal penetration with only appressoria formation; type2, IH with 1 or 2 short branches; type3, IH with at least 3 branches but the IH are short and extending within a plant cell; type 4, IH that has numerous branches and fully occupies the plant cell or even extended to an adjacent plant cell.). The arrow points out the appressorium (Ap) and the invasive hyphae (IH); asterisks indicate IH extended to surrounding cells. Error bars represent SD from three independent experiments. Scale bar: 10 μm.