RBF1 is a virulence determinant in Magnaporthe oryzae.
(A) Representative symptoms on the 6th leaf blades at 5 days after inoculation. Rice plants were sprayed with a conidial suspension of the wild-type strain (WT), an RBF1-knockout line (Δrbf1-1; KO), and a gene complementation line (KO+RBF1). Bar = 5 mm. (B) Evaluation of lesion types in leaf blades. Lesions formed at 5 days after spray-inoculation were counted according to the classifications displayed. Data are represented as the mean percentages ± SE (n = 5 plants). (C) Comparison of the development of invasive hyphae in rice leaf sheaths between WT and Δrbf1-1 (KO). Infection levels in leaf sheaths were assessed for each appressorium under a microscope and categorized as no invasion (S0), short invasive hyphae in one cell (S1), highly-branched invasive hyphae in one cell (S2), and multiple cell invasion (S3). To illustrate each category, typical images using a WT line transformed with TEFp::mCherry are displayed. Data are represented as the mean percentages ± SE [n = 14 plants (24 h post inoculation; hpi) and 23 plants (42 hpi)]. Student’s t-test was performed on arcsine-transformed data between WT and KO (*, P < 0.05; **, P < 0.005). The total numbers of appressoria observed per line were ~ 1,500 (24 hpi) and 3,000 (48 hpi). (D) Confocal images of rice epidermal cells in inoculated leaf blades. Transgenic rice plants constitutively expressing GFP under the CaMV 35S promoter were inoculated with the WT (left) or Δrbf1-2 line (KO; right) transformed with TEFp::mCherry. GFP and mCherry signals were merged. Note that the disappearance of the GFP signal (green) indicates host cell death. Arrows indicate invasive hyphae. Ap, appressorium. Bar = 20 μm. (E) Transverse sections of inoculated rice leaf blades at 6 dpi. To visualize invasive hyphae, the WT (upper) and Δrbf1-1 (KO; lower) were transformed with TEFp::GUS. Spot-inoculated rice leaf blades were stained for β-glucuronidase activity, hand-sectioned, and observed by light microscopy. Bar = 0.1 mm.