Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens
(A) MC69 is required for pathogenicity of M. oryzae strain Ina72. Conidial suspension of the wild-type strain Ina72 (Ina72 WT) and the mc69 mutants (mc69-9, -12, -87) were inoculated on barley (cv. Nigrate) and rice (cv. Shin No. 2) leaves, and incubated for 4 and 7 days, respectively. (B) Germination, appressorium formation and appressorial penetration of Ina72 WT and the mc69 mutants. The ratio of germination was calculated as the mean percentage of conidia germinated after 24 h on rice (cv. Shin No. 2) leaf sheath cells. The mean percentage of appressorium formation on rice leaf sheath cells among the germinated conidia is presented. Three replicates of ∼50 conidia were counted for each observation. The mean percentage of appressorial penetration by the mc69 mutants 32 h after inoculation is presented. Standard errors are indicated by the vertical bars. (C) Appressorial penetration assays on rice leaf sheath cells. Conidia from Ina72 WT and mc69-87 germinated and formed melanized appressoria. All appressoria formed by Ina72 WT penetrated and produced infectious hyphae but no appressoria formed by mc69-87 produced infectious hyphae in most of area. Photographs were taken 32 h after inoculation. Scale bar = 40 µm. (D) Invasive growth rating of rice leaf sheath cells 32 h after inoculating with Ina72 WT and mc69-87. The levels for invasive growth rating are given above. For details of the invasive growth levels and rating see Materials and Methods. Scale bar = 20 µm. (E), (F) Complementation of mc69 mutant with the wild type allele of MC69. (E) Appressorial penetration by Ina72 WT, mc69-87 (mc69) and the MC69 re-introduced strain (mc69+MC69). Mean percentage of appressorial penetration is recorded 32 h after inoculation in rice leaf sheath cells. Four replicates of ∼50 appressoria were counted for each observation. (F) Blast symptoms caused by Ina72 WT, mc69 and mc69+MC69 on barley (cv. Nigrate) and on rice (cv. Shin No 2) 3 and 4 days after inoculation, respectively.