Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease

Abstract

One of the first responses of plants to microbial attack is the production of extracellular superoxide surrounding infection sites. Here, we report that Magnaporthe grisea, the causal agent of rice blast disease, undergoes an oxidative burst of its own during plant infection, which is associated with its development of specialized infection structures called appressoria. Scavenging of these oxygen radicals significantly delayed the development of appressoria and altered their morphology. We targeted two superoxide-generating NADPH oxidase-encoding genes, Nox1 and Nox2, and demonstrated genetically, that each is independently required for pathogenicity of M. grisea. Deltanox1 and Deltanox2 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. The initiation of rice blast disease therefore requires production of superoxide by the invading pathogen.

Fig. 1.

ROS production during infection-related development of M. grisea. (A) Detection of superoxide by NBT staining during germination and appressorium formation in M. grisea strain Guy11. Conidia were inoculated on glass coverslips and incubated in a moist chamber at 26°C for 0, 3, 8, and 24 h before being stained with a 0.3 mM NBT aqueous solution for 20 min and viewed by bright-field microscopy. Representative bright-field images at each time point are shown. (B) Inhibition of M. grisea superoxide production by NADPH oxidase inhibitor diphenylene iodonium (DPI). Conidia were preincubated in a 25 μM DPI solution for 20 min before NBT staining. (C) Detection of ROS by H₂DCFDA staining during infection-related development. Conidia were inoculated onto glass coverslips and incubated in a moist chamber at 26°C for 0, 3, and 8 h before being stained with a 2.5 μg·ml⁻¹ carboxy-H₂DCFDA aqueous solution for 20 min and viewed by epifluorescence microscopy. (Scale bars, 10 μm.)

Fig. 2.

Exposure to antioxidants inhibits infection-related development by M. grisea. (A–D) Bar charts showing the percentage of conidia able to germinate and form appressoria in the presence of 10 mM, 100 mM, and 1 M ascorbic acid, 2, 4, 6, 8, and 24 h after inoculation (A and B); 1 μM, 10 μM, and 100 μM MnTMPyP, 2, 4, 6, 8, and 24 h after inoculation (C and D). (E) Bar chart showing the percentage of deformed appressoria resulting from exposure of germinating conidia to 100 mM ascorbic acid, 10 mM ascorbic acid, and 1 μM MnTMPyP for a 24-h time period. (F) Light micrographs showing appressorial deformities resulting from exposure to 100 mM ascorbic acid, 10 mM ascorbic acid, and 1 μM MnTMPyP for 24 h. (Scale bar, 10 μm.) (G) Bar chart showing the percentage of conidia able to germinate in the presence of 2.5 mM, 0.25 mM, and 25 μM DPI after 24 h. (H) Light micrographs showing germ tube deformities resulting from exposure of conidia to 25 μM DPI for 24 h.

Fig. 3.

Superoxide production is significantly altered in ΔnoxΔ1nox2 mutants. (A) Cellular localization of Nox1-Gfp in M. grisea. A Guy 11 transformant expressing a NOX1-GFP fusion under the control of the NOX1 promoter was inoculated onto glass coverslips and observed by epifluorescence microscopy. A faint fluorescence was observed at the appressorium periphery from 4 h. After 24 h, GFP was also detected in the central appressorium vacuole. (B) Detection of superoxide in the appressorium of Guy 11, and ΔnoxΔ1nox2 mutants, by NBT staining. Conidia were inoculated on glass coverslips and incubated in a moist chamber at 26°C for 5 h before being stained with a 0.5% (wt/vol) NBT solution for 20 min and viewed by bright-field microscopy. (C) Detection of DPI-sensitive superoxide production in the hyphal tips of M. grisea strain, Guy 11, and ΔnoxΔ1nox2 mutants by NBT staining. Colony mycelia were grown on CM media for 3 days and stained with 0.05% (wt/vol) NBT solution for 2 h in the presence and absence of 25 μM DPI. (D) Bar chart showing mean pixel intensity in the appressorium and hyphal tips of Guy 11 and ΔnoxΔ1nox2 mutants stained with NBT to detect superoxide (increased staining results in reduced pixel intensity and shorter bars, n = 10). Error bars indicate ± 2 SE. (Scale bars, 10 μm.)

Fig. 4.

Δnox1 and Δnox2 mutants are unable to cause rice blast disease. (A) Seedlings of rice cultivar CO-39 were inoculated with M. grisea conidial suspensions of identical concentration (1 × 10⁵ conidia per ml⁻¹) of Guy11, the nox1 mutant TM02.18, the nox2 mutant N2.6, and the respective complemented strains TM18C.1 and N2C.2. Seedlings were incubated for 4 days for development of blast disease. (B) Mycelium from Guy11, Δnox1, Δnox2, and ΔnoxΔ1nox2 mutants was inoculated onto complete medium containing calcofluor white at a concentration 10, 100, and 200 μg·ml⁻¹ and incubated at 24°C for 5 d with a 12-h light/dark cycle. (C–F) Transmission electron micrographs showing penetration hypha development (C) and cuticle penetration (D) by Guy 11 after 24 h on onion epidermis compared with appressorium of Δnox1 mutant (E) and attempted penetration point of Δnox1 mutant (F). (Scale bars, 1.0 μm.) (G–J) Light micrographs representing the same developmental stages in Guy 11 (G), Δnox1 mutants (H), Δnox2 mutants (I), and Δnox1Δnox2 mutants (J). (Scale bars, 10 μm.)