Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific, toxin-producing fungus Alternaria alternataJapanese pear pathotype

Black spot disease, Alternaria alternataJapanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plantpathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. noxA and noxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the noxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the noxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the noxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the noxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A.alternata.