NADPH Oxidases Are Required for Appressorium-Mediated Penetration in Colletotrichum scovillei-Pepper Fruit Pathosystem

NADPH oxidase (Nox) complexes are known to play essential roles in differentiation and proliferation of many filamentous fungi. However, the functions of Noxs have not been elucidated in Colletotrichum species. Therefore, we set out to characterize the roles of Nox enzymes and their regulators in Colletotrichum scovillei, which causes serious anthracnose disease on pepper fruits in temperate and subtropical and temperate region. In this study, we generated targeted deletion mutants for CsNox1, CsNox2, CsNoxR, and CsNoxD via homologous recombination. All deletion mutants were normal in mycelial growth, conidiation, conidial germination, and appressorium formation, suggesting that CsNox1, CsNox2, CsNoxR, and CsNoxD are not involved in those developmental processes. Notably, conidia of ΔCsnox2 and ΔCsnoxr, other than ΔCsnox1 and ΔCsnoxd, failed to cause anthracnose on intact pepper fruits. However, they still caused normal disease on wounded pepper fruits, suggesting that Csnox2 and CsnoxR are essential for penetration-related morphogenesis in C. scovillei. Further observation proved that ΔCsnox2 and ΔCsnoxr were unable to form penetration peg, while they fully developed appressoria, revealing that defect of anthracnose development by ΔCsnox2 and ΔCsnoxr resulted from failure in penetration peg formation. Our results suggest that CsNox2 and CsNoxR are critical for appressorium-mediated penetration in C. scovillei-pepper fruit pathosystem, which provides insight into understanding roles of Nox genes in anthracnose disease development.