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

Abstract

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.

Keywords: Colletotrichum scovillei; NADPH oxidase; pepper fruit anthracnose.

Fig. 1

Phylogenetic analysis and domain prediction of NADPH oxidases (Noxs) among several fungi. The phylogenetic tree was generated using maximum likelihood estimation with 1,000 bootstraps. Domains of Noxs, including ferric reductase transmembrane component-like domain (IPR013130), FAD-binding 8 (IPR013112), ferric reductase, NAD binding domain (IPR013121), and PB1 domain (IPR000270), were predicted through InterPro.

Fig. 2

Targeted deletion and complementation of Nox genes (CsNox1, CsNox2, CsNoxR, and CsNoxD). (A) Targeted gene deletion. Each Nox gene was replaced with the HPH cassette via homologous recombination. (B) Verification of deletion mutants. Genomic DNA of wild-type and candidates of deletion mutant was digested with restriction enzymes and hybridized to a probe (about 500 bp). (C) Expression of targeted gene in deletion mutant and complemented strain. Expression of each targeted gene was detected in wild-type and corresponded complemented strain, but not deletion mutant.

Fig. 3

Mycelial growth and conidiation assays. (A) Photographs of mycelial growth. Three-day old mycelial agar plugs (5 mm in diameter) from minimal media agar were inoculated onto potato dextrose agar (PDA) and incubated at 25°C for 5 days. (B) Measurement of mycelial growth. Diameters of colony growth on PDA were measured after 5-day incubation at 25°C.

Fig. 4

Appressorium formation assay. (A) Observation of appressorium development. Conidia obtained from 7-day-old oatmeal agar were placed on the hydrophobic surface of coverslips to form appressoria. Photographs were taken after 16-h incubation in humid box at 25°C. Black and white triangle indicates the presence and absence of internal light spot (ILS), respectively. Scale bars = 10 μm.

Fig. 5

Pathogenicity assay. Conidial suspensions were inoculated onto intact and wounded pepper fruits and incubated in a humid box at 25°C. The photographs of intact pepper were taken after 7 days, and wounded pepper were taken after 5 days.

Fig. 6

Observation of appressorium-mediated penetration. The conidial suspensions were inoculated onto intact pepper fruits and incubated in a humid box at 25°C for 3 days. Thin segments of inoculated area were sliced and observed. Co, Ap, and DS indicates conidium, appressorium, and dendroid structure, respectively. Scale bar = 10 μm.

Fig. 7

Penetration on host and artificial surfaces. (A) Penetration onto pepper fruits. Conidial suspensions were inoculated onto intact pepper fruits and incubated in a humid box at 25°C for 3 days. The samples were observed using a scanning electron microscope (Analytical HR-SEM). Ap indicates appressorium. Black and white triangle indicates presence and absence of holes generated by penetration peg, respectively. Scale bar = 1 μm. (B) Penetration onto cellophane membrane. Conidial suspensions were inoculated onto cellophane membranes, which are placed on potato dextrose agar (PDA). The cellophane membranes were removed after a 3-day incubation at 25°C. The PDA medium was additionally cultured for 2 days.