Genome-wide functional characterization of putative peroxidases in the head blight fungus Fusarium graminearum

Abstract

Reactive oxygen species (ROS) are associated with various developmental processes and host-pathogen interactions in pathogenic fungi. Peroxidases are a group of ROS-detoxifying enzymes that are involved in the oxidative stress response and in a variety of physiological processes. In this study, we performed a genome-wide functional characterization of putative peroxidase genes in Fusarium graminearum, a head blight pathogen of cereal crops. We identified 31 putative peroxidase genes and generated deletion mutants for these genes. Twenty-six of the deletion mutants showed developmental phenotypes indistinguishable from that of the wild-type, and five deletion mutants exhibited phenotypic changes in at least one phenotypic category. Four deletion mutants, fca6, fca7, fpx1 and fpx15, showed increased sensitivity to extracellular H₂ O₂ . Deletion mutants of FCA7 also exhibited reduced virulence and increased trichothecene production compared with those of the wild-type strain, suggesting that Fca7 may play an important role in the host-pathogen interaction in F. graminearum. To identify the transcription factors (TFs) regulating FCA6, FCA7, FPX1 and FPX15 in response to oxidative stress, we screened an F. graminearum TF mutant library for growth in the presence of H₂ O₂ and found that multiple TFs co-regulated the expression of FCA7 under oxidative stress conditions. These results demonstrate that a complex network of transcriptional regulators of antioxidant genes is involved in oxidative stress responses in this fungus. Moreover, our study provides insights into the roles of peroxidases in developmental processes and host-pathogen interactions in plant-pathogenic fungi.

Keywords: Fusarium graminearum; oxidative stress response; peroxidase.

Figure 1

Phylogenetic and transcriptional analysis of peroxidase genes. Phylogenetic trees based on the amino acid sequences of 23 putative haem peroxidases (A) and eight putative non‐haem peroxidases (B) in Fusarium graminearum. The phylogenetic trees were constructed using the mega program (version 6.06) by the neighbour‐joining method with 2000 bootstrap replicates (Tamura et al., 2013). The numbers at the nodes represent the bootstrap percentages. (C, D) Heatmap visualization of the peroxidase gene transcriptional profiles during asexual and sexual development (C) and plant infection (D). The heatmap depicts peroxidase gene transcript abundances during various asexual and sexual developmental stages based on log₂‐based relative transcript abundances compared with the 0‐day (sexual development), 3‐h (asexual development) and mock‐inoculated (plant infection) samples. Red and blue represent higher and lower expression, respectively. The rows represent transcriptional units. The expression data were obtained from previous studies (Harris et al., 2016; Sikhakolli et al., 2012; Son et al., 2013, 2016) and visualized using ClustVis (Metsalu and Vilo, 2015).

Figure 2

Sexual development and virulence of NADPH oxidase deletion mutants. (A) Perithecium formation by the NADPH oxidase deletion mutants. Cirrhi (indicated by white arrows) were observed in the wild‐type (WT) and in the noxB and noxC deletion mutants 10 days after sexual induction. Scale bar, 500 µm. (B) Virulence on wheat heads. The centre spikelet of each wheat head was injected with 10 µL of a conidial suspension, and photographs were taken at 21 days after inoculation. Arrowheads indicate the inoculated spikelets. ‘Mock’ indicates wheat heads that were mock inoculated with 0.01% Tween 20.

Figure 3

Peroxidases involved in the oxidative stress responses in Fusarium graminearum. (A) Oxidative stress sensitivity of F. graminearum strains. The mycelial growth of four peroxidase deletion mutants was evaluated on complete medium (CM) with and without supplementation with 10 mm H₂O₂. Photographs were taken 5 days after inoculation. (B) Peroxidase enzyme activities of the F. graminearum strains. (C) Hydrogen peroxide detection in mycelia of the F. graminearum strains. (D) Virulence on wheat heads. The centre spikelet of each wheat head was injected with 10 µL of a conidial suspension, and photographs were taken 21 days after inoculation. Arrowheads indicate the inoculated spikelets. ‘Mock’ indicates wheat heads that were mock inoculated with 0.01% Tween 20. (E) Total trichothecene production by the F. graminearum strains. Each strain was grown in minimal medium containing 5 mM agmatine (MMA) for 7 days. Trichothecenes were analysed by gas chromatography‐mass spectrometry (GC‐MS) and quantified based on the biomass of each strain. (F) Transcript levels of TRI5 and TRI6 in the wild‐type (WT) and fca7 deletion mutant strains. The transcript levels were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR) 4 days after inoculation in MMA. Asterisk indicates significant difference: P < 0.01, Tukey's test.

Figure 4

Characterization of transcription factors (TFs) involved in the oxidative stress response. (A) Oxidative stress sensitivity of eight TF mutants. The mycelial growth of the TF deletion mutants was evaluated on complete medium (CM) and on CM supplemented with 10 mm H₂O₂, 0.1 mm menadione or 1 mm diamide. Photographs were taken 5 days after inoculation. (B) Transcript abundances of peroxidase genes in the TF mutants. The transcript levels of FCA6, FCA7, FPX1 and FPX15 were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). Total RNA was isolated from wild‐type (WT) and TF deletion mutant strains grown for 30 min in CM only or in CM supplemented with 5 mm H₂O₂.

Figure 5

Oxidative stress sensitivity of eight transcription factor (TF) mutants carrying an FCA7 deletion or FCA7 overexpression (FCA7oe). The mycelial growth of TF mutant strains on complete medium (CM) supplemented with 10 mm H₂O₂ is shown. Photographs were taken 4 and 6 days after inoculation. WT, wild‐type.

Figure 6

Genetic network of transcription factor (TF) genes involved in the oxidative stress response in Fusarium graminearum. (A) Fold change values in the expression of TF genes after H₂O₂ treatment in the wild‐type strain. The transcript levels of the TF genes were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). Total RNA was isolated from the wild‐type strain grown for 30 min in complete medium (CM) only or in CM supplemented with 5 mm H₂O₂. (B) Heatmap of selected TF genes that were up‐regulated in the wild‐type strain in response to H₂O₂. The log base ratio of the fold change in gene expression in each deletion mutant compared with that in the wild‐type strain was converted to a heatmap using ClustVis (Metsalu and Vilo, 2015). (C) Proposed genetic network of TFs and FCA7 in the oxidative stress response. ?, unknown antioxidant components.