The novel fungal-specific gene FUG1 has a role in pathogenicity and fumonisin biosynthesis in Fusarium verticillioides

Abstract

Fusarium verticillioides is a globally important pathogen of maize, capable of causing severe yield reductions and economic losses. In addition, F. verticillioides produces toxic secondary metabolites during kernel colonization that pose significant threats to human and animal health. Fusarium verticillioides and other plant-pathogenic fungi possess a large number of genes with no known or predicted function, some of which could encode novel virulence factors or antifungal targets. In this study, we identified and characterized the novel gene FUG1 (Fungal Unknown Gene 1) in F. verticillioides through functional genetics. Deletion of FUG1 impaired maize kernel colonization and fumonisin biosynthesis. In addition, deletion of FUG1 increased sensitivity to the antimicrobial compound 2-benzoxazolinone and to hydrogen peroxide, which indicates that FUG1 may play a role in mitigating stresses associated with host defence. Transcriptional profiling via RNA-sequencing (RNA-seq) identified numerous fungal genes that were differentially expressed in the kernel environment following the deletion of FUG1, including genes involved in secondary metabolism and mycelial development. Sequence analysis of the Fug1 protein provided evidence for nuclear localization, DNA binding and a domain of unknown function associated with previously characterized transcriptional regulators. This information, combined with the observed transcriptional reprogramming in the deletion mutant, suggests that FUG1 represents a novel class of fungal transcription factors or genes otherwise involved in signal transduction.

Keywords: DIMBOA; Fusarium ear rot; benzoxazinoids; gene regulation; mycotoxin.

Figure 1

Phylogenetic reconstruction reveals broad conservation of Fug1 within the Ascomycota. Branches having bootstrap values ≥70% are presented in bold. Protein identity represents global amino acid sequence identity for each protein compared with Fug1.

Figure 2

Deletion of FUG1 (Fungal Unknown Gene 1) in Fusarium verticillioides impairs pathogenesis in maize kernels. Strains 7600, FV020 and FV022 were assessed for growth in maize kernels. (A) Visual assessment of external kernel colonization. (B) Visual assessment of internal kernel colonization. Arrows indicate location of germ tissue.

Figure 3

RNA‐sequencing (RNA‐seq) identifies numerous differentially regulated fungal genes in the kernel environment following FUG1 (Fungal Unknown Gene 1) deletion. (A) Hierarchical clustering of the 1241 differentially expressed genes illustrates similar patterns in gene expression among biological replications. (B) Significantly over‐represented functional terms in up‐ and down‐regulated genes in the FUG1 deletion strain compared with the wild type. Functional terms are represented as Gene Ontology (GO) terms. Significantly over‐represented terms were reduced to most specific terms. Black bars represent GO terms associated with biological processes, and white bars represent GO terms associated with molecular functions. Vertical broken lines represent a statistical cut‐off of P < 0.05 based on false discovery rate (FDR)‐corrected P values. (C) Volcano plot comparing gene expression [log₂(fold change)] and significance (–log₁₀ of FDR‐corrected P values) in the FUG1 deletion strain compared with the wild type. Significantly down‐regulated genes are shown in green. Significantly up‐regulated genes are shown in red. A statistical cut‐off of P < 0.01 based on FDR‐corrected P values is represented as a horizontal broken line. Fold changes of 2.0 and −2.0 are represented as vertical broken lines. Blue triangles represent fumonisin biosynthetic genes. The polyketide synthase (FUM1) and cluster‐specific transcription factor (FUM21) are labelled. (D) Expression of FUM1 and FUM21 was determined by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Bars indicate standard deviation. (E) Unclustered heatmap illustrating differential expression of genes in the fumonisin biosynthetic cluster following the deletion of FUG1. Expression values are presented as ‘Row Z‐score’.

Figure 4

Deletion of FUG1 (Fungal Unknown Gene 1) in Fusarium verticillioides reduces fumonisin accumulation in maize kernels. Strains 7600, FV020 and FV022 were assessed for growth and fumonisin accumulation in maize kernels. (A) Quantification of fumonisin B₁ (FB₁; left), fumonisin B₂ (FB₂; centre) and fumonisin B₃ (FB₃; right) in infected maize kernels. (B) Quantification of growth in maize kernels as determined by ergosterol accumulation. (C) Determination of FB₁ (left), FB₂ (centre) and FB₃ (right) in infected maize kernels relative to fungal growth. All bars indicate standard deviation. (D) Determination of percentage fumonisin analogue relative to total fumonisin content in infected maize kernels. Black bars represent FB₁, grey bars represent FB₂ and white bars represent FB₃.

Figure 5

Deletion of FUG1 (Fungal Unknown Gene 1) alters tolerance to the antimicrobial 2‐benzoxazolinone (BOA) and hydrogen peroxide, but not cell wall stress. (A) Strains 7600, FV020 and FV022 were assessed for tolerance to the antimicrobial compound BOA. PDA, potato dextrose agar. (B) Strains 7600, FV020 and FV022 were assessed for tolerance to oxidative stress. Visual assessment of growth inhibition 24 h after exposure to H₂O₂. (C) Zone of inhibition, measured as the diameter of growth inhibition, in response to H₂O₂. Bars indicate standard error. (D) Strains 7600, FV020 and FV022 were assessed for tolerance to cell wall stress. CR, Congo Red; SDS, sodium dodecylsulfate; YMA, sucrose yeast extract malt extract agar.

Figure 6

Deletion of FUG1 (Fungal Unknown Gene 1) in Fusarium verticillioides causes defects in asexual reproduction and mycelial hydrophobicity. (A) Strains 7600, FV020 and FV022 were assessed for differences in conidiation. Bars indicate standard deviation. (B) Strains 7600 and FV020 were assessed for the production of conidial chains. (C) Strains 7600, FV020 and FV022 were assessed for the development of aerial hyphae. (D) Strains 7600, FV020 and FV022 were assessed for the maintenance of mycelial hydrophobicity 10 min after the addition of water or detergent solution (DET). (E) Expression of hydrophobin‐encoding genes (HYD1–8) was determined by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Bars indicate standard deviation; nd, not detected (Ct value ≥ 37).