Involvement of FgERG4 in ergosterol biosynthesis, vegetative differentiation and virulence in Fusarium graminearum

Abstract

The ergosterol biosynthesis pathway is well understood in Saccharomyces cerevisiae, but currently little is known about the pathway in plant-pathogenic fungi. In this study, we characterized the Fusarium graminearum FgERG4 gene encoding sterol C-24 reductase, which catalyses the conversion of ergosta-5,7,22,24-tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant ΔFgErg4-2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of ΔFgErg4-2 revealed increased resistance to cell wall-degrading enzymes. Fungicide sensitivity tests revealed that ΔFgErg4-2 showed increased resistance to various sterol biosynthesis inhibitors (SBIs), which is consistent with the over-expression of SBI target genes in the mutant. ΔFgErg4-2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild-type progenitor. All of these phenotypic defects of ΔFgErg4-2 were complemented by the reintroduction of a full-length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the Saccharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F. graminearum.

Figure 1

Deletion of FgERG4 resulted in a complete depletion of ergosterol. Comparative production of ergosterol in the wild‐type progenitor HN9‐1 (a), the deletion mutant ΔFgErg4‐2 (b) and the complemented strain ΔFgErg4‐9C (c) as determined by high‐performance liquid chromatography (HPLC) assays. (d) Commercial standard of ergosterol was used as the control. The retention time for ergosterol is marked with arrows.

Figure 2

Reduced mycelial growth and conidiation in the FgERG4 deletion mutant. (a) Comparison of mycelial growth rates among the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented strain ΔFgErg4‐9C on potato dextrose agar (PDA) medium. (b) Comparison of conidiation among HN9‐1, ΔFgErg4‐2 and ΔFgErg4‐9C in mung bean liquid (MBL) medium. Bars denote standard errors from three repeated experiments.

Figure 3

Deletion of FgERG4 led to changes in conidial morphology. (a) Compared with the wild‐type progenitor HN9‐1 and the complemented strain ΔFgErg4‐9C, the deletion mutant ΔFgErg4‐2 produced shorter conidia in mung bean liquid (MBL) medium. (b) The conidia of ΔFgErg4‐2 showed less septation (top), but germinated normally (bottom). Conidia were examined after incubation in 2% sucrose (wt/vol) for the times indicated in the figure, followed by staining with calcofluor white.

Figure 4

Deletion of FgERG4 led to changes in conidial and hyphal cell length. (a) Comparison of the percentage of conidia with different numbers of septa among HN9‐1, ΔFgErg4‐2 and ΔFgErg4‐9C. (b) Comparisons of hyphal cell length among HN9‐1, ΔFgErg4‐2 and ΔFgErg4‐9C.

Figure 5

Exogenous application of ergosterol could not restore normal mycelial growth of ΔFgErg4‐2. The wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented strain ΔFgErg4‐9C were cultured on potato dextrose agar (PDA) medium supplemented with ergosterol at the concentrations indicated in the figure under aerobic (a) and anaerobic (b) conditions.

Figure 6

FgERG4 is important for full virulence of Fusarium graminearum. (a) Flowering wheat heads were point inoculated with a conidial suspension of the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented mutant ΔFgErg4‐9C. Infected wheat heads were photographed 10 days after inoculation. (b) Tomatoes were inoculated with a conidial suspension of each strain and infected fruits were photographed 3 days after inoculation.

Figure 7

Reduced deoxynivalenol (DON) production in the deletion mutant ΔFgErg4‐2. The amounts of DON (mg/mg of fungal DNA) produced by the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented stain ΔFgErg4‐9C in infected wheat kernels. Bars denote standard errors from three repeated experiments.

Figure 8

Comparison of the sensitivity to metal cations among the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented strain ΔFgErg4‐9C on minimal medium amended with each metal cation at the concentration indicated in the figure. Bars denote standard errors from three repeated experiments.

Figure 9

Comparison of the sensitivity to cell stress among the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented strain ΔFgErg4‐9C on potato dextrose agar (PDA) medium amended with various cell stress agents. Bars denote standard errors from three repeated experiments.

Figure 10

Comparison of the sensitivity to different fungicides among the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented mutant ΔFgErg4‐9C on potato dextrose agar (PDA) medium amended with various fungicides. Bars denote standard errors from three repeated experiments.

Figure 11

Deletion of FgERG4 resulted in increased resistance of Fusarium graminearum to cell wall‐degrading enzymes. Fresh hyphae of the wild‐type progenitor HN9‐1, the deletion mutant ΔFgErg4‐2 and the complemented strain ΔFgErg4‐9C were digested with a mixture of cellulase, lysozyme and snailase for 3 h at 30 °C. Circular protoplasts are marked with arrows.

Figure 12

FgERG4 partially complements the Saccharomyces cerevisiae ERG4 mutant. Comparison of ergosterol production in the wild‐type strain BY4741 (a) and the ERG4 deletion mutant BY4741ΔERG41(b) transformed with empty pYES2 vector, and the complemented strain BY4741ΔERG4 + pYES2‐FgERG4 (c) determined by high‐performance liquid chromatography (HPLC) assays. A commercial standard of ergosterol was used as control (d). The retention time for ergosterol is marked with arrows.