Involvement of a velvet protein FgVeA in the regulation of asexual development, lipid and secondary metabolisms and virulence in Fusarium graminearum

Abstract

The velvet protein, VeA, is involved in the regulation of diverse cellular processes. In this study, we explored functions of FgVeA in the wheat head blight pathogen, Fusarium graminearum,using a gene replacement strategy. The FgVEA deletion mutant exhibited a reduction in aerial hyphae formation, hydrophobicity, and deoxynivalenol (DON) biosynthesis. Deletion of FgVEA gene led to an increase in conidial production, but a delay in conidial germination. Pathogencity assays showed that the mutant was impaired in virulence on flowering wheat head. Sensitivity tests to various stresses exhibited that the FgVEA deletion mutant showed increased resistance to osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and fludioxonil fungicides. Ultrastructural and histochemical analyses revealed that conidia of FgVeA deletion mutant contained an unusually high number of large lipid droplets, which is in agreement with the observation that the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Serial analysis of gene expression (SAGE) in the FgVEA mutant confirmed that FgVeA was involved in various cellular processes. Additionally, six proteins interacting with FgVeA were identified by yeast two hybrid assays in current study. These results indicate that FgVeA plays a critical role in a variety of cellular processes in F. graminearum.

Figure 1. Schematic representation of the FgVEA deletion strategy. (A)

FgVEA and hygromycin resistance cassette (HPH) are denoted by large black and gray arrows, respectively. Annealing sites of PCR primers are indicated with arrows (see supplementary Table 1 for the primer sequences). (B) A 1,162-bp fragment of FgVEA was used as a probe in Southern blot hybridization analysis. Genomic DNA preparations of the wild-type PH-1, the FgVEA deletion mutant ΔFgVeA-9, and the complement strain ΔFgVeA-9C were digested with Pst I.

Figure 2. Impact of FgVEA on colony morphology and pigment formation.

(A) The wild-type strain PH-1, FgVEA deletion mutant ΔFgVeA-9, and complemented strain ΔFgVeA-9C were grown on solid media, PDA, CM, MM for 4 days at 25°C. (B) On each of the fungal colonies, 20 µl of water or 2.5% bromophenol blue solution was pipetted on the colony surface and photographed 10 min later. Spherical water droplets formed on colonies of PH-1 and ΔFgVeA-9C, whereas the droplet dispersed immediately on colonies of ΔFgVeA-9.

Figure 3. Relative expression levels of PKS12 and AURJ in the FgVEA deletion mutant ΔFgVeA-9.

RNA samples were extracted from mycelia of each strain after grown in potato dextrose broth for 2 days. The relative expression of PKS12 and AURJ in ΔFgVeA-9 is the relative amount of cDNA of each gene in the wild-type strain. Line bars in each column denote standard errors of three experiments.

Figure 4. Impact of FgVEA on conidiation and conidial germination of F. graminearum. (A)

Conidia were quantified after incubation of the wild-type strain PH-1, FgVEA deletion mutant ΔFgVeA-9, and complemented strain ΔFgVeA-9C in 10 ml mung bean liquid medium for 4 days in a shaker. (B) Percentages of germinated conidia of PH-1, ΔFgVeA-9, and ΔFgVeA-9C after incubated in 2% glucose for 6 h or 12 h. Bars denote standard errors from three repeated experiments.

Figure 5. Ultrastructural and histochemical analyses of lipid droplets within conidia and hyphae of the mutant ΔFgVeA-9.

(A) Differential interference contrast (DIC) images of conidia were captured with an electronic microscope. (B) Lipid drops within conidia of the wild type PH-1 and the ΔFgVeA-9 mutant examined with a transmission electronic microscope. (C) Lipid drops in conidia (top), germinating conidia (middle), and hyphae (bottom) were stained with Nile Red and examined under a microscope with episcopic fluorescence.

Figure 6. Sensitivity of the PH-1, ΔFgVeA-9 and ΔFgVeA-9C to osmotic stresses and fungicides.

Osmotic stresses were mediated by addition of 1.2 M NaCl or 1.2 M KCl in potato dextrose agar (PDA) medium. The fungicides iprodione and fludioxonil were added into PDA at 10 µg/ml and 0.1 µg/ml, respectively. Bars denote standard errors from three repeated experiments.

Figure 7. Effects of FgVeA on the glycerol biosynthesis.

Intracellular glycerol concentration (nmol/mg dried mycelia) in mycelia of the wild-type strain PH-1, FgVEA deletion mutant ΔFgVeA-9, and complemented strain ΔFgVeA-9C were analyzed after incubation in PDB for 2 days. Bars denote standard errors from three repeated experiments.

Figure 8. Effects of FgVeA on cell wall integrity of F. graminearum.

(A) Sensitivity of the wild type PH-1, FgVEA deletion mutant ΔFgVeA-9, and complemented strain ΔFgVeA-9C to cell wall damaging agents. (B) Relative expression levels of FgSLT1 and FgMKK1 in PH-1 and mutant ΔFgVeA-9. The relative expression of FgSLT1 and FgMKK1 in ΔFgVeA-9 is the relative amount of cDNA of each gene in the wild-type strain. Line bars in each column of each figure denote standard errors of three repeated experiments.

Figure 9. Effects of FgVeA on the biosynthesis of DON. (A)

Amount of DON (per mg fungal DNA) produced by FgVEA deletion mutant ΔFgVeA-9, and complemented strain ΔFgVeA-9C was detected in infected wheat kernels after 20 days of incubation. Line bars in each column denote standard errors of three repeated experiments. (B) Relative expression levels of TIR5 and TRI6 in PH-1 and ΔFgVeA-9. The relative expression of TIR5 or TRI6 in ΔFgVeA-9 is the relative amount of cDNA of each gene in the wild-type progenitor. Line bars in each column denote standard errors of three repeated experiments.

Figure 10. Virulence of the wild type PH-1, FgVEA deletion mutant ΔFgVeA-9, complemented strain ΔFgVeA-9C on flowering wheat heads.

Wheat heads were point-inoculated with conidial suspension of each strain, and infected wheat heads were examined 15 days after inoculation.

Figure 11. Pie chart grouping the genes up- and down-regulated in expression in ΔFgVeA compared with PH-1. (A)

A total of 1,215 genes were up-regulated more than 5 folds in the mutant ΔFgVeA-9 compared with wild-type PH-1. (B) A total of 354 genes were down-regulated more than 5 folds in the mutant ΔFgVeA-9 compared with wild-type PH-1. The expressions of genes were detected by the serial analysis of gene expression method.

Figure 12. Yeast two-hybrid analysis of the interaction between FgVeA, FgVelB and FgLaeA1 or six FgVeA interacting proteins (named FgVIP1-6).

The pair of plasmids pGBKT7-53 and pGADT7 was served as a positive control. The pairs of plasmids pGBKT7-Lam and pGADT7, pGADT7 and pGBKT7-FgVeA were used as negative controls. Growth of the transformed yeast was assayed on the medium containing 5 mM 3-aminotriazole (3-AT), but lacking His, Leu and Trp. Columns in each panel represent serial decimal dilution.