Mitogen-activated protein kinase hog1 in the entomopathogenic fungus Beauveria bassiana regulates environmental stress responses and virulence to insects

Abstract

Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. However, its insecticide efficacy in the field is often influenced by adverse environmental factors. Thus, understanding the genetic regulatory processes involved in the response to environmental stress would facilitate engineering and production of a more efficient biocontrol agent. Here, a mitogen-activated protein kinase (MAPK)-encoding gene, Bbhog1, was isolated from B. bassiana and shown to encode a functional homolog of yeast HIGH-OSMOLARITY GLYCEROL 1 (HOG1). A Bbhog1 null mutation was generated in B. bassiana by targeted gene replacement, and the resulting mutants were more sensitive to hyperosmotic stress, high temperature, and oxidative stress than the wild-type controls. These results demonstrate the conserved function of HOG1 MAPKs in the regulation of abiotic stress responses. Interestingly, DeltaBbhog1 mutants exhibited greatly reduced pathogenicity, most likely due to a decrease in spore viability, a reduced ability to attach to insect cuticle, and a reduction in appressorium formation. The transcript levels of two hydrophobin-encoding genes, hyd1 and hyd2, were dramatically decreased in a DeltaBbhog1 mutant, suggesting that Bbhog1 may regulate the expression of the gene associated with hydrophobicity or adherence.

FIG. 1.

Disruption of Bbhog1 influences the growth of B. bassiana. Wild-type strain Bb0062 (WT) and the Bbhog1 disruption mutant (ΔBbhog1) were incubated on CZP agar plates at 26°C for 10 days (A) or in CZP broth at 26°C on a rotary shaker (180 rpm) for 12 h (B) (control). The test strains were incubated on CZP agar plates supplemented with 0.4 M NaCl at 26°C for 10 days (C), in CZP broth containing 0.4 M NaCl at 26°C on a rotary shaker (180 rpm) for 12 h (D), on CZP agar plates supplemented (E) or not supplemented (F) with 0.4 M NaCl at 32°C for 10 days, or on CZP agar plates containing 20 mM H₂O₂ (G) or 2.5 μg ml⁻¹ fludioxonil (H) at 26°C for 10 days. Scale bars in panels B and D, 20 μm.

FIG. 2.

Compatible solute production in mycelia of the wild-type strain and the ΔBbhog1 mutant. Mycelia were grown for 48 h in SDY broth before they were transferred to SDY broth or SDY broth containing 0.4 M NaCl and incubated for a further 24 h. Carbohydrates were extracted and quantified by gas-liquid chromatography (25). (A) Mannitol; (B) glycerol; (C) erythritol; (D) arabitol. The error bars indicate standard deviations from three repeats of the experiment. WT, wild type.

FIG. 3.

Trends for mean survival of M. persicae after spraying. M. persicae peach aphids were sprayed with 1-ml portions of conidial suspensions containing 5 × 10⁷ conidia ml⁻¹ (A) and 1 × 10⁷ conidia ml⁻¹ (B), and the survival was recorded every day after inoculation. Control insects were sprayed with a conidial suspension prepared with the ectopic integration transformant T270 and sterilized water. For each treatment there were three replicates with 30 to 40 aphids each, and the experiments were repeated three times.

FIG. 4.

Determination of conidial germination, adherence, and appressorium formation. (A) Conidial germination. Conidia were inoculated onto CZP agar plates, and germinated conidia were counted hourly beginning 8 h after inoculation. (B) Conidial adherence on cicada hind wings. The assay was performed by using a previously described method (48). (C) Frequency of appressorium formation on cicada hind wings after 22 h of incubation. All experiments were repeated three times with three replicates for each repeat. WT, wild type.

FIG. 5.

(A) Appressorium morphology. Appressorium formation was induced on cicada hind wings using a method described previously (54). AP, appressorium; GE, germ tube; CO, conidium. Bar = 20 μm. (B) Hyphal body differentiation at 3 days after injection of conidia into P. brassicae larva. Bar = 20 μm. WT, wild type.

FIG. 6.

Expression of the hydrophobin-encoding genes hyd1 and hyd2 in the ΔBbhog1 mutant and the wild-type strain. Real-time RT-PCR was used to determine the relative levels of expression of hyd1 and hyd2 using Bgpd, β-tubulin, and 18S rRNA as loading controls to normalize samples by a method described previously (52) when the fungal cells were grown in the presence of cicada cuticle for 3 days. The error bars indicate standard deviations.