Requirement of a mitogen-activated protein kinase for appressorium formation and penetration of insect cuticle by the entomopathogenic fungus Beauveria bassiana

Abstract

Beauveria bassiana is an important insect-pathogenic fungus that invades insects by direct penetration of the host cuticle. To delineate the molecular mechanisms involved in fungal infection, a mitogen-activated protein kinase (MAPK) gene, Bbmpk1, which encodes a YERK1 family MAPK was isolated and characterized. Targeted gene disruption of Bbmpk1 resulted in a complete loss of virulence when applied topically to host insects but did not affect growth of the fungus when conidia were injected directly into the hemocoel. Hyphae of the mutant strain growing in the insect hemocoel were unable to penetrate the cuticle growing outwards and consequently failed to sporulate on the cadaver surface. These data suggest that BbMPK1 is essential for penetration of the insect cuticle both from the outside and from the inside-out in order to escape and disperse from the host. Inactivation of BbMPK1 also caused a significant decrease in fungal adhesion to insect cuticles and eliminated their ability to form appressoria. In order to identify downstream genes regulated by BbMPK1, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Thirty-one genes screened from the SSH library were determined to be expressed in the wild-type strain but either significantly reduced or not expressed in the mutant. Ten genes showed high or medium similarity to known protein encoding genes, including proteins involved in cell surface hydrophobicity, lipid metabolism, microtubule dynamics, mitochondrial electron transport, chromatin remodeling, transcription, rRNA processing, small nucleolar RNA accumulation, oxidation of aldehydes, translation, and likely other cellular processes.

FIG. 1.

(A) Conidial yield of WT, ΔBbmpk1, or reverse complementation transformant (RC) strains produced on Sabouraud dextrose agar (Difco Laboratories) supplemented with 1% (wt/vol) yeast extract. Conidial production was measured as described earlier (53). The error bars indicate the standard deviations from five repeats of the experiment. (B) Hyphal growth on the surface of a drop of liquid. Drops of Sabouraud dextrose broth (Difco Laboratories) supplemented with 1% (wt/vol) yeast extract containing conidia of the WT strain (left) or the Bbmpk1 disruption mutant (right) were placed in a petri dish and incubated for 96 h at 100% humidity.

FIG. 2.

(A) Symptoms of Pieris brassicae larva after topical application of 10⁷ conidia/ml suspensions of WT, ΔBbmpk1, or reverse complementation transformant (RC) strains (control insects were dipped in water). (B) Symptoms of insects following injection into the second proleg with 3 μl of 10⁷ conidia/ml suspensions (control insects were injected with 3 μl of water).

FIG. 3.

(A) Hyphal body differentiation at 3 days after injection of conidia into P. brassicae larva. Bar, 20 μm. (B) Conidial adherence to cicada hind wings. The assay was performed as described previously (43). The experiment was repeated three times with three replicates for each repeat. (C) Appressorial differentiation. Appressorium formation was induced on cicada hind wings as described previously (44). AP, appressorium; GE, germ tube; CO, conidium. Bar, 20 μm.

FIG. 4.

RT-PCR analysis of 31 selected SSH library genes. RNAs were extracted from conidia of the WT and ΔBbmpk1 mutant strains that were inoculated on cicada hind wings to induce appressorium formation (22 h after inoculation) and used for RT-PCR. All RT-PCR analysis was performed for 25 cycles using Bbgpd and β-tubulin as references. Lane 1, WT; lane 2, ΔBbmpk1 mutant.