The ASH1-PEX16 regulatory pathway controls peroxisome biogenesis for appressorium-mediated insect infection by a fungal pathogen

Abstract

Entomopathogenic fungi infect insects by penetrating through the cuticle into the host body. To breach the host cuticle, some fungal pathogens produce specialized infection cells called appressoria, which develop enormous turgor pressure to allow cuticle penetration. However, regulatory mechanisms underlying appressorium turgor generation are poorly understood. Here, we show that the histone lysine methyltransferase ASH1 in the insecticidal fungus Metarhizium robertsii, which is strongly induced during infection of the mosquito cuticle, regulates appressorium turgor generation and cuticle penetration by activating the peroxin gene Mrpex16 via H3K36 dimethylation. MrPEX16 is required for the biogenesis of peroxisomes that participate in lipid catabolism and further promotes the hydrolysis of triacylglycerols stored in lipid droplets to produce glycerol for turgor generation, facilitating appressorium-mediated insect infection. Together, the ASH1-PEX16 pathway plays a pivotal role in regulating peroxisome biogenesis to promote lipolysis for appressorium turgor generation, providing insights into the molecular mechanisms underlying fungal pathogenesis.

Keywords: appressorium turgor generation; entomopathogenic fungi; histone methylation; host–microbe interactions; mosquitoes.

Fig. 1.

MrASH1, an H3K36me2 methyltransferase in M. robertsii, affects fungal pathogenicity. (A) qPCR analysis of Mrash1 transcription during topical infection of A. stephensi by the WT strain. Data are shown as the mean ± SD of three technical replicates. Significant differences compared with that at 12 h after topical infection were determined by Student's t test. *P < 0.05, **P < 0.01, and ***P < 0.001. gpd was used as a reference gene. The experiments were repeated twice with similar results. (B) Survival of female adult A. stephensi mosquitoes following topical application of conidial suspension (6 × 10⁶ conidia/ml) of the WT, ΔMrash1, and com-Mrash1 strains. The control mosquitoes were treated with 0.01% Triton X-100. Each treatment was replicated three times, with 50 mosquitoes per replicate. Significant differences compared with those in WT were determined by the log-rank (Mantel–Cox) test. ***P < 0.001. The experiments were repeated twice with similar results. (C) Survival of female adult A. stephensi mosquitoes after injection of conidial suspension (138 nL of 1 × 10⁶ conidia/mL) of the WT, ΔMrash1, and com-Mrash1 strains. The control mosquitoes were injected with 0.01% Triton X-100 in PBS. Fifty mosquitoes were used in each treatment. Significant differences compared with those in WT were determined by the log-rank (Mantel–Cox) test. ns, not significant. (D) Protein domain structures of MrASH1 and its homologues in other fungal species. aa, amino acid. (E) Western blotting analysis of histone modifications in the WT and two ΔMrash1 mutants. Histones extracted from M. robertsii mycelia grown in MM2 medium were resolved by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with specific antibodies against H3K36me1, H3K36me2, H3K36me3, and a C-terminal peptide of histone H3.

Fig. 2.

ΔMrash1 exhibits defects in cuticle penetration, appressorium turgor generation, glycerol accumulation, and lipid droplet degradation in M. robertsii. (A) The percentage of germinated conidia with appressoria relative to the total germinated conidia at 16 h and 24 h after induction in MM-Gly medium on hydrophobic plates. Data are shown as the mean ± SD of three biological replicates. The experiments were repeated twice with similar results. (B) The penetration capacity of WT, ΔMrash1, and com-Mrash1 was assayed using cicada wings. Mycelial blocks inoculated on the cicada wings were incubated for 2 d (Left). Then, the cicada wings were removed, and the plates were incubated for an additional 5 d (Right). (C) Colony diameters of WT, ΔMrash1, and com-Mrash1 at 5 d after removing the cicada wings in the cuticle penetration assay. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. **P < 0.01. The experiments were repeated twice with similar results. (D) A schematic model of the incipient cytorrhysis assay. Black and red arrows in the upper image indicate appressorium turgor pressure and exogenous pressure exerted on the appressorium cell wall by PEG8000, respectively. The appressorium collapsed when the pressure of PEG8000 exceeded the appressorium turgor pressure, as shown in the lower image. (E) Microscopic images of WT, ΔMrash1, and com-Mrash1 appressoria induced on cicada wings after immersion in PEG8000 for 10 min in an incipient cytorrhysis assay. (F) Percentage of appressoria induced on cicada wings that underwent incipient cytorrhysis after immersion in PEG8000 for 10 min. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. *P < 0.05. The experiments were repeated twice with similar results. (G) Intracellular glycerol levels in WT and ΔMrash1 appressoria induced on hydrophobic plates. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. *P < 0.05. (H) Distribution of lipid droplets in appressoria of WT and ΔMrash1 induced on cicada wings. Lipid droplets were stained with BODIPY. CO, conidium; AP, appressorium.

Fig. 3.

MrASH1 positively regulates the peroxin gene Mrpex16 in M. robertsii. The mycelia of WT and ΔMrash1 incubated in MM2 medium supplemented with adult locust cuticle for 8 h were analyzed by a combined RNA-seq and H3K36me2 ChIP-seq analysis. (A) Volcano plot of DEGs (fold change ≥ 2, P < 0.05) between WT and ΔMrash1 identified by transcriptome analysis. Significantly up-regulated, down-regulated, and unchanged genes in ΔMrash1 compared with those in WT are marked in red, blue, and gray, respectively. Lipid metabolism–related genes are marked in black. (B) DEGs by gene ontology category (primary metabolism). Red and blue columns represent the number of genes up-regulated and down-regulated in ΔMrash1 compared with that in WT, respectively. (C) qPCR analysis of lipid metabolism–related genes in the mycelium of WT and ΔMrash1 cultured in MM2 medium supplemented with adult locust cuticle. Data are shown as the mean ± SD of three technical replicates. Statistical significance was determined with Student's t test. *P < 0.05, **P < 0.01, and ***P < 0.001. gpd was used as a reference gene. The experiments were repeated twice with similar results. (D) Representative genome browser view of the enrichment of H3K36me2 in two WT replicates and mRNA signals in WT and ΔMrash1. (E) H3K36me2 levels at the Mrpex16 gene locus in WT and ΔMrash1. The upper image shows H3K36me2 enrichment at the Mrpex16 gene locus in WT, as determined by ChIP-seq analysis, and the black arrows indicate the position and polarity (5′-3′) of the primers used for ChIP–qPCR. The lower image shows ChIP–qPCR validation of ChIP-seq shown as the percentage of the signal from immunoprecipitation over the input in WT and ΔMrash1. Data are shown as the mean ± SD of three technical replicates. Statistical significance was determined with Student's t test. *P < 0.05 and **P < 0.01. The experiments were repeated twice with similar results.

Fig. 4.

Mrpex16 affects cuticle penetration, appressorium turgor generation, glycerol accumulation, lipid droplet degradation, and fungal pathogenicity in M. robertsii. (A) The penetration capacity of the WT and two ΔMrpex16 strains was assayed using cicada wings. Mycelial blocks inoculated on the cicada wings were incubated for 2 d (Left). Then, the cicada wings were removed, and the plates were incubated for an additional 5 d (Right). (B) Colony diameters of the WT and two ΔMrpex16 strains at 5 d after removing the cicada wings in the cuticle penetration assay. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. **P < 0.01. The experiments were repeated twice with similar results. (C) Percentage of germinated conidia with appressoria relative to the total germinated conidia at 16 h and 24 h after induction in MM-Gly medium on hydrophobic plates. Data are shown as the mean ± SD of three biological replicates. The experiments were repeated twice with similar results. (D) Microscopic images of the WT and two ΔMrpex16 strains appressoria induced on cicada wings after immersion in PEG8000 for 10 min in an incipient cytorrhysis assay. (E) Percentage of appressoria induced on cicada wings that underwent incipient cytorrhysis after immersion in PEG8000 for 10 min. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. **P < 0.01 and ***P < 0.001. (F) Intracellular glycerol levels in WT and ΔMrpex16 appressoria induced on hydrophobic plates. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. *P < 0.05. The experiments were repeated twice with similar results. (G) Distribution of lipid droplets in appressoria of WT and ΔMrpex16 induced on cicada wings. Lipid droplets were stained with BODIPY. CO, conidium; AP, appressorium. (H) Survival of female adult A. stephensi mosquitoes following topical application of conidial suspensions (6 × 10⁶ conidia/mL) of the WT and two ΔMrpex16 strains. The control mosquitoes were treated with 0.01% Triton X-100. Each treatment was replicated three times, with 50 mosquitoes per replicate. Significant differences compared with those in WT were determined by the log-rank (Mantel–Cox) test. *P < 0.05 and ***P < 0.001. The experiments were repeated twice with similar results. (I) Survival of female adult A. stephensi mosquitoes after injection of conidial suspensions (138 nL of 1 × 10⁶ conidia/mL) of the WT and two ΔMrpex16 strains. The control mosquitoes were injected with 0.01% Triton X-100 in PBS. Fifty mosquitoes were used in each treatment. Significant differences compared with those in WT were determined by the log-rank (Mantel–Cox) test. ***P < 0.001. The experiments were repeated twice with similar results.

Fig. 5.

MrASH1 activates Mrpex16 to regulate peroxisome biogenesis and fatty acid utilization in M. robertsii. (A) Subcellular locations of PEX16 fused with GFP and peroxisomes marked with RFP–PTS1 in the appressorium of WT induced in MM-Gly medium on hydrophobic plates for 20 h. CO, conidium; AP, appressorium. (B) Subcellular locations of peroxisomes marked with RFP–PTS1 in the appressoria of the WT and two ΔMrpex16 strains induced in MM-Gly medium on hydrophobic plates for 20 h. CO, conidium; AP, appressorium. (C) Quantification of peroxisomes in the appressoria of the WT and two ΔMrpex16 strains induced in MM-Gly medium on hydrophobic plates for 20 h. In total, 50 to 100 appressoria were detected for each strain. Horizontal lines represent the medians. Significant differences in the median compared with those in WT were determined by Student's t test. ***P < 0.001. The experiments were repeated twice with similar results. (D) Subcellular locations of peroxisomes marked with RFP–PTS1 in the appressoria of the WT, ΔMrash1, com-Mrash1, and two ΔMrash1::pex16 strains induced in MM-Gly medium on hydrophobic plates for 20 h. CO, conidium; AP, appressorium. (E) Quantification of peroxisomes in the appressoria of the WT, ΔMrash1, com-Mrash1, and two ΔMrash1::pex16 strains induced in MM-Gly medium on hydrophobic plates for 20 h. In total, 50 to 100 appressoria were detected for each strain. Horizontal lines represent the medians. Significant differences in the median compared with those in WT were determined by Student's t test. ***P < 0.001. ns, not significant. The experiments were repeated twice with similar results. (F) Fungal colonies of the WT, two ΔMrpex16 strains, ΔMrash1, and com-Mrash1 strains grown on MM plates supplied with 2.5 mM oleic acid (a long-chain fatty acid) or 50 mM NaAc (sodium acetate, a short-chain fatty acid) for 7 d. (G) Colony diameters of the WT, two ΔMrpex16 strains, ΔMrash1, and com-Mrash1 strains on fatty acid or acetate plates. Data are shown as the mean ± SD of three biological replicates. Significant differences compared with those in WT were determined by Student's t test. *P < 0.05, **P < 0.01, and ***P < 0.001. The experiments were repeated twice with similar results. (H) Schematic model showing the regulatory mechanism of the ASH1–PEX16 regulatory pathway in appressorium turgor generation and cuticle penetration in the insect pathogenic fungus. Upon host cuticle exposure, the up-regulated epigenetic regulator MrASH1 activates the target gene Mrpex16 via H3K36me2 modification on chromatin to control peroxisome biogenesis in the appressorium. Efficient degradation of fatty acids in the peroxisome via β-oxidation prevents their accumulation from inhibiting lipolysis and in turn promotes the hydrolysis of TAGs within lipid droplets to produce large amounts of glycerol for appressorium turgor pressure generation.