A novel cascade allows Metarhizium robertsii to distinguish cuticle and hemocoel microenvironments during infection of insects

Abstract

Pathogenic fungi precisely respond to dynamic microenvironments during infection, but the underlying mechanisms are not well understood. The insect pathogenic fungus Metarhizium robertsii is a representative fungus in which to study broad themes of fungal pathogenicity as it resembles some major plant and mammalian pathogenic fungi in its pathogenesis. Here we report on a novel cascade that regulates response of M. robertsii to 2 distinct microenvironments during its pathogenesis. On the insect cuticle, the transcription factor COH2 activates expression of cuticle penetration genes. In the hemocoel, the protein COH1 is expressed due to the reduction in epigenetic repression conferred by the histone deacetylase HDAC1 and the histone 3 acetyltransferase HAT1. COH1 interacts with COH2 to reduce COH2 stability, and this down-regulates cuticle penetration genes and up-regulates genes for hemocoel colonization. Our work significantly advances the insights into fungal pathogenicity in insects.

Fig 1. Expression and regulation of the Coh1 gene.

(A) Agarose gel electrophoresis of quantitative reverse transcription PCR (qRT-PCR) products of Coh1. 1: Saprophytic growth in Sabouraud dextrose broth supplemented with 1% yeast extract (SDY) medium; 2: cuticle penetration; 3: colonization of plant roots; 4: growth in the hemocyte-containing hemolymph; 5: live insects infected with M. robertsii; 6: insect cadavers mummified with M. robertsii mycelium; M: DNA ladder. Images are representative of 3 independent experiments. Upper panel: the gene Coh1; lower panel: the reference gene Gpd encoding glyceraldehyde 3-phosphate dehydrogenase. (B) LT₅₀ (time taken to kill 50% of insects) values when the insects were inoculated by topical application of conidia on the cuticle. ΔCoh1#1, ΔCoh1#2, and ΔCoh1#3 are 3 independent isolates of the deletion mutant ΔCoh1. WT, wild type. The bioassays were repeated 3 times with 40 insects per repeat. Data are expressed as mean ± SE. Values with different letters are significantly different (n = 3, P < 0.05, Tukey’s test in one-way ANOVA). (C) Agarose gel electrophoresis of qRT-PCR products of Coh1 in WT strain and deletion mutants of the histone deacetylase gene Hdac1 (ΔHdac1) and the histone acetyltransferase gene Hat1 (ΔHat1) during saprophytic growth. Note: No qRT-PCR product was seen in the WT strain. (D) qRT-PCR analysis of the expression of Hdac1 and Hat1 in the WT strain during the surrogate hemocoel colonization (Hemolymph) and cuticle penetration (Cuticle) relative to saprophytic growth (SDY). (E) GFP signal in the fungal cells on the cuticle (Cuticle) and in the real hemocoel (Hemocoel) of G. mellonella larvae. The strain with the gfp gene was driven by the promoter of Hat1 (upper panels) or Hdac1 (lower panels). F, fungal cells; H, hemocyte. Scale bar: 10 μm. Images are representative of 3 independent experiments. The mean gray value (MGV) shows the GFP fluorescence intensity in the fungal hyphae. (F) qRT-PCR analysis of Coh1 expression in the WT strain and the mutants ΔHat1 and ΔHdac1 during the surrogate hemocoel colonization. All qRT-PCR experiments in this study were repeated 3 times. For qRT-PCR analyses in this figure, the values represent the fold-change of expression of a gene in treatment compared with expression in its respective control, which is set to 1. The data underlying all the graphs shown in this figure can be found in S1 Data.

Fig 2. HDAC1 and HAT1 control Coh1 expression by regulating histone H3 acetylation in its promoter region.

(A) Immunoblot analysis of acetylation levels of 7 lysine residues on histone H3 protein in the wild-type (WT) strain and the mutants ΔHdac1 and ΔHat1. All Western blot images shown in this study are representatives of at least 3 independent experiments. (B) The acetylation level of H3K56 in the Coh1 promoter in the mutant ΔHdac1, its complemented strain C-ΔHdac1, the Hdac1-overexpressing strain Hdac1^OE, and the WT strain during saprophytic growth in Sabouraud dextrose broth supplemented with 1% yeast extract (SDY) and surrogate hemocoel colonization (Hemolymph). (C) The acetylation levels of histone H3, H3K56, and H3K4 in the Coh1 promoter in the mutant ΔHat1 and its complemented strain C-ΔHat1 relative to the WT strain during saprophytic growth. (D) qRT-PCR analysis of Hdac1 expression in the WT strain, the mutant ΔHat1, and its complemented strain C-ΔHat1 during saprophytic growth and surrogate hemocoel colonization. (E) The acetylation levels of histone H3 and H3K4 in the Hdac1 promoter in the mutant ΔHat1 and its complemented strain C-ΔHat1 relative to the WT strain during saprophytic growth. (F) The acetylation levels of histone H3 and H3K4 in the Hdac1 promoter in the WT strain during surrogate hemocoel colonization relative to saprophytic growth. For chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analyses in this figure, the values represent the fold-change of the acetylation level of histone H3, H3K4, or H3K56 compared with the level in its respective control, which is set to 1. All ChIP-qPCR experiments were repeated at least 3 times. The data underlying all the graphs shown in this figure can be found in S1 Data.

Fig 3. COH1 physically interacts with the transcription factor COH2 to reduce COH2 stability.

(A) Yeast 2-hybrid analysis confirms the physical interaction of COH1 with COH2. Left panel: colonies were grown in SD/−Ade/−His/−Leu/−Trp + X-α-gal + AbA. Right panel: COH1 lacks autoactivation activity. The Y2HGold cells with pGBKT7-COH1 cannot grow in SD/−Ade/−His/−Trp + X-α-gal. BD, binding domain; NC, negative control (yeast cells containing the plasmid pGADT7-T and pGBKT7-Lam); PC, positive control (yeast cells containing the plasmid pGADT7-T and pGBKT7-53). (B) Coimmunoprecipitation confirmation of the physical interaction of COH1 with COH2. The fusion proteins COH1::HA and COH2::Myc (molecular weight = 44.6 kDa) were simultaneously expressed in the strain COH1-HA/COH2-Myc. The control was the strain WT-COH2-Myc expressing the protein COH2::Myc. Immunoprecipitation was conducted with anti-HA antibody. Proteins were detected by immunoblot (IB) analysis with anti-HA or anti-Myc antibodies. The dimer COH2::Myc is indicated by red arrows. M, protein ladder. (C) Differential accumulation of the COH2::FLAG protein in 2 isolates of the strain WT-COH2-FLAG and 5 isolates of the strain Coh1^OE-COH2-FLAG. Equal loading of proteins was confirmed by the β-tubulin protein that was detected by the anti-β-tubulin antibody. Numbers indicate band intensity for COH2::FLAG relative to β-tubulin. The values of the #1 isolate of the strain WT-COH2-FLAG were set to 1. (D) Histoimmunochemical staining of the COH2::FLAG protein in fungal cells in the real hemocoel of G. mellonella larvae. Top panel: the strain WT-COH2-FLAG; middle panel: ΔCoh1-COH2-FLAG; bottom panel: Coh1^OE-COH2-FLAG. Scale bar represents 10 μm. Images are representative of 3 independent experiments. F, fungal cells; FITC, fluorescein isothiocyanate; H, hemocyte; MGV, mean gray value. (E) Confirmation of degradation of the COH2::FLAG protein by the proteasome pathway. The mycelium grown in Sabouraud dextrose broth supplemented with 1% yeast extract (SDY) medium was treated with the 26S proteasome inhibitor MG132. (F) The ubiquitination level of the COH2::FLAG protein increased due to its interaction with COH1. The COH2::FLAG protein was pulled down from the MG132-treated mycelium with an anti-FLAG antibody and immunoblotted with an anti-ubiquitin (Ubi) antibody (left) and the anti-FLAG antibody (right). IP, immunoprecipitation.

Fig 4. Identification of hemocoel-colonizing genes regulated by COH1 and COH2.

(A) Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis identified the DNA motif COH2-BM that was bound by the transcription factor COH2. (B) Electrophoretic mobility shift assay (EMSA) confirms the in vitro binding of the biotin-labeled motif COH2-BM (Bio-probe) to the recombinant protein COH2-DBD (COH2 DNA binding domain). The binding activity was demonstrated by the shift of the labeled DNA band prior to the addition of the specific competitor (Cold-probe: the unlabeled motif COH2-BM) in 50-, 100-, 150-, 200-, or 300-fold excess. The tested DNA motif COH2-BM is from the promoter of the gene MAA_04430, which was shown to have the motif COH2-BM by the ChIP-Seq analysis. (C) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis confirms that COH2 in vivo binds to the motif COH2-BM in the MAA_04430 promoter in the strain WT-COH2-FLAG, expressing the fusion protein COH2::FLAG. WT-FLAG: a strain expressing the tag FLAG only. (D) ChIP-qPCR confirmation of the in vivo binding of COH2 to the promoters of the 4 destruxin biosynthesis genes (DtxS1, DtxS2, DtxS3, and DtxS4). (E) ChIP-qPCR analysis shows that during surrogate hemocoel colonization, deleting the Coh1 gene increased the binding of COH2 to the promoters of the 4 destruxin biosynthesis genes. ΔCoh1-COH2-FLAG: a strain expressing the protein COH2::FLAG in the mutant ΔCoh1. (F) ChIP-qPCR analysis confirms that during saprophytic growth, COH1 reduced the in vivo binding of COH2 to the promoters of the 4 destruxin biosynthesis genes. Coh1^OE-COH2-FLAG: a strain expressing the protein COH2::FLAG and overexpressing the Coh1 gene. The data underlying all the graphs shown in this figure can be found in S1 Data.

Fig 5. COH1 inactivates the COH2-mediated induction of cuticle-degrading genes during hemocoel colonization.

(A) Quantitative reverse transcription PCR (qRT-qPCR) analysis of the expression of cuticle-degrading genes during cuticle penetration in the mutant ΔCoh2 relative to the wild-type (WT) strain. The chitinase MAA_10456 and the proteases MAA_10199 and MAA_10350 are used as representatives. (B) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis shows the occupancy of the COH2::FLAG protein in the promoters of the chitinase and protease genes in the strain WT-COH2-FLAG relative to occupancy in the control strain WT-FLAG, which is set to 1. The fungal strains were grown in the cuticle medium. (C) qRT-PCR analysis of the expression levels of the 3 genes during surrogate hemocoel colonization relative to the expression level during cuticle penetration, which is set to 1. (D) ChIP-qPCR analysis of the occupancy of COH2 in the promoters of the 3 genes during surrogate hemocoel colonization (Hemolymph) relative to the growth in the cuticle medium (Cuticle medium), which is set to 1. The strain WT-COH2-FLAG was used. (E) qRT-PCR analysis of the expression levels of the 3 genes during surrogate hemocoel colonization in the mutant ΔCoh1 relative to the expression level in the WT strain, which is set to 1. (F) ChIP-qPCR analysis of the occupancy of the COH2::FLAG protein in the promoters of the 3 genes during surrogate hemocoel colonization in the strain ΔCoh1-COH2-FLAG (the mutant ΔCoh1 with COH2::FLAG expressed) relative to occupancy in the strain WT-COH2-FLAG, which is set to 1. (G) GFP signal in the fungal cells of 2 strains (WT-PMAA_10199-GFP and ΔCoh1-PMAA_10199-GFP) with the gfp gene driven by the promoter of the protease gene MAA_10199. Top panel: WT-PMAA_10199-GFP on the cuticle (Cuticle); middle panel: WT-PMAA_10199-GFP in the real hemocoel of G. mellonella larva infected by a fungal strain (Hemocoel); bottom panel: ΔCoh1-PMAA_10199-GFP in the real hemocoel. F, fungal cells; H, hemocyte; MGV, mean gray value. Scare bar, 10 μm. The data underlying all the graphs shown in the figure can be found in S1 Data.