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

['Xing Zhang', 'Moe Key Laboratory Of Biosystems Homeostasis', 'Protection', 'Institute Of Microbiology', 'College Of Life Science', 'Zhejiang University', 'Hangzhou', 'Yamin Meng', 'Yizhou Huang', 'Dan Zhang']

Date: 2021-08

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.

In this study, we discovered a novel regulatory cascade that controls the response of M. robertsii to the 2 microenvironments in its pathogenesis. On the insect cuticle, the transcription factor COH2 (colonization of hemocoel 2) activates the expression of genes involved in cuticle penetration. Once the fungus enters the hemocoel, the regulatory protein COH1 (colonization of hemocoel 1) is expressed, which physically contacts the transcription factor COH2 to reduce its stability, resulting in the inhibition of the expression of genes for cuticle penetration and the up-regulation of genes for hemocoel colonization. We further found that the expression of COH1 in the hemocoel results from the reduction in epigenetic repression conferred by the histone deacetylase HDAC1 and the histone acetyltransferase HAT1.

The mechanisms for cuticle penetration have been extensively investigated, and many cuticle-degrading genes and important regulators that control cuticle penetration have been reported [ 9 ]. Recently, some factors have also been found to play important roles in hemocoel colonization, including the sterol carrier Mr-NPC2a, which is responsible for acquisition of host sterols to maintain the integrity of the cell membrane of the fast-proliferating hyphal bodies [ 10 ]. The collagen-like protein MCL1 and toxic secondary metabolites such as destruxins facilitate the evasion of the host innate immune system [ 11 , 12 ]. The siderophore for iron metabolism is also an important factor for hemocoel colonization [ 13 ]. RNA sequencing (RNA-Seq) analysis in our recent work showed that many genes switch off during the microenvironmental transition from the insect cuticle to the hemocoel, including cuticle-degrading enzymes [ 9 ]. Some of these cuticle-degrading enzymes, such as Pr1 proteases and the metalloproteases MrMep1 and MrMep2, are important virulence factors [ 14 , 15 ], and their expression needs to be down-regulated in the hemocoel, where they would otherwise activate insect immunity [ 16 ]. Despite the extensive characterization of fungal functional genes for the infection of insects, the detailed regulatory mechanisms underlying the response of M. robertsii to different microenvironments remain to be explored.

Fungal pathogens of insects, plants, and mammals usually encounter dynamic microenvironments during infection of their hosts, but the mechanisms for them to respond and adapt to microenvironments are not well understood [ 1 ]. The entomopathogenic and endophytic fungus Metarhizium robertsii has been used as a model to study fungal pathogenesis in insects [ 2 ]. Infection occurs when conidia adhere to the cuticle of a susceptible insect host and produce germ tubes that differentiate into infection structures called appressoria. The appressoria produce infection pegs, which penetrate the cuticle via a combination of mechanical pressure and cuticle-degrading enzymes. Once reaching the insect hemocoel, the fungus undergoes dimorphism from hyphae to yeast-like cells (i.e., blastospores), and the insect is killed by a combination of fungal growth and toxins. Finally, the fungus grown in the hemocoel reemerges from the dead insect. In this pathogenesis progression, M. robertsii encounters 2 different microenvironments: the insect cuticle and the insect hemocoel. The insect cuticle is the first barrier against fungal infection, and the mechanisms for M. robertsii to breach this barrier are similar to those of some plant pathogenic fungi such as Magnaporthe oryzae and Colletotrichum lagenarium [ 3 , 4 ], which all form appressorial infection structures. Pathogenicity factors such as protein kinase A and hydrophobins are functionally conserved in the development of the appressorium between M. oryzae and M. robertsii [ 5 , 6 ]. The pathogenesis of M. robertsii also resembles that of mammalian pathogenic fungi in many aspects, such as the ability to evade the host innate immune system that has been conserved between insects and mammals [ 7 ]. Therefore, M. robertsii can be used as a representative fungus to study broad themes of fungal pathogenicity. In addition, Metarhizium species are being developed as environmentally friendly alternatives or supplements to chemical insecticides in biocontrol programs for agricultural pests and vectors of disease [ 8 ]. Detailed mechanistic knowledge of fungal pathogenicity in insects is therefore needed for optimal mycoinsecticide development and improvement.

Results

Identification of the regulatory protein COH1 In our previous RNA-Seq analysis [9], we found that a gene (MAA_08820, designated as Coh1, as it is involved in colonization of the hemocoel) was highly expressed during hemocoel colonization. In this study, hemolymph collected from last instar Galleria mellonella larvae (used for all assays in this study) was treated with an anticoagulant, and fungal growth in the resulting hemocyte-containing hemolymph (hereafter called hemolymph) was used as an approximation of hemocoel colonization, hereafter called surrogate hemocoel colonization. Penetration of the cuticle was achieved by incubating the fungus on the cuticle of G. mellonella larvae. Unless otherwise indicated, the insect cuticle used in this study was from G. mellonella larvae. Saprophytic growth was achieved by growing the fungus in the liquid medium Sabouraud dextrose broth supplemented with 1% yeast extract (SDY) or on potato dextrose agar (PDA) plates. Root colonization refers to the growth of M. robertsii on the roots of Arabidopsis thaliana. When quantitative reverse transcription PCR (qRT-PCR) analysis was conducted with RNA samples from cuticle penetration, saprophytic growth, and root colonization, the Cq values (the PCR cycle number at which a sample reaction curve intersects the threshold line) were approximately 38 cycles, and no PCR products were detected on the agarose gel (Fig 1A), suggesting that the transcription level of Coh1 was extremely low or was not detectable. However, qRT-PCR analysis and subsequent detection of PCR products on agarose gel confirmed that the Coh1 transcript was expressed during the surrogate hemocoel colonization (Fig 1A). The PCR product of Coh1 was also detected with RNA prepared from live G. mellonella larvae infected by M. robertsii, suggesting that this gene was expressed in the real hemocoel of the insects. When RNA from the insect cadavers that were mummified with the mycelium was used, no PCR product was detected, indicating that Coh1 was not expressed during the necrotrophic stage (Fig 1A). PPT PowerPoint slide

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larger image TIFF original image Download: 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) LT 50 (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. https://doi.org/10.1371/journal.pbio.3001360.g001 Coh1 is a single-copy gene with a 525-bp open reading frame (ORF) that encodes a protein containing 174 amino acid residues. COH1 contains an Ecl1 domain (PFAM12855) that was also identified in the ECL1 proteins of the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe; these proteins are considered regulators though their functions have not been characterized [17]. However, BLASTp analysis using the full-length protein sequence of COH1 as a query identified no significant similarity (>e−5) between M. robertsii COH1 and the yeast ECL1 proteins. No putative signal peptide or nuclear localization signal (NLS) was predicted in COH1. In the chromosome of M. robertsii, Coh1 was distant from its adjacent genes, with its ORF being 111,146 bp away from that of the upstream gene (MAA_10633) and 7,498 bp away from that of the downstream gene (MAA_08821).

Regulation of Coh1 by a histone deacetylase and a histone acetyltransferase To identify the mechanisms that regulate Coh1 expression, we compared the expression level of Coh1 in the WT strain with that in mutants with regulator genes deleted [2,9,19]. Since it was impossible to collect sufficient hemolymph from the G. mellonella larvae for screening a large number of mutants, we searched for the regulators that suppress the expression of Coh1 during saprophytic growth in the medium SDY, where Coh1 was not expressed (Fig 1A). We found that Coh1 was expressed in 11 epigenetic mutants with deletion of histone acetyltransferases, deacetylases, and methyltransferases, indicating that these epigenetic regulators negatively controlled Coh1 expression during saprophytic growth (Figs 1C and S4A). Compared with during saprophytic growth, only a histone H3 deacetylase gene (MAA_02098, designated as Hdac1) and a histone H3 acetyltransferase gene (MAA_02282, designated as Hat1) were significantly down-regulated during surrogate hemocoel colonization (Figs 1D and S4B). No significant differences in the expression levels of Hat1 and Hdac1 were found between cuticle penetration and saprophytic growth (Fig 1D). We further compared the expression levels of Hdac1 and Hat1 during cuticle penetration with that during colonization of the real hemocoel (i.e., the hemocoel of the insects infected by M. robertsii). As we could not obtain enough fungal biomass from the real hemocoel to prepare sufficient RNA for qRT-PCR analysis, we analyzed the expression of Hdac1 and Hat1 by tracing the GFP (green fluorescent protein) signal in 2 strains (WT-PHdac1-GFP and WT-PHat1-GFP), in which the gfp gene was driven by Hdac1 or Hat1 promoter in the WT strain (S1J Fig). Consistent with the results obtained from the qRT-PCR analysis with the surrogate hemocoel colonization, in both strains the GFP fluorescent intensity in the fungal cells on the cuticle was stronger than in the real hemocoel (Fig 1E). In a previous study, we constructed a deletion mutant of Hat1, ΔHat1, and its complemented strain C-ΔHat1 [19]. In this study, we generated an Hdac1 deletion mutant, ΔHdac1, and its complemented strain C-ΔHdac1 (S1G and S1H Fig). During the surrogate hemocoel colonization, the expression level of Coh1 was increased 27.6-fold in the mutant ΔHat1 and 15.2-fold in the mutant ΔHdac1 (Fig 1F). We thus postulated that the reduction in the expression of Hat1 and Hdac1 during hemocoel colonization derepressed their negative regulation of Coh1, resulting in the expression of Coh1. To test this postulation, we first tried to identify the target sites of HAT1 and HDAC1 by immunoblot analysis of the acetylation levels of 7 lysine residues in histone H3. Compared with the WT strain, the acetylation level of histone H3 on lysine 4 (H3K4) was significantly reduced in the mutant ΔHat1, whereas the acetylation level of histone H3 on lysine 56 (H3K56) was increased in the deletion mutants ΔHdac1 and ΔHat1 (Fig 2A). PPT PowerPoint slide

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larger image TIFF original image Download: 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 Hdac1OE, 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. https://doi.org/10.1371/journal.pbio.3001360.g002 We then investigated whether Hat1 and Hdac1 regulated the acetylation of histone H3 in the Coh1 promoter. Using chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis, we found that the acetylation level of histone H3 in the Coh1 promoter in the WT strain during surrogate hemocoel colonization was 13.8-fold higher than during saprophytic growth. More specifically, the acetylation level of H3K56 in the Coh1 promoter during surrogate hemocoel colonization was 3.5-fold higher than during saprophytic growth, but no significant difference in the acetylation level of H3K4 was observed between saprophytic growth and surrogate hemocoel colonization. During saprophytic growth, the acetylation level of H3K56 in the Coh1 promoter in the deletion mutant ΔHdac1 was 3.3-fold higher than in the WT strain, but no significant difference was found between the WT strain, the complemented strain C-ΔHdac1, and the Hdac1-overexpressing strain Hdac1OE (Fig 2B). Overexpression of Hdac1, driven by the constitutive promoter Ptef from A. pullulans [18], was confirmed by qRT-PCR (S1I Fig). During surrogate hemocoel colonization, H3K56 in the promoter of Coh1 in the WT strain was 5-fold more acetylated than in the strain Hdac1OE, but no difference was found among the WT strain, the deletion mutant ΔHdac1, and the strain C-ΔHdac1 (Fig 2B). As described above, HAT1 is a histone acetyltransferase. Unexpectedly, the acetylation levels of histone H3, H3K4, and H3K56 in the Coh1 promoter in the WT strain were all lower than in the mutant ΔHat1; however, no significant differences were observed between the WT strain and the strain C-ΔHat1 (Fig 2C). It is likely that HAT1 does not directly acetylate histone H3 in the promoter of Coh1 but instead could regulate other components, which in turn alter the histone H3 acetylation level in the Coh1 promoter. Since the target of HDAC1 is H3K56, and HAT1 negatively regulated the global acetylation level of H3K56 (Fig 2A), we investigated whether HAT1 regulated Hdac1 expression by controlling the acetylation of histone H3 in its promoter. qRT-PCR analysis showed that the expression level of Hdac1 in the WT strain was significantly higher than in the mutant ΔHat1 during saprophytic growth and surrogate hemocoel colonization (Fig 2D), suggesting that HAT1 positively regulated Hdac1 transcription. ChIP-qPCR analysis further showed that the acetylation levels of histone H3 and H3K4 in the Hdac1 promoter in the mutant ΔHat1 were both lower than in the WT strain (Fig 2E). In the WT strain, the acetylation levels of histone H3 and H3K4 in the promoter of Hdac1 during saprophytic growth were significantly higher than during surrogate hemocoel colonization (Fig 2F). To further confirm that HAT1 regulated the expression of Hdac1, which in turn controlled Coh1 expression, we constructed the strain ΔHat1-Hdac1OE with Hdac1 overexpressed in the mutant ΔHat1 (S1I Fig). qRT-PCR analysis showed that the expression level of Coh1 in ΔHat1 during surrogate hemocoel colonization was 3.3-fold higher than that of ΔHat1-Hdac1OE (S4C Fig).

COH1 physically interacts with the transcription factor COH2 As a putative regulatory protein, COH1 could interact with other proteins to control hemocoel colonization. Using a pull-down assay with the strain WT-COH1-HA expressing the fusion protein COH1::HA (a protein with COH1 tagged with HA [hemagglutinin]) in the WT strain (S5B Fig), we failed to identify the proteins that interact with COH1. Unless otherwise indicated, all genes encoding fusion proteins were driven by the constitutive promoter Ptef from A. pullulans in this study. Previous studies showed that alteration of expression of a gene can change the expression pattern of its interacting components [20–23], so we used RNA-Seq to profile the differentially expressed genes (DEGs) between the Coh1-overexpressing strain Coh1OE and the WT strain to identify candidates interacting with COH1. There were 1,893 DEGs, with 1,227 genes up-regulated and 666 genes down-regulated in the Coh1OE strain. As a regulatory protein, it is more likely that COH1 interacts with other regulators to control gene expression. Compared with the WT strain, 5 transcription factors were up-regulated in the strain Coh1OE. Yeast 2-hybrid assays showed that COH1 interacted with 1 transcription factor (MAA_07838) (Fig 3A), which is designated as COH2, as it also regulates colonization of the hemocoel (see below). Coh2 has an 849-bp ORF that encodes a protein containing 282 amino acid residues with a deduced molecular weight of 30.9 kDa. COH2 is a bZIP transcription factor with a DNA binding domain (COH2-DBD; Ser-30 to Thr-50), NLS (Ser-16 to Asp-39), and dimer interface domain (DID) (His-53 to Leu-88). PPT PowerPoint slide

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larger image TIFF original image Download: 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, DNA ladder. (C) Differential accumulation of the COH2::FLAG protein in 2 isolates of the strain WT-COH2-FLAG and 5 isolates of the strain Coh1OE-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: Coh1OE-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. https://doi.org/10.1371/journal.pbio.3001360.g003 We further conducted a coimmunoprecipitation (Co-IP) assay using a strain (COH1-HA/COH2-Myc) constitutively expressing COH1::HA and COH2::Myc (a protein with COH2 tagged with Myc) and confirmed that COH1 physically interacted with COH2 in vivo (Fig 3B). In the Co-IP assay, the Western blot analysis with an anti-Myc antibody always showed that the mass of the detected protein was approximately 2-fold greater than the predicted molecular weight (44.6 kDa) of the protein COH2::Myc (Fig 3B), suggesting that COH2 formed a homodimer. To confirm this, a further Co-IP assay using a strain (COH2-FLAG/COH2-Myc) constitutively expressing COH2::FLAG (a protein with FLAG fused to COH2) and COH2::Myc showed that these 2 fusion proteins formed a dimer in vivo (S6A Fig). The DID in COH2 contains 7 leucine residues that could be responsible for the dimerization of this transcription factor. To test this, we constructed a strain (COH2ΔDID-Myc) expressing the protein COH2ΔDID::Myc with the leucine residues in the DID substituted to alanine in the fusion protein COH2::Myc. Western blot analysis showed that the fusion protein COH2ΔDID::Myc did not form dimers (S6B Fig). We then assayed the region of COH2 that physically contacted COH1 using Co-IP assays. COH2 was divided into the N-terminus section (Met-1 to Thr-100) and the C-terminus section (Ser-101 to Arg-282). The N-terminus section contained the predicted DNA binding domain COH2-DBD, the DID, and the NLS. For Co-IP assays, we constructed 2 strains: COH1-HA/COH2-N-Myc, expressing HA-tagged COH1 and the fusion protein with the N-terminus of COH2 (COH2-N) fused with Myc, and COH1-HA/COH2-C-Myc, expressing the HA-tagged COH1 and the COH2 C-terminus (COH2-C) fused with Myc. Co-IP assays showed that COH1 can physically interact with the N-terminus of COH2, but did not with the C-terminus (S6C and S6D Fig). As with the protein COH2::Myc, COH2-N::Myc also formed dimers (S6C Fig). The protein COH2::FLAG was used as a representative of the COH2 fusion proteins used in this study (see below) to assay whether the COH2 protein fused with a tag retained its WT activity. To this end, we constructed a COH2::FLAG expression cassette with its coding sequence driven by the Coh2 promoter, which was then transformed into the mutant ΔCoh2 (see below) to produce the strain ΔCoh2-PCoh2-COH2-FLAG. No differences in saprophytic growth and pathogenicity were found between the WT strain, ΔCoh2-PCoh2-COH2-FLAG, and the complemented strain C-ΔCoh2, indicating that the COH2::FLAG protein functioned as COH2 (S7A and S7B Fig). However, this method could not be used to investigate whether the COH1 fusion proteins used in this study retained their WT activity because the native chromosomal position of Coh1 is essential for its promoter activity (S1D and S1E Fig). However, the Coh1-overexpressing strain Coh1OE was not different in colony growth and pathogenicity from the strain constitutively expressing the fusion protein COH1׃׃Myc (S3C Fig), indirectly showing that COH1 and COH1׃׃Myc had the same functions.

The COH1 and COH2 interaction reduces COH2 stability The impacts of the physical interaction between COH1 and COH2 on the activity of the transcription factor COH2 were then investigated. We first assayed the impact of the interaction on COH2 stability. To this end, the protein level of the fusion protein COH2::FLAG in the strain WT-COH2-FLAG (the protein COH2::FLAG expressed in the WT strain) was compared with the strain Coh1OE-COH2-FLAG (COH2::FLAG expressed in the Coh1-overexpressing strain Coh1OE). Five isolates of the strain Coh1OE-COH2-FLAG and 2 isolates of the strain WT-COH2-FLAG were selected, as they had the same transcription level of the fusion gene encoding the protein COH2::FLAG (S8A Fig). In the SDY medium, where COH1 was expressed in the strain Coh1OE-COH2-FLAG but not in WT-COH2-FLAG, 2 isolates of the strain WT-COH2-FLAG both accumulated much higher levels of the protein COH2::FLAG than all 5 isolates of the strain Coh1OE-COH2-FLAG (Fig 3C). We further assayed whether COH1 reduced the amount of the COH2::FLAG protein in the real hemocoel of insects infected by M. robertsii. With histoimmunochemical staining of the fungal cells collected from the real hemocoel with anti-FLAG antibody, the COH2::FLAG protein was found to be more abundant in the strain ΔCoh1-COH2-FLAG than the strain WT-COH2-FLAG (Coh1 expressed in the real hemocoel), which in turn had more COH2::FLAG protein than the Coh1-overexpressing strain Coh1OE-COH2-FLAG (Fig 3D). To investigate whether reduction of the COH2::FLAG protein caused by the expression of COH1 was due to degradation by the proteasome, the strains WT-COH2-FLAG and Coh1OE-COH2-FLAG were grown in the SDY medium supplemented with MG132, a specific inhibitor of the 26S proteasome. As shown in Fig 3E, MG132 treatment increased the level of the protein COH2::FLAG in the strain Coh1OE-COH2-FLAG, but not in the strain WT-COH2-FLAG, indicating that the interaction between COH1 and COH2 induces the degradation of the COH2::FLAG protein by the proteasome. We further found that the ubiquitination level of the COH2::FLAG protein in the strain Coh1OE-COH2-FLAG was higher than in WT-COH2-FLAG, indicating that the ubiquitin–proteasome pathway was responsible for the COH2 degradation (Fig 3F). We then tested the impact of COH1 on COH2 dimerization. To do this, we constructed a strain (Coh1OE-COH2-FLAG/COH2-Myc) with the fusion proteins COH2::FLAG and COH2::Myc expressed in the strain Coh1OE (S5 Fig). As in the strain COH2-FLAG/COH2-Myc, with the fusion proteins COH2::FLAG and COH2::Myc expressed in the WT strain (S5 Fig), COH2 dimerized in the strain Coh1OE-COH2-FLAG/COH2-Myc (S8B Fig), showing that COH1 did not impact COH2 dimerization. We also assayed the impact of COH1 on the entry of COH2 into the nucleus. To this end, we tried to construct a strain expressing the fusion protein COH2::GFP in the strain Coh1OE, but this attempt was not successful because the protein COH2::GFP was always separated into the 2 proteins: COH2 and GFP. However, we successfully expressed a fusion protein COH2-N::GFP (GFP fused to the COH2 N-terminus containing the NLS, COH2-DBD, and DID) in the WT strain and the strain Coh1OE to produce WT-COH2-N-GFP and Coh1OE-COH2-N-GFP (S5C Fig), respectively. In these 2 strains during saprophytic growth in SDY medium and during real hemocoel colonization, the GFP fluorescence intensity was strongest in the nucleus (S8C Fig). However, using the strain COH1-HA/COH2-N-GFP that simultaneously expressed COH1::HA and COH2-N::GFP, a Co-IP analysis showed that these 2 fusion proteins could not physically contact each other (S8D Fig). In addition, the protein COH2-N::GFP did not undergo dimerization (S5C Fig). Therefore, COH2-N::GFP is not suitable for assaying the impact of COH1 on the entry of COH2 into the nucleus.

COH2 is involved in pathogenicity To investigate the biological functions of Coh2, we first assayed its expression pattern. qRT-PCR analysis showed that Coh2 was constitutively expressed during saprophytic growth, cuticle penetration, and surrogate hemocoel colonization (S9A Fig). For the strain WT-PCoh2-GFP, with gfp driven by Coh2 promoter in the WT strain, the GFP fluorescent signal in the fungal cells on the insect cuticle was as strong as that in the real hemocoel of infected insects (S9B Fig), showing that Coh2 was constitutively expressed during cuticle penetration and real hemocoel colonization. We then constructed a Coh2 deletion mutant (ΔCoh2) and its complemented strain C-ΔCoh2 (S1G and S1H Fig). On PDA plates, no significant differences in colony growth, colony phenotype, or conidial yield were seen between ΔCoh2, C-ΔCoh2, and the WT strain (S9C and S9D Fig). With topical application of conidia on the insect cuticle, the LT 50 value of ΔCoh2 (17.4 ± 0.59 d) was 2-fold higher than that of the WT strain (8.7 ± 0.05 d) (P < 0.05), but no significant difference (P > 0.05) was found between the WT strain and the complemented strain C-ΔCoh2 (8.6 ± 0.07 d). With direct injection, the LT 50 value of ΔCoh2 (4.5 ± 0.24 d) was slightly higher than that of the WT strain (3.6 ± 0.09 d) (P < 0.05), and again the strain C-ΔCoh2 (3.7 ± 0.13 d) was not significantly different (P > 0.05) from the WT strain. On the hydrophobic surfaces of plastic petri dishes, appressorial formation was delayed in the mutant ΔCoh2, whereas no significant difference was observed between the WT strain and C-ΔCoh2 (S9E Fig). Compared with the WT strain, the ability of the mutant ΔCoh2 to penetrate the cuticle was decreased, and again no difference was found between the WT strain and C-ΔCoh2 (S9F Fig). To investigate how Coh1 interacted with Coh2 to regulate pathogenicity, we constructed the double gene deletion mutant ΔCoh1::ΔCoh2 (S1G Fig). WT-COH2-FLAG and ΔCoh1-COH2-FLAG were strains that overexpressed Coh2 in the WT strain and the mutant ΔCoh1, respectively. With topical application, the LT 50 value of the mutant ΔCoh1::ΔCoh2 was not significantly different from those of ΔCoh1 and ΔCoh2, but was significantly higher than those of the strains WT-COH2-FLAG and ΔCoh1-COH2-FLAG. Compared to the WT strain, the virulence of the strains WT-COH2-FLAG and ΔCoh1-COH2-FLAG was also significantly reduced (S9G Fig). With direct injection, the LT 50 value of the strain ΔCoh1::ΔCoh2 was not significantly different from those of ΔCoh1, ΔCoh2, WT-COH2-FLAG, and ΔCoh1-COH2-FLAG (S9H Fig). With both inoculation methods, no significant difference in fungal burden was found between the insects infected by ΔCoh1, ΔCoh2, ΔCoh1::ΔCoh2, WT-COH2-FLAG, and ΔCoh1-COH2-FLAG, which had significantly lower fungal burden than insects infected with the WT strain (S2F and S2G Fig). Compared to insects infected by the WT strain, the phenoloxidase activity in insects infected by the mutant ΔCoh2 was increased 2.3-fold (P < 0.05). No significant difference in phenoloxidase activity was found between the insects infected by the WT strain, ΔCoh1, C-ΔCoh2, WT-COH2-FLAG, and ΔCoh1-COH2-FLAG (S2J Fig). No significant differences in the expression levels of the antimicrobials gallerimycin and defensin were found between the insects infected by the WT strain and the mutants ΔCoh1, ΔCoh2, WT-COH2-FLAG, and ΔCoh1-COH2-FLAG (S2K Fig).

Determination of the DNA motif bound by COH2 To investigate how the transcription factor COH2 regulates pathogenicity genes with the regulatory protein COH1, we first used ChIP-Seq analysis with the strain WT-COH2-FLAG to identify the genes directly regulated by COH2. In the ChIP-Seq analysis, the peak caller MACS (Model-based Analysis of ChIP-Seq) identified a total of 562 peaks that were bound by COH2 (S10A Fig), 66.4% of which contained a consensus 7-nucleotide motif (TGA[C/G]T[C/A][G/A]) with multiple possible nucleotides at position 4, 6, and 7 (Figs 4A and S10B). This motif is designated as COH2-BM (COH2 binding motif). Consistent with COH2 forming a dimer, the motif COH2-BM contains a palindrome sequence: TGA and TCA spaced by 1 nucleotide at the fourth position. Electrophoretic mobility shift assay (EMSA) was then used to confirm the binding of COH2 to the motif COH2-BM (TGAGTCT) in the promoter of the gene MAA_04430, a representative of genes with the motif COH2-BM in their promoters determined by the ChIP-Seq analysis. We failed to express the whole protein of COH2 in Escherichia coli, but the portion (Met-1 to Thr-100) containing the predicted DNA binding domain, designated as COH2-DBD, was successfully expressed and the recombinant protein was then purified for EMSA (S10C and S10D Fig). The gel shift assay showed the DNA probe containing the motif COH2-BM (biotin-labeled) bound to the recombinant COH2-DBD protein (Fig 4B). The specific competitor (unlabeled DNA probe) almost completely abolished the DNA band shift (Fig 4B). To assay the importance of each position in the motif COH2-BM for binding to COH2-DBD, unlabeled COH2-BM mutants were constructed by replacing the consensus base with each of the other 3, and these mutants were subsequently used as competitors for the binding of the biotin-labeled DNA probe to the recombinant protein COH2-DBD. Consistent with the data obtained from the ChIP-Seq analysis, only the probe with G to A mutation at position 7 almost completely abolished the band shift of the labeled DNA probe, and the probe with C to A mutation at position 6 and all 3 mutated probes at the space nucleotide (at position 4) also significantly altered the band shift (S10E and S10F Fig). Mutations at other positions also impacted the band shift, but to a lesser extent than the mutated probes described above (S10F Fig). PPT PowerPoint slide

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larger image TIFF original image Download: 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. Coh1OE-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. https://doi.org/10.1371/journal.pbio.3001360.g004 To validate the ChIP-Seq and EMSA results, we performed quantitative ChIP-qPCR with primers designed to cover the motif COH2-BM in the promoter of the gene MAA_04430. After immunoprecipitation, qPCR analysis showed that the copy number of the DNA fragment containing the motif COH2-BM from the strain WT-COH2-FLAG was 29.4-fold higher than that from the strain WT-FLAG, which expressed the FLAG tag only (Fig 4C).

The COH1 and COH2 interaction derepresses COH2-mediated repression of hemocoel colonization genes As COH1 was only expressed during hemocoel colonization, we thus investigated how COH1 and COH2 interacted to regulate this infection stage. To this end, RNA-Seq analysis was first used to compare the transcriptomes of the WT strain and the mutants ΔCoh1 and ΔCoh2 during surrogate hemocoel colonization. Compared with the WT strain, 68 genes were up-regulated and 207 genes down-regulated in the mutant ΔCoh2 (S11A Fig). Compared with the WT strain, 52 genes were down-regulated and 97 genes up-regulated in the mutant ΔCoh1 (S11A Fig). RNA-Seq analysis showed that neither Coh1 nor Coh2 regulated Hat1expression, and subsequent qRT-PCR analysis further confirmed that there was no significant difference in the expression level of Hat1 between the WT strain and the mutants ΔCoh1 and ΔCoh2, and between the Coh1-overexpressing strain Coh1OE and the Coh2-overexpressing strain WT-COH2-FLAG (S11C Fig). Remarkably, among the 15 genes that were down-regulated in ΔCoh1 but up-regulated in ΔCoh2 (S11B Fig), 10 were involved in hemocoel colonization, including 4 genes in the destruxin biosynthesis pathway, a siderophore iron transporter (MAA_10457), and a laccase (MAA_00990). To investigate whether these 15 genes are directly regulated by COH2, we searched for the motif COH2-BM in their promoters, which were determined as the approximately 2-kb DNA fragments upstream of the ORF start sites or as the regions, if shorter than 2 kb, between the ORFs of the 15 genes and the ORFs of their respective adjacent genes. The motif COH2-BM was found in the promoters of all the 15 genes. Using the 4 destruxin biosynthesis genes (DtxS1, DtxS2, DtxS3, and DtxS4) as representatives, we investigated the mechanisms for COH1 and COH2 to regulate the genes involved in hemocoel colonization. qRT-PCR further confirmed that during surrogate hemocoel colonization, compared with the WT strain, these 4 destruxin biosynthesis genes were down-regulated in the mutant ΔCoh1 and up-regulated in the mutant ΔCoh2 (S11D Fig). Using the gene DtxS3 as a representative, we assayed the impact of Coh1 and Coh2 on the expression of the destruxin biosynthesis genes in the real hemocoel of insects infected by M. robertsii. To this end, an expression cassette with gfp driven by the DtxS3 promoter PDtxS3 was transformed into the WT strain and the mutants ΔCoh1 and ΔCoh2 to produce the strains WT-PDtxS3-GFP, ΔCoh1-PDtxS3-GFP, and ΔCoh2-PDtxS3-GFP (S1J Fig). GFP fluorescent intensity in ΔCoh2-PDtxS3-GFP cells collected from the real hemocoel appeared to be stronger than in WT-PDtxS3-GFP, which was in turn stronger than in ΔCoh1-PDtxS3-GFP (S11E Fig). ChIP-qPCR analysis showed that COH2 occupancy at DtxS1, DtxS2, DtxS3, and DtxS4 promoters in the strain WT-COH2-FLAG was 102.8-, 29.3-, 62-, and 13.9-fold higher, respectively, than in the strain WT-FLAG (Fig 4D). Based on the qRT-PCR and ChIP-qPCR data described above, we postulated that COH2 is a repressor of the destruxin biosynthesis genes and that this repression is mitigated by COH1 when the fungus enters the insect hemocoel. To test this, we assayed the impact of the presence of COH1 on the binding of COH2 to the promoters of the 4 genes. To this end, we constructed a strain (ΔCoh1-COH2-FLAG) with the fusion protein COH2::FLAG expressed in the mutant ΔCoh1 (S5A Fig). During surrogate hemocoel colonization, the enrichments of the protein COH2::FLAG in the promoters of Dtxs1, Dtxs2, Dtxs3, and Dtxs4 in the strain ΔCoh1-COH2-FLAG were all higher than in WT-COH2-FLAG (Fig 4E). Conversely, compared with the strain WT-COH2-FLAG, the enrichment of COH2::FLAG at the promoter of Dtxs1, Dtxs2, Dtxs3, and Dtxs4 was reduced 5.3-, 3.4-, 5.3-, and 5.3-fold, respectively, in the strain Coh1OE-COH2-FLAG (Fig 4F).

COH2 induces the expression of cuticle-degrading genes As described above, in addition to regulating hemocoel colonization along with the regulator COH1, COH2 also controlled cuticle penetration. To investigate how COH2 regulates cuticle penetration, we used RNA-Seq to compare the transcriptomes of the mutant ΔCoh2 and the WT strain when they were grown on locust hindwings. Compared with the WT strain, 549 genes were down-regulated and 236 genes up-regulated in ΔCoh2 (S11A Fig). The COH2 binding motif COH2-BM was found in 368 genes out of the 549 down-regulated genes, and 171 out of the 236 up-regulated genes. In this study, the G. mellonella larva is used as the host for pathogenicity analysis. Therefore, by qRT-PCR analysis of 5 representative genes, we validated the results obtained with locust hindwings on the cuticle of G. mellonella larva, and no difference in their expression was found between the locust hindwings and the G. mellonella cuticle (S11G Fig). Remarkably, 44 cuticle-degrading genes were down-regulated in the deletion mutant ΔCoh2, including 30 proteases, 3 chitinases, 2 lipases, and 9 cytochrome P450 enzymes (S11F Fig). Among the 30 protease genes, 27 had the motif COH2-BM in their promoters. The COH2-BM motif was also found in the promoters of the 3 chitinase genes, 1 lipase gene, and 7 cytochrome P450 genes. Two protease genes (MAA_10199 and MAA_10350) and 1 chitinase gene (MAA_10456) were used as representatives to confirm that COH2 directly regulated the cuticle-degrading genes described above. During penetration of the G. mellonella cuticle, qRT-PCR confirmed that these 3 genes were down-regulated in the mutant ΔCoh2 compared with the WT strain (Fig 5A). Since it was not possible to obtain enough biomass to get sufficient DNA for ChIP-qPCR analysis during cuticle penetration, i.e., growing M. robertsii on the insect cuticle, an approximation of cuticle penetration was prepared by growing the fungus in a cuticle medium with the cuticle of G. mellonella larvae as the sole carbon and nitrogen source (called cuticle medium hereafter). When fungus was grown in the cuticle medium, ChIP-qPCR analysis showed that in the strain WT-COH2-FLAG, the COH2::FLAG protein bound to the promoters of these 3 genes (Fig 5B). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001360.g005 We further investigated the impact of the cuticle-degrading genes regulated by COH2 on the ability of the fungus to degrade the insect cuticle. To this end, we quantified cuticle degradation products following 12 h of fungal growth in the cuticle medium. Although the growth rates of the WT strain and the mutant ΔCoh2 in the cuticle medium were similar, the mutant secreted significantly less total extracellular protease than the WT strain (P < 0.05) (S11H Fig), and it thus released significantly fewer amino acids (S11I Fig) and peptides (S11J Fig) from the cuticle (P < 0.05). Likewise, the WT strain produced significantly (P < 0.05) more chitinase than ΔCoh2 (S11K Fig).

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