Fig 1.
Gene and protein expression of BbEng1 in B. bassiana.
A. Transcript levels of BbEng1 in different morphological cells and cultures grown in different media and under stress conditions. AHY, aerial hyphae. CO, conidia. LHY, submerged hyphae. BLS, blastospores. HB, hyphal bodies (in vivo blastospores). BS, the basic salt broth. Cuticle and hemolymph, BS + 0.167 g / L silkworm cuticle or 5 mL / L hemolymph. Chitin, glucan or trehalose, used as sole carbon source in CZB as a substitute for sucrose at 2%. NaCl, sorbitol, H2O2, MND, t-BHP or lactic acid, CZB containing 0.5 M NaCl, 1.0 M sorbitol, 5.76 mM H2O2, 37 μM menadione or 0.78 mM tert-Butyl hydroperoxide or 2% (v/v) lactic acid. LO, low oxygen condition (with ~6% of initial O2 concentration). All fungal cells were cultured 6 h in different media and under different stress conditions. Error bars denote standard deviations (SDs) from three biological replicates. Different letters (a–f) indicate statistically significant differences (P < 0.01 in LSD test). B. Microscopic images (scale: 5 μm) of GFP fluorescence expressed in fungal cells of PBbEng1::GFP strain. GFP was driven by BbEng1 promoter. C. Microscopic images (scale: 5 μm) of GFP fluorescence in BbEng1::GFP hyphal bodies and cells cultured in insect-derived nutrients. Hyphal bodies were examined in hemolymph samples at the indicated time points after intra-hemocoel injection of conidia into G. mellonella larvae (left). Fungal cells grown in insect-derived nutrients (5 mL / L hemolymph or 0.167 g / L cuticle from silkworm) were collected at the indicated time points (right) of incubation. D. Microscopic images (scale: 5 μm) of GFP fluorescence and FM4-64 (stained cell membrane) in BbEng1::GFP hyphal bodies.
Fig 2.
Distribution and secretion of BbEng1 in B. bassiana.
A. Indirect immunofluorescence (IIF) images (scale: 5 μm) examining subcellular localization of BbEng1 in B. bassiana wild-type, ΔBbEng1, BbEng1OE and complementation (Comp) hyphal bodies probed with anti-BbEng1 polyclonal antibody and FITC-conjugated-goat anti-rabbit IgG. Fungal cell wall was stained with CFW (calcofluor white). B. Immuno-transmission electron images of BbEng1 in hyphal bodies of BbEng1OE probed with anti-BbEng1 polyclonal antibody and anti-Rabbit IgG (Whole molecule)-Gold. C. Microscopic images (scale: 5 μm) of BbEng1::GFP expressed in hyphal bodies after 12-h treatment with 2 mM dithiothreitol (DTT). Left, GFP fluorescence in fungal cells. Right, fluorescence density in the supernatant. The BbEng1::GFP cells and the supernatant were probed with anti-GFP antibody. Control (-), not treated with DTT. Error bars: SDs. Different letters indicate significant differences pairs (P < 0.01, t-test). D. Western blotting detection of secreted BbEng1 by anti-BbEng1 polyclonal antibody. Upper, wild-type incubated 12 h in insect-derived nutrient-contained broth (0.167 g / L silkworm cuticle; 5 mL / L silkworm hemolymph) and 1/4 SDY broth. Lower, insect hemolymph of larvae after 48-h injection with wild-type and BbEng1OE conidia. The digital values denote the amount of BbEng1 secreted by the tested strains in the host hemolymph.
Fig 3.
Morphological characterization and conidial germination of B. bassiana wild type and mutant strains.
A. Colony edge and aerial hyphae morphology. B. Conidial morphology and size (scale: 10 μm). C, D. Morphology of conidial germination (scale: 20 μm) and ratio of bidirectional apical growth from conidia. Error bars in (D) denote SDs. Different letters indicate significant differences (P < 0.01 in LSD test).
Fig 4.
Cell wall structures of B. bassiana wild type and mutant strains.
A, B. TEM micrographs (scale: 0.2 μm) for cell walls of hyphal bodies and cell wall thickness (n = 30). C, D. Confocal microscopic images (scale: 5 μm) for cell wall β-1,3-glucan and chitin and measurements of their contents. β-1,3-glucan was labeled with monoclonal β-1,3-glucan specific antibody and goat anti-mouse IgG-FITC. Chitin was labeled with WGA or stained with CFW as detailed in the Methods section. Mean fluorescence intensities were quantified densitometrically using the ImageJ software. Error bars: SDs. Different letters indicate significant differences (P < 0.01 in LSD test).
Fig 5.
Binding of purified BbEng1 to various substrates.
A. Western blots probed with anti-BbEng1 antibody after pull-down assays with indicated polysaccharides and silkworm cuticles as substrates (2%, w/v). The protein concentration of BbEng1 used in assays was 60 μg / mL. B, C. TLC and HPLC analyses for the carbohydrates released from the reaction of BbEng1 (2 μg, pH 6.0, 30°C for 1 h) with the extracts of polysaccharides (1%, w/v) from B. bassiana cell wall and silkworm cuticle. AS, alkali-soluble fraction. ASDN, WSDN and AIDN, alkali-soluble, water-soluble and alkali-insoluble fractions generated by nitrous deamination of fully de-N-acetylated alkali-insoluble fraction (AI).
Fig 6.
Carbohydrate epitopes and β-1,3-glucan levels on surfaces of B. bassiana wild type and mutant hyphal bodies.
A. Fluorescence images (scale: 5 μm) of hyphal bodies treated with monoclonal β-1,3-glucan specific antibody and goat anti-mouse IgG-FITC and Fluor 488-labeled lectins. BL, bright light. FL, fluorescent light. B. Mean fluorescence levels quantified from at least 100 individual cells of tested strains. Error bars: SDs. Different letters indicate significant differences (P < 0.01 in LSD test).
Fig 7.
Virulence of B. bassiana wild type and mutant strains and insect immune responses.
A, B. Survival trends and calculated LT50 values using G. mellonella larvae after topical application and intrahemocoel injection of conidial suspension. P < 0.05* or 0.01** in a log rank test. C. Symptoms of G. mellonella larvae infected by indicated strains and fungal outgrowth on cadavers at indicated time points. D. Microscopic images (scale: 5 μm) of fungal development and insect immune responses at indicated time points after injection. Arrows indicate fungal cells (hyphal bodies, HBs). E. Quantification of fungal biomass in hemolymph samples at 36, 48 and 60 h after injection as determined using qPCR analysis. Error bars: SDs. Different letters in A, B and E indicate significant differences (P < 0.01 in LSD test).
Fig 8.
Immune responses of G. mellonella after challenge by B. bassiana wild type and UV-killed cells with or without addition of purified BbEng1.
A. The phenoloxidase (PO) activities at indicated time points after injection. B. H2O2 (ROS) levels during fungal infection. C. Expression levels of genes involved in the Toll pathway, antimicrobial peptide (AMP) and proPO system in the larvae 12 h post-injection (β-actin used as a reference for normalized fold expression, NFE). D. Effects of added 0.5 μM BbEng1 or iBbEng1 (heated-inactivated BbEng1) on fungal cell wall (UKC, UV-killed B. bassiana conidia, 5 × 106 conidia / mL)-activated PO activity (total volume: 2 μL). Hemolymph samples taken at 12 h after injection of UKC, UKC + BbEng1 or UKC + iBbEng1 were used for PO assay. Larvae injected with 2 μL of 0.85% NaCl or UKC+ BSA (bovine serum albumin, 0.5 μM) were used as controls. E. Expression levels of genes in the Toll pathway components, AMP proPO-system-associated genes in the larvae at 12 h after injection of UKC or UKC + proteins (β-actin used as the reference). F, G. Microscopic images and fluorescence intensities of UKC and conidia (CO) (n > 100) labeled with FITC-labeled BbEng1. Error bars: SDs. Different letters indicate significant differences (P < 0.01 in LSD test).
Fig 9.
Insect bioassays of M. robertsii and M. acridum wild type and Eng1 overexpression strains.
A, B and D. Survival trends of G. mellonella larvae after topical application (A) or hemocoel injection (B and D). C. Survival trends of L. migratoria manilensis nymphs after topical inoculation. **P < 0.01 in log-rank test. Mr-BbEng1OE and MrEng1OE, M. robertsii strains overexpressing BbEng1 and MrEng1. Ma-BbEng1OE and MaEng1OE, M. acridum strains overexpressing BbEng1 and MaEng1. Marked LT50 estimates differ significantly (P < 0.01 in LSD test).