Fig 1.
Structure modulation of the LysM proteins encoded by B. bassiana.
ChtBD1, chitin-binding domain; Glyco_18, GH18 chitinase domain; Hce2, homolog of Cladopsorium fulvum Ecp2 effector domain.
Fig 2.
Gene and protein expression assays.
A. RT-PCR analysis of the expression of 12 putative LysM protein genes in B. bassiana after growth in different nutrients. CO, conidia harvested from PDA for ten days; PDA, mycelium sample harvested from the PDA medium for three days; BL, blastospores harvested from the SDB culture for seven days; SDB, mycelia harvested from the SDB for three days; HB, hyphal bodies harvested from insect hemolymph 36 hrs post injection of fungal spores. Tub, β-tubulin gene used as a reference. B. RT-PCR verification of gene deletion and genetic engineering for overexpression. WT and mutants were grown in SDB for three days and cultures were harvested for RNA extraction and gene expression assay. C. Western blotting verification of Blys2 secretion feature. BG, Blys2-GFP, Blys2 fused in frame with GFP; BcG, Blys2-SP-GFP, Blys2 without signal peptide in fusion with GFP protein; nc, non-concentrated; c, concentrated. The anti-GFP antibody was used for analysis.
Table 1.
Comparison of the virulence between the WT and mutants assayed against the wax moth larvae.
Fig 3.
Protein solubility, polysaccharide binding and cell wall protection assays.
A. Polyacrylamide gel analysis of protein solubility. The protein samples were centrifuged at a maximum speed for 5 min and aliquots of each sample (10 μg each) were analyzed on a 12% SDS-PAGE gel. M, protein marker. B. Protein binding assay. After incubation of WT Blys2, truncated forms of Blys2 and Blys5 proteins with various polysaccharides, the supernatant and polysaccharide precipitated samples were run through a 12% SDS-PAGE gel and stained with the Coomassie brilliant blue to examine the protein-binding features. Ch, chitin extracted from fungal walls; Chb, chitin beads; Chs, chitosan; Cel, cellulose. The truncated forms of Blys2 are indicated as, e.g., Blys2D1-2 for the truncated Blys2 only containing the first two LysM domains and Blys2D1-4 for the truncated Blys2 only containing the 1–4 LysM domains etc. C. Cell wall protection from chitinase hydrolysis. The germlings were pre-incubated with either Blys2 or Blys5 for 2 hrs before the treatment with a chitinase cocktail for 2 hrs. The release rate of protoplast was quantified and compared. The WT and WT::Slp1 germlings without the pre-incubation with either protein were treated with chitinase cocktails as controls. ***, P < 0.0001.
Fig 4.
A. Localization of Blys2 on the fungal cell walls of different type cells. Full length Blys2 was fused in frame with a GFP gene and the cassette was controlled by the GpdA gene promoter for transformation of the WT strain of B. bassiana. B. Cytosolic localization of Blys2 without signal peptide (Blys2-SP) in different cells. The truncated Blys2 without signal peptide sequence was fused in frame with a GFP gene and the cassette was controlled by the GpdA gene promoter for transformation of the WT strain of B. bassiana. C. Cytosolic localization of GFP protein. The GFP gene was controlled by the GpdA gene promoter for transformation of the WT strain of B. bassiana. D. Localization of Slp1 on the cells walls of B. bassiana cells. Full length Slp1 of M. oryzae was fused in frame with a GFP gene and the cassette was controlled by the GpdA gene promoter for transformation of the WT strain of B. bassiana. The cells of different mutants were examined and photographed under the 100 × field of a confocal microscope. CO, conidia; HY, hyphae harvested from the PDA plate for three days; BL, blastospore; MY, mycelia harvested from SDB for three days; HB, hyphal body harvested from insect hemocoels. CW, Calcofluor White. Bar, 2 μm.
Fig 5.
Insect hemocyte encapsulation and fungal developments in insect hemocoels on a time scale.
The last instar larvae of wax moth were injected with spores of WT and mutants and bled at different times (labeled on the left) for microscopic examination of insect cellular immune responses and fungal developments. Bar: 10 μm. Arrows in different panels point to fungal cells. For overexpression of Blys2 in the WT strains of B. bassiana, both the GpdA (to obtain the transformant WT::gp-Blys2) and laccase (WT::lp-Blys2) gene promoters were used to control gene transcription.
Fig 6.
Comparison of fungal cell propagation and antifungal gene expression in insects.
A. Comparison of fungal hyphal body (HB) numbers in insect hemocoel 48 hrs post injection. Ten insects were bled for each treatment, and five microscopic fields were observed for each insect. Unpaired t-test was conducted to compare the difference level between WT and mutants. ***, represents the difference level at P < 0.001. B. qRT-PCR analysis of Gal (for gallerimycin) gene expression by insects treated with spores of WT and Blys2-related mutants for 36 hrs. *, P < 0.05. C. qRT-PCR analysis of Gal gene expression by insects treated with spores of WT and Blys5-related mutants for 36 hrs. *, P < 0.05. The Slp1 gene of M. oryzae was used to complement the null mutants of Blys2 (ΔBlys2::Slp1) and Blys5 (ΔBlys5::Slp1), respectively. Overexpress of Blys2 in the WT strain of B. bassiana was performed by using two different constitutive promoters, i.e., the GpdA (to obtain the transformant WT::gp-Blys2) and laccase gene (to obtain the transformant WT::lp-Blys2) promoters.