Figure 1.
Phylogenetic relationship of MEST1 with its homologs.
The amino acid sequences were aligned with Clustal X and a neighbor-joining tree was generated with 1,000 boot strap replicates using the program MEGA v4.0.
Figure 2.
Disruption of the Mest1 gene in Metarhizium robertsii Mr2575.
(A) Schematic representation of the Mest1 wild type (WT) locus and the plasmid pBarGFP-Mest1 (containing two regions homologous to the Mest1 reading frame), that was used for gene disruption through double crossover recombination. Replacement- and WT-specific primer combinations and expected fragments are shown as grey lines. (B) Replacement-specific PCR analysis. Confirmation of the predicted gene targeting conducted with primer combinations that only amplify a signal in the recombinant locus (mu). The absence of a WT-specific signal in the clonal disruptants ΔMest1 (D1, D2) and plasmid pBarGFP-Mest1 (P) confirms the genetic homogeneity of the mutant isolates. (C) MEST1 expression in M. acridum Ma324 was verified by RT-PCR using cDNA from wild type Mr2575, Ma324, mutant (ΔMest1) and transgenic strain Ma324-Mest1. (D) MEST1 expression in M. acridum Ma324-Mest1, E. coli (pYES2-Mest1), and yeast cells (pET28a-Mest1) was confirmed by Western blot using a 6-Histidine Epitope Tag Antibody.
Figure 3.
Pathogenicity of Metarhizium robertsii strains.
(A) Survival of Manduca sexta larvae following topical application with suspensions of 107 conidia/ml of Mr2575, mutant ΔMest1, or overexpression strain Mr2575-gpd::Mest1 under control of the constitutive gpd promoter. (B) Survival of Galleria mellonella larvae following topical application with suspensions of 107 conidia/ml of Mr2575, mutant ΔMest1, or Mr2575-gpd::Mest1. (C) Survival of grasshopper Melanoplus femurrubrum following application of 3 µl of 108 conidia/ml of Mr2575, mutant ΔMest1, Mr2575-Mest1 under native promoter control, or Mr2575-gpd::Mest1 per grasshopper pronotum. Control insects were treated with 0.01% Tween-80. Error bars indicate standard errors.
Table 1.
List of Metarhizium strains used in this study.
Figure 4.
MEST1 regulates appressorial differentiation.
Growth of (A) wild type Metarhizium robertsii Mr2575, (B) mutant ΔMest1, (C) over-expressing strain Mr2575-gpd::Mest1, (D) transgenic strain Ma324-Mest1, and (E) wild type Metarhizium acridum Ma324 in YE+Manduca sexta cuticular lipids. Appressoria formed by transgenic strain Ma324-Mest1 on (F) Galleria mellonella and (G) M. sexta cuticles compared to differentiation of wild type Ma324 on (H) G. mellonella and (I) M. sexta cuticles 24 h post inoculation. CO, conidia; AP, appressorium. Bar, 10 µm. The fungal hyphae and infection structure were stained by Lactophenol Cotton Blue stain.
Table 2.
Effects of Manduca sexta cuticular lipids on conidial germination and appressorial differentiation of Metarhizium robertsii strains.
Table 3.
Effects of Manduca sexta cuticular lipids on conidial germination and appressorial differentiation of Metarhizium acridum strains.
Figure 5.
Pathogenicity of Metarhizium acridum strains.
(A) Survival of Manduca sexta or (B) Galleria mellonella larvae following topical application with suspensions of 107 Ma324 or Ma324-Mest1 conidia/ml. (C) Survival of Melanoplus femurrubrum following application of 3 µl of 108 conidia/ml of Ma324 or Ma324-Mest1 per grasshopper pronotum. Control insects were treated with 0.01% Tween-80. Error bars indicate standard errors.
Figure 6.
Mest1 expression in wild type Mr2575, as measured by quantitative real-time RT-PCR.
(A) Analysis of Mest1 expression by wild type Mr2575 grown for 6 h in Sabouraud dextrose broth (SDB), cell-free hemolymph (HE), basal medium, 1% grasshopper cuticle (Gcut), 1% Manduca sexta cuticle (Mcut), 0.1% bean root exudates (BRE), 0.01% yeast extract medium (YEM) and water. Quantitative RT-PCR time course analyses of Mest1 expression by wild type M. robertsii Mr2575 incubated in (B) water for up to 8 h, or (C) 1% (w/v) ground M. sexta cuticle for up to 12 h. (D) Effect of sugars on Mest1 expression by Mr2575. Quantitative RT-PCR analysis of Mest1 expression by wild type Mr2575 grown in SDB or basal medium (BM) complemented with 1% glucose (Glu), 1% galactose (Gal), 1% sorbose (Sor), 1% trehalose (Tre), 1% sucrose (Suc) or 1% alanine (Ala). Culture conditions and RNA extraction were as described previously [38]. gfp and tef were used as reference genes.
Figure 7.
Intracellular localization of MEST1 in Metarhizium robertsii Mr2575 and budding yeast Saccharomyces cerevisiae.
Co-localization of MEST1 and neutral lipids demonstrated by Nile red staining of GFP-MEST1 expressing cells in (A) an ungerminated M. anisopliae conidium, (B) a germinated M. anisopliae conidium and (C) a budding yeast (S. cerevisiae) cell. BF, bright field microscopy. GFP, fluorescence filter. NR, Nile red staining.
Figure 8.
Determination of lipid content.
(A) Lipid content in conidia freshly harvested from PDA plates (black bars), nutrient-stressed conidia soaked in water for 36 h (grey bars) and conidia germinated in 1% alanine for 12 h (white bars). (B) Glycerol content in the six strains grown for 48 h in BM plus 0.25 mg/ml Manduca cuticle lipids.
Figure 9.
Esterase activity of MEST1 is highest in the presence of short-chain esters (C4–C8).
Substrate specificity of MEST1 was determined by hydrolysis of p-nitrophenyl esters with different carbon chain lengths. The activity values are reported as means ± standard errors from three independent assays using crude cell extracts of E. coli Rosetta (DE3) expressing MEST1 as compared to extracts from E. coli transformed with the corresponding empty vector.