To determine whether Msp1 could damage fungal cells, we germinated the spores of M. robertsii (5 × 106 conidia/ml) in LB with the addition of Msp1 at a final concentration of 10 µg/ml in a Petri dish (60 mm in diameter). The mock group was included without the addition of Msp1. Each group had three replicates. After 12 h, the supernatants were carefully removed with a pipette, and fungal cells were fixed in 4% formaldehyde for 12 h. The samples were then dehydrated for observation using the Field-Emission Scanning Electron Microscope (Merlin Compact VP, Zeiss) [42 (link)].
We also performed ninhydrin staining assays after treating Metarhizium spores with Msp1 [43 (link)]. The supernatants collected above were centrifuged at 12,000 rpm for 30 min, transferred, and added with PBS containing 2% (w/v) ninhydrin. A reference control group was included by only containing Msp1 protein (10 µg/ml). The samples were boiled in a water bath for 15 min, and immediately cooled on ice. Sample absorbance was measured at a wavelength of 570 nm with a spectrophotometer (GENESYS 50™, Thermo Fisher Scientific) [43 (link)].
We also performed ninhydrin staining assays after treating Metarhizium spores with Msp1 [43 (link)]. The supernatants collected above were centrifuged at 12,000 rpm for 30 min, transferred, and added with PBS containing 2% (w/v) ninhydrin. A reference control group was included by only containing Msp1 protein (10 µg/ml). The samples were boiled in a water bath for 15 min, and immediately cooled on ice. Sample absorbance was measured at a wavelength of 570 nm with a spectrophotometer (GENESYS 50™, Thermo Fisher Scientific) [43 (link)].
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