Fig. 1

C. merolae transformation system using BS and BSD selectable marker. (a) Schematic diagram of the BSD selectable marker and MITmSCARLET expression cassette. To constitutively express BSD as a selectable marker in C. merolae, BSD orf of A. terreus was codon-optimized to the C. merolae nuclear genome and conjugated with the C. merolae CAB promoter and APX 3′-utr. To constitutively express MITmSCARLET (mSCARLET connected to the mitochondrion-targeted sequence of C. merolae EFTu) as a transgene, MITmSCARLET orf was conjugated with the C. merolae FBA promoter and β-tubulin 3′-utr. (b) Schematic diagram of the insertion of the BSD selectable marker and MITmSCARLET expression cassette into an intergenic region between CMD184C and CMD185C by homologous recombination. The first line indicates the introduced linear DNA, the second line indicates the genomic structure of the WT strain, and the third line indicates the expected genomic structure of the transformant (BSD-MITmS). (c) Selection of BS-resistant transformants in a liquid medium. After PEG-mediated transformation, cells were recovered in a drug-free MA2 liquid medium for 2 days in the light. Cells were transferred to the MA2 liquid medium supplemented with a series of BS concentrations and incubated for 16 days in the light in a 24-well plate. WT cells were also cultured as a negative control. (d) Fluorescent micrographs showing mSCARLET fluorescence detected in BS-resistant transformants in the medium supplemented with 1 mg/mL BS. Cells were observed 14 days after inoculation of cells into the medium. A schematic diagram of a C. merolae cell is also shown. The cell has a single disk-shaped mitochondrion and a single cup-shaped chloroplast. PC, phase-contrast; orange, fluorescence of mSCARLET; red, autofluorescence of the chloroplast. Bar, 5 μm