Living Colors in the Gray Mold Pathogen Botrytis cinerea: Codon-Optimized Genes Encoding Green Fluorescent Protein and mCherry, Which Exhibit Bright Fluorescence

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungusBotrytis cinerea because of low fluorescence intensity. The codon usage ofB. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of theB. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp ) for improved expression inB. cinerea . Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of theatrB gene encoding an ABC-type drug efflux transporter ofB. cinerea . In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence inB. cinerea .