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Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein
Michaela Leroch1, Dennis Mernke, Dieter Koppenhoefer
1Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany. mleroch@rhrk.uni-kl.de
Abstract:
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 fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. 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 the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. 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 the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.
Insights
Researchers developed a codon-optimized green fluorescent protein (GFP) gene for the fungus Botrytis cinerea, significantly enhancing its expression and fluorescence for advanced biological imaging and research applications.
Area of Science:
- Molecular Biology
- Mycology
- Biotechnology
Background:
- Green fluorescent protein (GFP) and its variants are crucial tools for live-cell imaging in biology.
- Expression of GFP in the gray mold fungus Botrytis cinerea has been limited due to low fluorescence intensity.
- Botrytis cinerea exhibits unique codon usage bias and lower GC content compared to other fungi, posing challenges for heterologous gene expression.
Purpose of the Study:
- To develop a codon-optimized enhanced GFP (eGFP) gene for improved expression in Botrytis cinerea.
- To enhance fluorescence intensity and utility of reporter genes in this fungal species.
- To create tools for visualizing fungal processes and studying gene expression.
Main Methods:
- Codon optimization of the enhanced GFP (eGFP)-encoding gene for Botrytis cinerea, creating Bcgfp.
- Inclusion of an intron to further enhance gene expression.
- Construction of promoter-Bcgfp fusions for gene expression analysis.
- Development of a codon-optimized mCherry gene for red fluorescence.
Main Results:
- The codon-optimized Bcgfp gene showed significantly higher mRNA levels and increased fluorescence in Botrytis cinerea compared to non-optimized versions.
- Codon optimization was the primary driver of enhanced expression, with a smaller contribution from intron insertion.
- Bcgfp enabled visualization of nuclear localization in germinating spores and host penetration.
- Promoter-Bcgfp fusions facilitated quantitative assessment of toxic compound induction of the atrB gene.
- A codon-optimized mCherry gene also demonstrated bright red fluorescence in Botrytis cinerea.
Conclusions:
- Codon optimization is an effective strategy to overcome expression barriers for reporter genes like GFP in Botrytis cinerea.
- The developed Bcgfp provides a valuable tool for live-cell imaging, gene expression studies, and functional analysis in Botrytis cinerea.
- The codon-optimized mCherry expands the toolkit for multicolor imaging in this fungus.
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