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

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.The green fluorescent protein (GFP), originally isolated from the jellyfish Aequorea victoria, has been developed into a widely used reporter system, allowing the observation of a variety of cellular and molecular events in living cells. While the original jellyfish GFP yielded only weak or no fluorescence when expressed in other organisms, several amino acid substitutions in the chromophore region have been discovered which lead to improved fluorescence yields. Most of the commonly used GFP derivatives carry the S65T substitution, which leads to a red-shifted excitation maximum and strongly increased fluorescence (5, 19). A further improvement was the use of synthetic GFP (sGFP)-encoding genes, with a codon usage adapted to the host organisms (5, 18). Probably the most widely used GFP variant is enhanced GFP (eGFP), with the substitutions F64L and S65T in the chromophore region, encoded by a gene with a codon usage optimized for expression in human cells (7,8,18,56). The eGFP-encoding gene has been demonstrated to be well expressed in various eukaryotes, including fungi (30). In basidiomycetes, satisfying fluorescence has been found to require the presence of an intron in the GFP-encoding gene (2, 31).In filamentous fungi, expression of GFP has been first reported for Ustilago maydis (48), Aspergillus nidulans (13) Botrytis cinerea is a necrotrophic fungus that attacks more than 200 host plants, causing great damage to a variety of economically important fruits, vegetables, and ornamental flowers (55). In the last years, significant progress has been made in understanding the molecular mechanisms of infection and in the identification of genes that contribute to the pathogenicity of B. cinerea (52). A few reports describe the use of GFP in B. cinerea. Transformants carrying a pls1-egfp fusion were shown to express GFP fluorescence in conidia during germination and host c...

Transcriptome Profiling of Botrytis cinerea Conidial Germination Reveals Upregulation of Infection-Related Genes during the Prepenetration Stage

“…The cda1-GFP and cutB-GFP promoter reporter strains were constructed by using a plasmid encoding a B. cinerea codon-optimized GFP (Bc-GFP1) and a hygromycin resistance cassette for selection (36). For expression of Bc-gfp1 under the control of either cda1 or cutB promoter, fragments covering 1,313 bp (cda1) and 1,281 bp (cutB) upstream of the start codons were amplified (primers are listed in Table S1 in the supplemental material) and used to replace the constitutive oliC promoter fragment.…”

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6

“…To study the cellular localization of BcBot6 in living hyphae, we transformed the DBcbot6 71.3 with a cassette containing Bcbot6 under the control of a constitutive promoter PoliC and fused to the codonoptimized GFP ( Fig. S1; Leroch et al, 2011). The functionality of the BcBot6-GFP hybrid protein in the resulting strain (…”

Different ROS-Scavenging Properties of Flavonoids Determine Their Abilities to Extend Shelf Life of Tomato

“…The B05.10-derived strain OliCBcGFP constitutively expresses BcGFP1, a synthetic variant of enhanced GFP with codon usage optimized for B. cinerea (Leroch et al, 2011), was kindly provided by M. Leroch and M. Hahn (University of Kaiserslautern) and has been used for in vitro growth assays in liquid medium.…”