The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.

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...

In French and German vineyards, Botrytis cinerea isolates with multiple fungicide resistance phenotypes have been observed with increasing frequencies. Multidrug resistance (MDR) results from mutations that lead to constitutive overexpression of genes encoding drug efflux transporters. In MDR2 and MDR3 strains, overexpression of the major facilitator superfamily transporter gene mfsM2 has been found to result from a rearrangement in the mfsM2 promoter (type A), caused by insertion of a retroelement (RE)-derived sequence. Here, we report the discovery of another, similar RE-induced rearrangement of the mfsM2 promoter (type B) in a subpopulation of French MDR2 isolates. MDR2 isolates harboring either type A or type B mutations in mfsM2 show the same resistance phenotypes and similar levels of mfsM2 overexpression. RE sequences similar to those in mfsM2 were found in low copy numbers in other but not all B. cinerea strains analyzed, including non-MDR2 strains. Population genetic analyses support the hypothesis that the two rearrangement mutations have only occurred once, and are responsible for the appearance and subsequent spread of all known MDR2 and MDR3 strains in French and German wine-growing regions.