A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi
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To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.
KeywordsBiFC Bimolecular fluorescence complementation Co-localization Colletotrichum graminicola Plasmid Protein localization
We thank Liane Freitag and Carolin Müller for their excellent technical assistance. Funding for this study was provided by a Grant from the Deutsche Forschungsgemeinschaft (DFG PE1500/2-1) within the Research Unit FOR 666 (project A3) and by the Land Sachsen-Anhalt.
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