Skip to main content
Log in

A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi

Current Genetics Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Baubet V, Mouellic HL, Campbell AK, Lucas-Meunier E, Fossier P, Brulet P (2000) Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc Natl Acad Sci USA 97:7260–7265

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Bowman BJ, Draskovic M, Freitag M, Bowman EJ (2009) Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa. Eukaryot Cell 8:1845–1855

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Castillo MB, Celio MR, Andressen C, Gotzos V, Rülicke T, Berger MC, Weber J, Berchtold MW (1995) Production and analysis of transgenic mice with ectopic expression of parvalbumin. Arch Biochem Biophys 317:292–298

    Article  PubMed  CAS  Google Scholar 

  • Demaegd D, Foulquier F, Colinet A-S, Gremillon L, Legrand D, Mariot P, Peiter E, van Schaftingen E, Matthijs G, Morsomme P (2013) A newly characterized Golgi-localized family of proteins is involved in calcium and pH homeostasis in yeast and human cells. Proc Natl Acad Sci USA 110:6859–6864

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Doehlemann G, van der Linde K, Assmann D, Schwammbach D, Hof A, Mohanty A, Jackson D, Kahmann R (2009) Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells. PLoS Pathog 5:e1000290

    Article  PubMed  PubMed Central  Google Scholar 

  • Elmayan T, Tepfer M (1995) Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Transgenic Res 4:388–396

    Article  PubMed  CAS  Google Scholar 

  • Forgey WM, Blanco M, Loegering WQ (1978) Differences in pathological capabilities and host specificity of Colletotrichum graminicola on Zea mays. Plant Dis Rep 62:573–575

    Google Scholar 

  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  PubMed  CAS  Google Scholar 

  • Hoff B, Kück U (2005) Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum. Curr Genet 47:132–138

    Article  PubMed  CAS  Google Scholar 

  • Kopke K, Hoff B, Bloemendal S, Katschorowski A, Kamerewerd J, Kück U (2013) Members of the Penicillium chrysogenum velvet complex play functionally opposing roles in the regulation of penicillin biosynthesis and conidiation. Eukaryot Cell 12:299–310

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Lange M, Müller C, Peiter E (2014) Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses. Anal Biochem 453:58–60

    Article  PubMed  CAS  Google Scholar 

  • Mazur P, Baginsky W (1996) In vitro activity of 1, 3-beta-D-glucan synthase requires the GTP-binding protein Rho1. J Biol Chem 271:14604–14609

    Article  PubMed  CAS  Google Scholar 

  • Michielse CB, Pfannmüller A, Macios M, Rengers P, Dzikowska A, Tudzynski B (2014) The interplay between the GATA transcription factors AreA, the global nitrogen regulator and AreB in Fusarium fujikuroi. Mol Microbiol 91:472–493

    Article  PubMed  CAS  Google Scholar 

  • Peiter E, Montanini B, Gobert A, Pedas P, Husted S, Maathuis FJM, Blaudez D, Chalot M, Sanders D (2007) A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance. Proc Natl Acad Sci USA 104:8532–8537

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Pöggeler S, Masloff S, Hoff B, Mayrhofer S, Kück U (2003) Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi. Curr Genet 43:54–61

    PubMed  Google Scholar 

  • Purschwitz J, Müller S, Fischer R (2009) Mapping the interaction sites of Aspergillus nidulans phytochrome FphA with the global regulator VeA and the White Collar protein LreB. Mol Genet Genomics 281:35–42

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2006) The condensed protocols from molecular cloning: a laboratory manual, 1st edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008) Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 56:505–516

    Article  PubMed  CAS  Google Scholar 

  • Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438

    Article  PubMed  CAS  Google Scholar 

  • Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual, 1st edn. Cold Spring Harbor Laboratory, New York, pp 168–169

    Google Scholar 

  • Werner S, Sugui JA, Steinberg G, Deising HB (2007) A chitin synthase with a myosin-like motor domain is essential for hyphal growth, appressorium differentiation, and pathogenicity of the maize anthracnose fungus Colletotrichum graminicola. Mol Plant Microbe Interact 20:1555–1567

    Article  PubMed  CAS  Google Scholar 

  • Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Edgar Peiter.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 2242 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lange, M., Oliveira-Garcia, E., Deising, H.B. et al. A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi. Curr Genet 60, 343–350 (2014). https://doi.org/10.1007/s00294-014-0429-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-014-0429-y

Keywords

Navigation