Skip to main content

Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes

Abstract

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5′ fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

References

  • Arvidsson S, Kwasniewski M, Riaño-Pachón DM, Mueller-Roeber B (2008) QuantPrime -a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinform 9:465

    Article  CAS  Google Scholar 

  • Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2(4):229–236

    CAS  PubMed  Article  Google Scholar 

  • Bantscheff M, Lemeer S, Savitski MM, Kuster B (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404(4):939–965

    CAS  PubMed  Article  Google Scholar 

  • Baumgartner K, Fujiyoshi P, Foster GD, Bailey AM (2010) Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea. Appl Environ Microbiol 76(24):7990–7996

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Bensimon A, Heck AJ, Aebersold R (2012) Mass spectrometry-based proteomics and network biology. Annu Rev Biochem 81:379–405

    CAS  PubMed  Article  Google Scholar 

  • Bialecka-Fornal M, Makushok T, Rafelski SM (2016) A review of fluorescent proteins for use in yeast. Methods Mol Biol 1369:309–346

    CAS  PubMed  Article  Google Scholar 

  • Bicknell AA, Ricci EP (2017) When mRNA translation meets decay. Biochem Soc Trans 45(2):339–351

    CAS  PubMed  Article  Google Scholar 

  • Bielska E, Higuchi Y, Schuster M, Steinberg N, Kilaru S, Talbot NJ, Steinberg G (2014) Long-distance endosome trafficking drives fungal effector production during plant infection. Nat Commun 5:5097

    CAS  PubMed  Article  Google Scholar 

  • Boisnard-Lorig C, Colon-Carmona A, Bauch M, Hodge S, Doerner P, Bancharel E, Dumas C, Haseloff J, Berger F (2001) Dynamic analyses of the expression of the HISTONE:YFP fusion protein in arabidopsis show that syncytial endosperm is divided in mitotic domains. Plant Cell 13(3):495–509

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Bondy SC (1971) The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain. Biochem J 123(3):465–469

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Bourdon V, Harvey A, Lonsdale DM (2001) Introns and their positions affect the translational activity of mRNA in plant cells. EMBO Rep 2(5):394–398

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Brenna A, Montanini B, Muggiano E, Proietto M, Filetici P, Ottonello S, Ballario P (2014) Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation. AMB Express 4:43

    PubMed  PubMed Central  Article  Google Scholar 

  • Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD (1988) Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 85(3):836–840

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  • Burns C, Gregory KE, Kirby M, Cheung MK, Riquelme M, Elliott TJ, Challen MP, Bailey A, Foster GD (2005) Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genet Biol 42(3):191–199

    CAS  PubMed  Article  Google Scholar 

  • Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1(10):1183–1200

    CAS  PubMed  Article  Google Scholar 

  • Campoy S, Pérez F, Martín JF, Gutiérrez S, Liras P (2003) Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer. Curr Genet 43(6):447–452

    CAS  PubMed  Article  Google Scholar 

  • Chen X, Stone M, Schlagnhaufer C, Romaine CP (2000) A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66(10):4510–4513

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6(3):325–330

    CAS  PubMed  Article  Google Scholar 

  • Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90(3):1103–1163

    CAS  PubMed  Article  Google Scholar 

  • Cormack B (1998) Green fluorescent protein as a reporter of transcription and protein localization in fungi. Curr Opin Microbiol 1(4):406–410

    CAS  PubMed  Article  Google Scholar 

  • Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ (1997) Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology 143(Pt 2):303–311

    CAS  PubMed  Article  Google Scholar 

  • Curi GC, Chan RL, Gonzalez DH (2005) The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency. J Exp Bot 56(419):2563–2571

    CAS  PubMed  Article  Google Scholar 

  • Dagdas YF, Yoshino K, Dagdas G, Ryder LS, Bielska E, Steinberg G, Talbot NJ (2012) Septin-mediated plant cell invasion by the rice blast fungus Magnaporthe oryzae. Science 336(6088):1590–1595

    CAS  PubMed  Article  Google Scholar 

  • Dahan O, Gingold H, Pilpel Y (2011) Regulatory mechanisms and networks couple the different phases of gene expression. Trends Genet 27(8):316–322

    CAS  PubMed  Article  Google Scholar 

  • Dalla Venezia N, Vincent A, Marcel V, Catez F, Diaz JJ (2019) Emerging role of eukaryote ribosomes in translational control. Int J Mol Sci 20(5):E1226

    PubMed  Article  CAS  Google Scholar 

  • del Poeta M, Toffaletti DL, Rude TH, Sparks SD, Heitman J, Perfect JR (1999) Cryptococcus neoformans differential gene expression detected in vitro and in vivo with green fluorescent protein. Infect Immun 67(4):1812–1920

    PubMed  PubMed Central  Article  Google Scholar 

  • Di Battista C, Selosse MA, Bouchard D, Stenström E, Le Tacon F (1996) Variation in symbiotic efficiency, phenotypic characters and ploidy level among different isolates of the ectomycorrhizal basidiomycete Laccaria bicolor strain S238. Mycol Res 100:1315–1324

    Article  Google Scholar 

  • Ding DQ, Matsuda A, Okamasa K, Nagahama Y, Haraguchi T, Hiraoka Y (2016) Meiotic cohesin-based chromosome structure is essential for homologous chromosome pairing in Schizosaccharomyces pombe. Chromosoma 125(2):205–214

    CAS  PubMed  Article  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(2):e1000290

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Dvir S, Velten L, Sharon E, Zeevi D, Carey LB, Weinberger A, Segal E (2013) Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast. Proc Natl Acad Sci 110(30):E2792–E2801

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  • Ferretti MB, Ghalei H, Ward EA, Potts EL, Karbstein K (2017) Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements. Nat Struct Mol Biol 24(9):700–707

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Ford KL, Baumgartner K, Henricot B, Bailey AM, Foster GD (2016) A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea. Sci Rep 6:29226

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Fraser ST, Hadjantonakis AK, Sahr KE, Willey S, Kelly OG, Jones EA, Dickinson ME, Baron MH (2005) Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice. Genesis 42(3):162–171

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Furger A, O'Sullivan JM, Binnie A, Lee BA, Proudfoot NJ (2002) Promoter proximal splice sites enhance transcription. Genes Dev 16(21):2792–2799

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Gallegos JE, Rose AB (2015) The enduring mystery of intron-mediated enhancement. Plant Sci 237:8–15

    CAS  PubMed  Article  Google Scholar 

  • Gallegos JE, Rose AB (2017) Intron DNA sequences can be more important than the proximal promoter in determining the site of transcript initiation. Plant Cell 29(4):843–853

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Godio RP, Fouces R, Gudiña EJ, Martín JF (2004) Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium. Curr Genet 46(5):287–294

    CAS  PubMed  Article  Google Scholar 

  • Gong X, Hurtado O, Wang B, Wu C, Yi M, Giraldo M, Valent B, Goodin M, Farman M (2015) pFPL vectors for high-throughput protein localization in fungi: detecting cytoplasmic accumulation of putative effector proteins. Mol Plant Microbe Interact 28(2):107–121

    PubMed  Article  CAS  Google Scholar 

  • Grzegorski SJ, Chiari EF, Robbins A, Kish PE, Kahana A (2014) Natural variability of Kozak sequences correlates with function in a zebrafish model. PLoS ONE 9(9):e108475

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Hadjantonakis AK, Papaioannou VE (2004) Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice. BMC Biotechnol 4:33

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Hamilton R, Watanabe CK, De Boer HA (1987) Compilation and comparison of the sequence context around the AUG start codons in Saccharomyces cerevisiae mRNAs. Nucleic Acids Res 15:3581–3593

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Haruyama N, Cho A, Kulkarni AB (2009) Overview: engineering transgenic constructs and mice. Curr Protoc Cell Biol 42(1):19.10.1–19.10.9

    Google Scholar 

  • Hinnebusch AG (2017) Structural insights into the mechanism of scanning and start codon recognition in eukaryotic translation initiation. Trends Biochem Sci 42(8):589–611

    CAS  PubMed  Article  Google Scholar 

  • Huang MT, Gorman CM (1990) Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleic Acids Res 18(4):937–947

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Huch S, Nissan T (2014) Interrelations between translation and general mRNA degradation in yeast. Wiley Interdiscip Rev RNA 5(6):747–763

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Jackson RJ, Hellen CU, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11:113–127

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Jiang D, Berger F (2017) Histone variants in plant transcriptional regulation. Biochim Biophys Acta Gene Regul Mech 1860(1):123–130

    CAS  PubMed  Article  Google Scholar 

  • Jiang L, Huang C, Sun Q, Guo H, Cheng T, Peng Z, Dang Y, Liu W, Xu G, Xia Q (2015) The 5'-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms. Insect Biochem Mol Biol 63:1–6

    CAS  PubMed  Article  Google Scholar 

  • Kanda T, Sullivan KF, Wahl GM (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 8(7):377–385

    CAS  PubMed  Article  Google Scholar 

  • Kemppainen M, Pardo A (2010) pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA—triggering in the mycorrhizal fungus Laccaria bicolor. Microb Biotechnol 3:178–200

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Kemppainen M, Circosta A, Tagu D, Martin F, Pardo AG (2005) Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N. Mycorrhiza 16(1):19–22

    CAS  PubMed  Article  Google Scholar 

  • Kemppainen M, Duplessis S, Martin F, Pardo AG (2008) T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor. Microb Biotechnol 1:258–269

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Kemppainen M, Duplessis S, Martin F, Pardo AG (2009) RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus. Environ Microbiol 11:1878–1896

    CAS  PubMed  Article  Google Scholar 

  • Khang CH, Berruyer R, Giraldo MC, Kankanala P, Park SY, Czymmek K, Kang S, Valent B (2010) Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement. Plant Cell 22(4):1388–1403

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Kilaru S, Schuster M, Studholme D, Soanes D, Lin C, Talbot NJ, Steinberg G (2015) A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici. Fungal Genet Biol 79:125–131

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Kozak M (1984) Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature 308(5956):241–246

    CAS  PubMed  Article  Google Scholar 

  • Kozak M (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196:947–950

    CAS  PubMed  Article  Google Scholar 

  • Kozak M (2002) Pushing the limits of the scanning mechanism for initiation of translation. Gene 299(1–2):1–34

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Lakshman D, Pandey R, Kamo K, Bauchan G, Mitra A (2012) Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus. Eur J Plant Pathol 133(3):729–738

    Article  Google Scholar 

  • Lange M, Oliveira-Garcia E, Deising HB, Peiter E (2014) A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi. Curr Genet 60(4):343–350

    CAS  PubMed  Article  Google Scholar 

  • Lanver D, Tollot M, Schweizer G, Lo Presti L, Reissmann S, Ma LS, Schuster M, Tanaka S, Liang L, Ludwig N, Kahmann R (2017) Ustilago maydis effectors and their impact on virulence. Nat Rev Microbiol 15(7):409–421

    CAS  PubMed  Article  Google Scholar 

  • Laxa M (2017) Intron-mediated enhancement: a tool for heterologous gene expression in plants? Front Plant Sci 7:1977

    PubMed  PubMed Central  Article  Google Scholar 

  • Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9:963–967

    CAS  PubMed  Article  Google Scholar 

  • Le Hir H, Gatfield D, Izaurralde E, Moore MJ (2001) The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J 20(17):4987–4997

    PubMed  PubMed Central  Article  Google Scholar 

  • Le Hir H, Nott A, Moore MJ (2003) How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci 28(4):215–220

    PubMed  Article  CAS  Google Scholar 

  • Leroch M, Mernke D, Koppenhoefer D, Schneider P, Mosbach A, Doehlemann G, Hahn M (2011) Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence. Appl Environ Microbiol 77(9):2887–2897

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Li J, Liang Q, Song W, Marchisio MA (2017) Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae. J Biol Eng 11:25

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408

    CAS  PubMed  Google Scholar 

  • Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, Rollins JA, Wolpert TJ, Johnson KB, Rodriguez RJ, Dickman MB, Ciuffetti LM (2001) Green fluorescent protein is lighting up fungal biology. Appl Environ Microbiol 67(5):1987–1994

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Lu S, Cullen BR (2003) Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA 9(5):618–630

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Luehrsen KR, Walbot V (1991) Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol Gen Genet 225(1):81–93

    CAS  PubMed  Article  Google Scholar 

  • Luger K (2006) Dynamic nucleosomes. Chromosome Res 14(1):5–16

    CAS  PubMed  Article  Google Scholar 

  • Lugones LG, Scholtmeijer K, Klootwijk R, Wessels JG (1999) Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol Microbiol 32(4):681–689

    CAS  PubMed  Article  Google Scholar 

  • Ma B, Mayfield MB, Gold MH (2001) The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium. Appl Environ Microbiol 67:948–955

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Maniatis T, Reed R (2002) An extensive network of coupling among gene expression machines. Nature 416(6880):499–506

    CAS  PubMed  Article  Google Scholar 

  • Martin F, Aerts A, Ahrén D, Brun A, Danchin EG, Duchaussoy F, Gibon J, Kohler A et al (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452(7183):88–92

    CAS  PubMed  Article  Google Scholar 

  • Martin F, Kohler A, Murat C, Veneault-Fourrey C, Hibbett DS (2016) Unearthing the roots of ectomycorrhizal symbioses. Nat Rev Microbiol 14(12):760–773

    CAS  PubMed  Article  Google Scholar 

  • Maruyama J, Nakajima H, Kitamoto K (2001) Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS. Biosci Biotechnol Biochem 65(7):1504–1510

    CAS  PubMed  Article  Google Scholar 

  • Moabbi AM, Agarwal N, El Kaderi B, Ansari A (2012) Role for gene looping in intron-mediated enhancement of transcription. Proc Natl Acad Sci USA 109(22):8505–8510

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  • Molden RC, Bhanu NV, LeRoy G, Arnaudo AM, Garcia BA (2015) Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications. Epigenet Chromatin 8:15

    Article  CAS  Google Scholar 

  • Müller T, Benjdia M, Avolio M, Voigt B, Menzel D, Pardo A, Frommer WB, Wipf D (2006) Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum. Mycorrhiza 16(6):437–442

    PubMed  Article  CAS  Google Scholar 

  • Murata H, Sunagawa M, Yamazaki T, Shishido K, Igasaki T (2006) Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation. Mycorrhiza 16(6):407–412

    CAS  PubMed  Article  Google Scholar 

  • Nakagawa S, Niimura Y, Gojobori T, Tanaka H, Miura K (2008) Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes. Nucleic Acids Res 36(3):861–871

    CAS  PubMed  Article  Google Scholar 

  • Nishikawa R, Yoshida M, Noda T, Okuhara T, Taguchi G, Inatomi S, Shimosaka M (2016) pFungiway: a series of plasmid vectors used for gene manipulation in fungi. Ann Microbiol 66(2):825–832

    CAS  Article  Google Scholar 

  • Okkema PG, Harrison SW, Plunger V, Aryana A, Fire A (1993) Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics 135(2):385–404

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Plett J, Kemppainen MJ, Kale S, Kohler A, Legue V, Brun A, Tyler BM, Pardo AG, Martin F (2011) A secreted effector protein of Laccaria bicolor is required for symbiosis development. Curr Biol 21:1197–1203

    CAS  PubMed  Article  Google Scholar 

  • Plett JM, Daguerre Y, Wittulsky S, Vayssières A, Deveau A, Melton SJ, Kohler A, Morrell-Falvey JL, Brun A, Veneault-Fourrey C, Martin F (2014) Effector MiSSP7 of the mutualistic fungus Laccaria bicolor stabilizes the Populus JAZ6 protein and represses jasmonic acid (JA) responsive genes. Proc Natl Acad Sci USA 111(22):8299–8304

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  • Plett JM, Tisserant E, Brun A, Morin E, Grigoriev IV, Kuo A, Martin F, Kohler A (2015) The mutualist Laccaria bicolor expresses a core gene regulon during the colonization of diverse host plants and a variable regulon to counteract host-specific defenses. Mol Plant Microbe Interact 28(3):261–273

    CAS  PubMed  Article  Google Scholar 

  • Plett JM, Yin H, Mewalal R, Hu R, Li T, Ranjan P, Jawdy S, De Paoli HC, Butler G, Burch-Smith TM, Guo HB, Ju Chen C, Kohler A, Anderson IC, Labbé JL, Martin F, Tuskan GA, Yang X (2017) Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis. Sci Rep 7(1):382

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111(2):229–233

    CAS  PubMed  Article  Google Scholar 

  • Radhakrishnan A, Green R (2016) Connections underlying translation and mRNA stability. J Mol Biol 428(18):3558–3564

    CAS  PubMed  Article  Google Scholar 

  • Rech C, Engh I, Kück U (2007) Detection of hyphal fusion in filamentous fungi using differently fluorescence-labeled histones. Curr Genet 52(5–6):259–266

    CAS  PubMed  Article  Google Scholar 

  • Rekangalt D, Verner MC, Kües U, Walser PJ, Marmeisse R, Debaud JC, Fraissinet-Tachet L (2007) Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum. FEMS Microbiol Lett 268(1):67–72

    CAS  PubMed  Article  Google Scholar 

  • Rodríguez-Tovar AV, Ruiz-Medrano R, Herrera-Martínez A, Barrera-Figueroa BE, Hidalgo-Lara ME, Reyes-Márquez BE, Cabrera-Ponce JL, Valdés M, Xoconostle-Cázares B (2005) Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius. J Microbiol Methods 63(1):45–54

    PubMed  Article  CAS  Google Scholar 

  • Rosa S, Ntoukakis V, Ohmido N, Pendle A, Abranches R, Shaw P (2014) Cell differentiation and development in Arabidopsis are associated with changes in histone dynamics at the single-cell level. Plant Cell 26(12):4821–4833

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Rose AB, Beliakoff JA (2000) Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing. Plant Physiol 122(2):535–542

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Samadder P, Sivamani E, Lu J, Li X, Qu R (2008) Transcriptional and post-transcriptional enhancement of gene expression by the 5' UTR intron of rice rubi3 gene in transgenic rice cells. Mol Genet Genomics 279(4):429–439

    CAS  PubMed  Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    CAS  Article  PubMed  Google Scholar 

  • Schoberle TJ, Nguyen-Coleman CK, May GS (2013) Plasmids for increased efficiency of vector construction and genetic engineering in filamentous fungi. Fungal Genet Biol 58–59:1–9

    PubMed  Article  CAS  Google Scholar 

  • Scholtmeijer K, Wösten HA, Springer J, Wessels JG (2001) Effect of introns and AT-rich sequences on expression of the bacterial hygromycin B resistance gene in the basidiomycete Schizophyllum commune. Appl Environ Microbiol 67(1):481–483

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342

    PubMed  Article  CAS  Google Scholar 

  • Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572

    CAS  PubMed  Article  Google Scholar 

  • Shipman EN, Jones K, Jenkinson CB, Kim DW, Zhu J, Khang CH (2017) Nuclear and structural dynamics during the establishment of a specialized effector-secreting cell by Magnaporthe oryzae in living rice cells. BMC Cell Biol 18(1):11

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Smith S, Read DJ (2008) Mycorrhizal symbiosis. Academic Press, London

    Google Scholar 

  • Spellig T, Bottin A, Kahmann R (1996) Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis. Mol Gen Genet 252(5):503–509

    CAS  PubMed  Google Scholar 

  • Sudbery P (2011) Fluorescent proteins illuminate the structure and function of the hyphal tip apparatus. Fungal Genet Biol 48(9):849–857

    CAS  PubMed  Article  Google Scholar 

  • Toews MW, Warmbold J, Konzack S, Rischitor P, Veith D, Vienken K, Vinuesa C, Wei H, Fischer R (2004) Establishment of mRFP1 as a fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in vitro (GATEWAY). Curr Genet 45(6):383–389

    CAS  PubMed  Article  Google Scholar 

  • Vanden Wymelenberg AJ, Cullen D, Spear RN, Schoenike B, Andrews JH (1997) Expression of green fluorescent protein in Aureobasidium pullulans and quantification of the fungus on leaf surfaces. Biotechniques 23(4):686–690

    CAS  PubMed  Article  Google Scholar 

  • Vayssières A, Pěnčík A, Felten J, Kohler A, Ljung K, Martin F, Legué V (2015) Development of the Poplar-Laccaria bicolor Ectomycorrhiza modifies root auxin metabolism, signaling, and response. Plant Physiol 169(1):890–902

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Walter D, Matter A, Fahrenkrog B (2010) Bre1p-mediated histone H2B ubiquitylation regulates apoptosis in Saccharomyces cerevisiae. J Cell Sci 123(Pt 11):1931–1939

    CAS  PubMed  Article  Google Scholar 

  • Wiegand HL, Lu S, Cullen BR (2003) Exon junction complexes mediate the enhancing effect of splicing on mRNA expression. Proc Natl Acad Sci USA 100(20):11327–11332

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  • Woudt LP, Pastink A, Kempers-Veenstra AE, Jansen AE, Mager WH, Planta RJ (1983) The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences. Nucleic Acids Res 11(16):5347–5360

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Yun CS, Nishida H (2011) Distribution of introns in fungal histone genes. PLoS ONE 6(1):e16548

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

Funding to M.K. and A.P. was provided by grants from National University of Quilmes, National Council of Scientific and Technical Research (CONICET), and National Agency for Scientific and Technological Promotion (ANPCyT), Argentina. J.C. was funded by the Kempe foundations grant (SMK15-33) and The Swedish Research Council FORMAS (942-2015-539). We thank L. Nilsson for the help with media preparations during experiments. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers MN781138, MN781139, MN781140 and MN781141.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Minna Kemppainen.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by M. Kupiec.

Electronic supplementary material

Below is the link to the electronic supplementary material.

294_2020_1060_MOESM1_ESM.pdf

Supplementary file1 Online Resource file 1 (Table S1) Primers used in this study for constructing the cloning vectors presented and the GOI-FP fusion constructs used in Laccaria ATMT. (Table S2) Primers used in quantitative RT-PCR analyses. HK: housekeeping gene used for normalization (PDF 303 kb)

294_2020_1060_MOESM2_ESM.pdf

Supplementary file2 Online Resource file 2 pN-GFP, pN-mCherry, pCEBN-GFP and pCEBN-mCherry cloning vector sequences (between M13F and M13R primer binding sites) (PDF 514 kb)

294_2020_1060_MOESM3_ESM.pdf

Supplementary file3 Online Resource file 3 Laccaria and Ustilago codon usage. Nucleotide and protein sequence analyses involving Laccaria histone H2B isoforms. Laccaria consensus Kozak sequence analysis (PDF 1182 kb)

294_2020_1060_MOESM4_ESM.xlsx

Supplementary file4 Online Resource file 4 Excel file containing information of Laccaria gene models used for establishing the consensus Kozak sequence (XLSX 21 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kemppainen, M., Chowdhury, J., Lundberg-Felten, J. et al. Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes. Curr Genet 66, 791–811 (2020). https://doi.org/10.1007/s00294-020-01060-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-020-01060-4

Keywords

  • Laccaria bicolor
  • Fluorescent proteins
  • Histone H2B
  • Ectomycorrhiza