TECHNICAL NOTE

Versatile EGFP reporter plasmids for cellular localizationof recombinant gene products in filamentous fungi

Received: 18 October 2002 / Revised: 19 December 2002 /Accepted: 19 December 2002 / Published online: 31 January 2003� Springer-Verlag 2003

Abstract The recent development of variants of thegreen fluorescent protein (GFP) with altered codoncomposition facilitated the efficient expression of thisreporter protein in a number of fungal species. In thisreport, we describe the construction and application of aseries of plasmids, which support the expression of anenhanced gfp (egfp) gene in filamentous fungi and assistthe study of diverse developmental processes. Includedwere a promoterless egfp vector for monitoring theexpression of cloned promoters/enhancers in fungal cellsand vectors for creating translation fusions to theN-terminus of EGFP. The vectors were further modifiedby introducing a variant hygromycin B phosphotrans-ferase (hph) gene, lacking the commonly found NcoI site.Instead, this site, which contained an ATG start codon,was placed in front of the egfp gene and thus was madesuitable for the cloning of translational fusions. Theapplicability of these vectors is demonstrated by ana-lyzing transcription regulation and protein localizationand secretion in two ascomycetes, Acremonium chrys-ogenum and Sordaria macrospora. In the latter, theheterologous egfp gene is stably inherited during meioticdivisions, as can easily be seen from fluorescentascospores.

Keywords Filamentous fungi Æ GFP Æ Proteinlocalization Æ Protein secretion

Introduction

The green fluorescent protein (GFP) is a spontaneouslyfluorescent polypeptide of 27 kDa, derived from the

jellyfish Aequorea victoria, which absorbs UV or bluelight and emits in the green region of the spectrum.Unlike the bacterial b-galactosidase and b-glucuroni-dase, which are widely used reporters in fungi, GFPdoes not rely on exogenous substrates or cofactorsother than oxygen (Prasher et al. 1992). Therefore,GFP can be used as a fusion tag in vivo to localizeproteins, to follow their movement, or to study thedynamics of the subcellular compartments to whichthese proteins are targeted (Chalfie et al. 1994; Prasher1995). Since wild-type GFP performs inefficiently indifferent cellular contexts, efforts were focused on theimprovement of GFP expression and/or fluorescencelevels. Enhanced GFP (EGFP) includes chromophoremutations that increase fluorescence intensity and op-timize codon usage for yeasts (yEGFP), plants (SGFP),the green alga Chlamydomonas reinhardtii (cgfp) andmammals (EGFP1; Chiu et al. 1996; Haas et al. 1996;Yang et al. 1996; Cormack et al. 1997; Fuhrmann et al.1999). Both SGFP and EGFP1 have been successfullysynthesized to high levels in a number of different fil-amentous fungi and are widely used to monitor thelocalization of tagged proteins in fungal cells (for areview, see Lorang et al. 2001). The selection of a gfpgene variant depends on codon preferences of thefungus to be transformed. Here, we report on a seriesof improved reporter gene vectors successfully used forstable expression of the egfp gene in Sordaria macro-spora and Acremonium chrysogenum. The egfp genecontains a serine-to-threonine substitution at aminoacid 65 (S65T), conferring a red-shifted excitationmaximum of 488 nm and an emission maximum of507 nm (Cormack et al. 1996). In addition, it harbors190 silent base mutations to be human codon-opti-mized for high expression levels in mammalian systems(Yang et al. 1996).

In this study, we demonstrate the successful expres-sion of recombinant egfp constructs in two filamentousfungi. One is S. macrospora, a model organism for in-vestigating fruiting body development in filamentousfungi (Masloff et al. 1999; Nowrousian et al. 1999).

Curr Genet (2003) 43: 54–61DOI 10.1007/s00294-003-0370-y

Stefanie Poggeler Æ Sandra Masloff

Birgit Hoff Æ Severine Mayrhofer Æ Ulrich Kuck

Communicated by S. Hohmann

S. Poggeler Æ S. Masloff Æ B. Hoff Æ S. Mayrhofer Æ U. Kuck (&)Department for General and Molecular Botany,Ruhr-University Bochum,44780 Bochum, GermanyE-mail: Ulrich.Kueck@ruhr-uni-bochum.de

During sexual propagation, this homothallic ascomyceteproduces meiotically derived ascospores, but no coni-diospores. The other organism tested is the cephalo-sporin C-producer, A. chrysogenum, which has beenused for investigations of the regulation of b-lactambiosynthesis (Brakhage 1998). Transformation of bothfungi with vectors carrying different egfp genes results inbrightly fluorescent vegetative hyphae. In S. macrospora,fluorescence is emitted during all stages of sexual de-velopment. In addition, we are able to show thatS. macrospora transformants stably inherit the egfp geneduring meiosis.

Promoterless derivatives of this vector can be appliedfor gene expression analysis. Translational fusions withthe egfp gene can be used either to monitor proteinlocation in living fungal cells or to follow proteinsecretion.

Materials and methods

Strains and culture conditions

Escherichia coli strain XL1-blue was used as host for plasmidamplification (Bullock et al. 1987). S. macrospora strain S17736(which has a wild-type phenotype) and S. macrospora spore colormutant fus (from our laboratory collection) were cultivated inBMM-medium or CM medium (Esser 1982; Nowrousian et al.1999). For experiments with A. chrysogenum, we used strainATCC 14553, which was grown on modified CCM media (Walzand Kuck 1991).

Transformation of S. macrospora and A. chrysogenum

Transformation of S. macrospora was performed according toNowrousian et al. (1999) and transformation of A. chrysogenumaccording to Walz and Kuck (1993). When using plasmid pcpcR1-egfp, for cotransformation experiments with S. macrospora andA. chrysogenum, we used plasmid pANsCOS1 (Osiewacz 1994) andplasmid pMW1 (Kuck et al. 1989), respectively, which carry thehph marker gene, encoding hygromycin B phosphotransferase.

Since transformed S. macrospora protoplasts are heterokary-otic, containing both transformed and untransformed nuclei, wecreated homokaryotic S. macrospora strains by selfing the primarytransformants

Construction of plasmids

For construction of EGFP vectors, we used plasmid pEGFP/gpd/tel (Inglis et al. 1999), which contains the egfp gene, GFPmut(Cormack et al. 1996), excised from pEGFP-N1 (BD Bioscience,France), under control of the Aspergillus nidulans gpd promoterand trpC terminator. Standard molecular techniques were em-ployed, according to Sambrook et al. (1989).

For construction of gfp vectors, we first cloned a 2.2-kb ApaI/XbaI fragment of pEGFP/gpd/tel containing the A. nidulans gpdpromoter and egfp gene into pBluescript II KS+ (Stratagene, UK),resulting in the generation of plasmid p77.15. In a second step, wecloned a 0.7-kb XbaI fragment of pEGFP/gpd/tel containing thetrpC terminator of A. nidulans into the XbaI site of plasmid p77.15,to yield plasmid p80.3. A 2.4-kb SacI fragment of plasmid p80.3was then cloned into vector pBluescript KS+ (Stratagene, UK) togenerate plasmid p82.9. This has a size of 5.3 kb and is thereforemuch easier to handle than plasmid pEGFP/gpd/tel (with a size of6.9 kb). For this reason, the smaller vector p82.9 was used for

further cloning experiments to generate pSM1. Insertion of the1.4-kb EcoRI hph cassette of pCB1003, under control of the A.nidulans trpC promoter (Carroll et al. 1994), into p82.9 resulted inplasmid pSM1 (Fig. 1A). The promoterless EGFP vector pSM2(Fig. 1B) was produced by first ligating a blunted 1.4-kb NcoI/SacIfragment of p82.9 containing the egfp gene and the A. nidulans trpCterminator into the SmaI site of pBluescript KS+ (Stratagene,UK) and then ligating the 1.4-kb SalI hph cassette of pCB1003(Carroll et al. 1994) into the SalI site (Fig. 1B). Plasmid pIG1783, aderivative of pEGFP/gpd/tel (Inglis et al. 1999) contains a modifiedhph gene without a restriction site for NcoI (Fig. 1C). It was con-structed as follows: two overlapping fragments of the hph cassetteof pCB1003 (Carroll et al. 1994) were PCR-amplified using primerpairs Hyg1 (5¢-AGG GCC CGT TAA CTG ATA TTG AAGGAG CAT T-3¢) and Hyg2 (5¢-CCA TCG CCT CCG CGA CCGGCT CGA GAA CAG-3¢), or Hyg3 (5¢-GTT CTC GAG CCGGTC GCG GAG GCG AGG AT-3¢) and Hyg4 (5¢-AGG GCCCGT TAA CGT TAA CTG GTT CCC GGT C-3¢). The mutationleading to the NcoI site deletion is a silent mutation. Codon 118(GCC) for Ala was changed into a GCG codon. Similarly, codon112 (CTG) for Leu was mutated to a CTC codon. The latter changeresulted into a new XhoI restriction site. The two PCR fragmentswere subcloned, sequenced and co-ligated as ApaI/XhoI fragmentsinto the ApaI site of plasmid pEGFP/gpd/tel (Inglis et al. 1999). Toachieve nuclear localization of EGFP, the full-length cpcR1 gene ofAcremonium chrysogenum (Schmitt and Kuck 2000) was transla-tionally fused to the egfp gene. A 2,598-bp DNA fragment wasPCR-amplified from plasmid pKSC1 (Schmitt and Kuck 2000),using primers cpcR1A (5¢-CAC ACC ATG GGA GCA GACGAG CCG GCC G-3¢) and cpcR1B (5¢-CAC ACC ATG GCTGCA GGA GCC GCC CAT TCT CG)3¢) that generate NcoI sitesat the respective 5¢ ends. After subcloning and sequencing, the2,598-bp NcoI fragment was cloned into the NcoI site of plasmidp82.9 to create pcpcR1-egfp, which encodes a CPCR1-EGFP fu-sion under control of the Aspergillus nidulans gpd promoter. Inorder to achieve secretion of EGFP, expression vector pSetp1 wasconstructed, containing a 69-bp secretion signal sequence from theputative extracellular aspartic proteinase gene (etp) from S. mac-rospora (accession number AJ507456). The signal sequence con-sisted of two complementary oligonucleotides: ETP3 (5¢-C ATGGTA GCC CTC ACC AAC CTC CTC CTC ACT ACC GTCCTC GCC TCT GCC GGC CTC GGT TCC GCC CTG CCAGC-3¢) and ETP4 (5¢-C ATG GCT GGC AGG CCG GAA CCGAGG CCG GCA GAG GCG AGG ACG GTA GTG AGG AGGAGG TTG GTG AGG GCT AC-3¢). They were fused in sense in-frame with the egfp gene by insertion into the NcoI restriction siteof vector p82.9. Subsequent cloning of the 1.4-kb EcoRI hph cas-sette of pCB1003 resulted in plasmid pSetp1 with the ETP secretionsignal in sense orientation and pSetp2 in antisense orientation.

Fluorescence, light microscopy and confocal laser microscopy

Fluorescence and light microscopy were performed using a ZeissAxiophot fluorescence microscope and a MC80DX camerawithout further manipulation of the object. Differential interfer-ence contrast optics were used for light microscopy. EGFP wasvisualized with the Zeiss filter set for fluorescein isothiocyanatefluorescence (BP 450–490 excitation filter, 510 nm dichoric filter,LP 520 emission filter). Staining of nuclei was performed by in-cubating the mycelium of S. macrospora and Acremoniumchrysogenum in 5 lM of Sytox orange nucleic acid stain(S-11368, Molecular Probes Europe, Leiden, The Netherlands) in1 M TrisHCl, pH 8.0, for 30 min. After removing the dye, themycelia were fixed for 30 min in 4% para-formaldehyde (Riedelde Haen, Germany). For confocal laser microscopy, S. macros-pora and A. chrysogenum mycelium was cultivated in CM me-dium and CCM medium, respectively. Prior to microscopicinvestigation, mycelia were fixed for 30 min in 4% para-formal-dehyde (Riedel de Haen, Germany). Confocal laser microscopywas carried out on a Zeiss LSM 510 META confocal systemver 3.0 (excitation/emission 488 nm blue, samples at 10% argon

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laser power, Ch2-1, BP505-550 filter, Ch2/72 lm pinhole, ZeissAxiovert 100 M, Plan Apo 63/1.4 oil lens). Fungal nuclei, pre-viously stained with Sytox orange, were observed using filterCh3-2 and a HeNe laser. For double-labeling, images in twochannels were simultaneously collected and then overlaid, usingthe Zeiss LSM5 image browser.

Immunodetection of secreted EGFP

For Western analysis of secreted EGFP, pSetp1 and pSetp2,S. macrospora transformants and the wild-type strain were grownfor 3–4 days in CM medium. Extracellular proteins in 1 ml of theculture medium were precipitated by adding 100 ll of 10% tri-chloracetic acid and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according tostandard procedures (Laemmli 1970). The proteins were transferredto polyvinylidene difluoride Western blotting membranes (Roche,Germany), using a semi-dry blotting system (Biometra, Germany).The detection was carried out with a (1:500) polyclonal anti-GFPliving-colors peptide antibody (BD Bioscience, France) and withthe chemiluminescence Western blotting kit (Roche, Germany), asdescribed by the manufacturers.

Results and discussion

Construction of egfp reporter plasmids

Direct visualization of a synthesized reporter protein infungal cells is a particularly helpful technique for theanalysis of gene regulation, signal transduction andprotein localization. Consequently, we tested the use ofthe jellyfish gene encoding EGFP as a marker to monitorgene expression.

Initially, we placed the wild-type sequence of the gfpgene (Prasher et al. 1992) and the sgfp gene, which wascodon-optimized for higher plants (Chiu et al. 1996),under the control of different strong fungal promoters.The DNA was integrated into the A. chrysogenum andS. macrospora genome, but isolated transformants nevershowed the expected green fluorescence when irradiatedwith blue light (data not shown).

In an attempt to overcome these problems, we usedthe EGFP1 gene, which has a codon-usage optimized forhumans (Yang et al. 1996). This improved version of gfphas been successfully expressed to high level in different

Fig. 1A–C Physical maps of enhanced green fluorescent protein(EGFP) plasmids. A pSM1, B pSM2 and multiple cloning site ofpSM2 (the ATG of the egfp gene is indicated in bold) C pIG1783

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filamentous fungi, such as Aspergillus flavus, Magna-porthe grisea, Podospora anserina and Trichoderma har-zianum (Inglis et al. 1999; Lorang et al. 2001).

On the basis of the plasmid pEGFP/gpd/tel (Ingliset al. 1999), we constructed the plasmid pSM1, whichcontains egfp under control of the strong constitutivegpd promoter of A. nidulans and the trpC terminator ofA. nidulans (Fig. 1A). In contrast to pEGFP/gpd/tel(Inglis et al. 1999), pSM1 contains the hygromycin Bphosphotransferase (hph) gene as a dominant selectablemarker for transformation of fungal protoplasts. Vec-tors based on resistance to hygromycin are widely ap-plied in a variety of mycelial fungi and are particularlyuseful when auxotrophic mutations are unavailable(Lemke and Peng 1995).

Plasmid pSM1 contains the previously improvedcompact hygromycin cassette of plasmid pCB1003(Carroll et al. 1994), in which four commonly used re-striction enzyme sites in the hph gene were eliminatedthrough single base-pair changes. A promoterless deriv-ative of pSM1, plasmid pSM2, can be applied to assessthe putative promoter strength and expression patternsof fungal genes (Fig. 1B). The vector contains five uniquesites of the commonly used restriction enzymes PstI,EcoRI, EcoRV, HindIII and ClaI. All sites are locatedupstream of the egfp gene and can be employed to inte-grate promoter sequences. The corresponding sequenceis shown in Fig. 1B and offers the opportunity to intro-duce coding sequences in a translational fusion into theegfp gene under control of an endogenous promoter. Wesuccessfully used pSM2 to monitor the expression ofpheromone precursor genes in S. macrospora (data notshown). Plasmid pSM2 should also prove beneficial inevaluating regulatory sequences that govern gene ex-pression in a variety of mycelial fungi.

To improve translational fusions of the egfp genewith protein coding sequences of fungal genes, we cre-ated plasmid pIG1783 (Fig. 1C). Plasmid pIG1783contains a modified hph cassette lacking the NcoI site,normally found in the hph gene. Further details of thein vitro mutagenesis are given in Material and methods.

The vector is extremely useful for the construction ofN-terminal translational fusions of any protein codingsequence, because these sequences can be placed directlyinto the single NcoI site, thereby forming the ATG startcodon, of the fusion gene in front of the egfp gene.Cloning into theNcoI site ensures proximity to theKozakconsensus sequence for Neurospora crassa and S. mac-rospora (Bruchez et al. 1993; Edelman and Staben 1994;Poggeler 1997), which promotes functional translationinitiation. The following sections gives examples for theapplication of the egfp reporter plasmids in the two as-comycetes, Acremonium chrysogenum and S. macrospora.

Cytoplasmic localization

In a first approach, we used plasmid pSM1 for trans-formation of S. macrospora and A. chrysogenum,

respectively. In this plasmid, expression of the egfp geneis under control of the strong constitutive gpd promoterof Aspergillus nidulans and thus ensures a high level ofexpression of the egfp gene. Several S. macrospora andAcremonium chrysogenum transformants were obtained,which grew on selective hygromycin-containing solidmedium. Subsequently, the transformants were analyzedby fluorescence confocal microscopy. In young, fastgrowing hyphae, fluorescence appeared uniformly dis-tributed throughout the cytoplasm of the hyphae. GFPfluorescence was not extensively concentrated in thenuclei or in other organelles and appeared to be ex-cluded from the vacuoles. Typical microscopic imagesare presented in Fig. 2. In control experiments with non-transformed mycelia, we were unable to detect anyhyphae showing fluorescence (Fig. 2). S. macrospora andA. chrysogenum transformants carrying plasmidpIG1783, which contains a modified hph gene without arestriction site for NcoI, exhibited the same GFP fluo-rescence (Fig. 2). The level of hygromycin resistanceinduced by the modified hph gene was identical to theone obtained with the non-modified hph gene. Recently,it was demonstrated that a derivative of pSM1, theplasmid p82.9 (see Materials and methods), could beused efficiently to express the egfp gene in the mulun-docandin producer Aspergillus sydowii (Schmitt et al.2002).

Meiotic segregation of egfp in S. macrospora

In filamentous fungi, expression of heterologous genes isoften prevented by gene silencing mechanisms, such asquelling and/or methylation (Selker 1997). Some of theseinactivation processes take place in ascogenous hyphaeand explain the difficulty in transmitting active recombi-nant DNA sequences through meiosis (Fincham 1989).To assess GFP expression during the sexual phase, ex-pression of a pSM1 transformant (T-EGFP) was ana-lyzed. As shown in Fig. 3A, even early developmentalstages, for example ascogonia, were shown to exhibitgreen fluorescence. In control experiments with untrans-formed wild-type strains, fluorescent ascogonia werenever observed (data not shown). To address the questionwhether or not the egfp gene is stably inherited throughmeiosis, we crossed transformant T-EGFP with the non-transformed spore color mutant fus. As shown in Fig. 3B,young recombinant asci contain four brightly coloredascospores carrying the fus mutation and four darker-appearing wild-type ascospores. The egfp gene segregatedas expected in a 4:4 Mendelian ratio and was expressed infour ascospores of each ascus. Note that the egfp expres-sion is not linked with the spore color mutation. In plant,insect and mammalian cells, expression of the GFP mes-sage in the cytosol is often followed by penetration intothe nucleoplasm. This penetration appears passive andreversible (Plautz et al. 1996; Grebenok et al. 1997).As shown in Fig. 3B, GFP seems to accumulate in thenucleoplasm of S. macrospora ascospores. A similar

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observation was made recently in the closely related asc-omycete N. crassa (Freitag et al. 2001).

Nuclear localization

Transcriptional fusions with nuclear localization signals(NLS) or transcription factors are widely used to targetGFP to nuclei of fungal hyphae (for example, Suelmannet al. 1997; Fernandez-Abalos et al. 1998). In plants, itwas shown that NLS-targeted GFP is too small to bephysically excluded from the nucleus (Grebenok et al.1997). Therefore, the C-terminal side of the transcriptionfactor CPCR1 (Schmitt and Kuck 2000), which belongsto the regulatory factor X family, was fused with theN-terminal side of EGFP, thus creating a 120 kDapolypeptide. The chimeric cpcr1-egfp gene was placedunder the control of the gpd promoter of A. nidulans andwas transformed together with the hygromycin resis-tance (hph) gene into either Acremonium chrysogenum orS. macrospora. As shown in Fig. 4, in both A. chrysog-enum and S. macrospora, the CPCR1-EGFP fusion istargeted to the nucleoplasm, producing a fluorescencepattern, which coincides with the staining pattern of theSytox orange nucleic acid dye. Organelle targeting ofGFP fusion proteins was recently tested in yeast withendogenous and strong promoter sequences to drivechimeric gene expression (Prein et al. 2002). It wasdemonstrated that subcellular localization patterns areidentical, albeit with different expression levels of thechimeric gfp gene. Nuclear localization of GFP offersthe opportunity to observe the behavior of differentiallylabeled nuclei in heterokaryotic mycelia or the fertiliza-tion process in homothallic and heterothallic fungi.Differential labeling of two nuclei can be performed forexample with a second fluorescent marker gene, such asthe DsRed gene (a red fluorescent reporter for eukaryoticcells), or color variants of the gfp gene encoding the cyanand the yellow fluorescent proteins (Heim and Tsien1995; Miyawaki et al. 1997; Matz et al. 1999). Hetero-karyon formation can easily be done in S. macrospora byconventional crossing experiments with two strainscarrying differently labeled nuclei.

Secretion studies

We recently isolated the etp gene, encoding a putativeaspartic protease. The encoded protein of 449 aminoacids was found to be closely related to the endothia-pepsin of Cryphonectria parasitica and to the podos-porapepsin from Podospora anserina. Both peptidasesbelong to the eukaryotic aspartyl protease family(Barkholt 1987; Choi et al. 1993; Paoletti et al. 1998).Since this enzyme was found to be secreted in C. par-asitica (Barkholt 1987), we used the SignalP ver 1.1program (Nielsen et al. 1997) to search for a hydro-phobic signal sequence in the predicted N-terminusof the S. macrospora ETP polypeptide. We detected a

hydrophobic signal sequence, which was predicted to becleaved between amino-acid positions 21 and 22. In or-der to achieve secretion of the EGFP, the 5¢ sequence ofthe etp gene, which encodes the hydrophobic signalpeptide, was constructed synthetically from oligonucle-otides. The synthetic fragment was translationally fusedto the 5¢ end of the egfp gene. For further details, refer tothe Material and methods section. The resulting vectors,pSetp1 and pSetp2, contained the secretion signal insense and antisense orientation, respectively. The vectorswere transformed in S. macrospora and A. chrysogenum.Analysis by confocal fluorescence microscopy revealed adifferent localization of GFP in S. macrospora pSetp1and pSetp2 transformants (Fig. 5). In pSetp1 transfor-mants, GFP fluorescence appeared as distinct patchesdistributed along the fungal hyphae, thus suggestinglocalization within cytoplasmic vesicles. A pronouncedbackground of cytoplasmic fluorescence was missing(Fig. 5A). The same localization of GFP was observedin A. chrysogenum pSetp1 transformants (data notshown). In contrast, analysis of S. macrospora trans-formants containing pSetp2 showed high expressionlevels of cytoplasmic GFP. Since the antisense orienta-tion of the etp signal sequence encoded no stop codon,transformants containing pSept2 should be able to ex-press the egfp gene. Similar to transformants carryingpSM1 (cf. Fig. 2), GFP was present throughout the cy-toplasm and appeared to be excluded from the vacuoles,which were visible as dark spots within the hyphalcompartments (Fig. 5B).

To investigate the possibility of the protein encodedby pSetp1 also being present in the culture medium,samples of culture medium were taken at different timesduring growth of S. macrospora transformants on liquidmedia. The presence of GFP in the extracellular culturebroth was analyzed by means of SDS-PAGE andWestern blotting with a polyclonal antibody against

Fig. 2A, B Fluorescence microscopy of Sordaria macrospora andAcremonium chrysogenum. A Left Hyphae of S. macrosporatransformed with the EGFP vector pSM1, right hyphae of theuntransformed wild-type strain of S. macrospora. B Left Hyphae ofA. chrysogenum transformed with EGFP vector pIG1783, righthyphae of the untransformed wild-type strain of A. chrysogenum.Arrows indicate vacuoles, which show reduced fluorescenceFig. 3A, B Expression of GFP during the sexual cycle of S. mac-rospora. A Early stages of sexual development (ascogonia) in the S.macrospora transformant (T-EGFP) carrying plasmid pSM1. BSegregation of egfp expression and fus in developing asci from aT-EGFP·fus cross. In fluorescent ascospores, the enhancedstaining of two nuclei can be observed. Left Differential interfer-ence contrast light micrographs, right fluorescence micrographsFig. 4A, B Colocalization of CPCR1-EGFP and Sytox orangenucleic acid stain. A Hyphae from S. macrospora carrying thechimeric cpcR1-egfp gene. B Hyphae from A. chrysogenum carryingthe chimeric cpcR1-egfp gene. Top GFP fluorescence, middle Sytoxorange staining of nuclei, bottom merged image of top and bottomFig. 5A, B Fluorescence microscopy of S. macrospora producingEGFP with a N-terminal signal peptide.AHyphae of S. macrosporatransformed with pSetp1. The fluorescence is visible as distinctpatches. B Hyphae of S. macrospora transformed with pSetp2,showing a strong cytoplasmic fluorescence

c

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GFP. Immunoblotting with the GFP antibody resultedin the detection of a 27-kDa polypeptide in the super-natant of pSetp1 primary transformants which, aftergrowth for 2 days, was absent in culture supernatantsfrom pSetp2-, pSM1- and pANsCos1 transformants(Fig. 6). This result confirmed that GFP was present inthe culture medium of pSetp1 transformants. Oftenfungal transformants are heterokaryotic and myceliacarry transformed and non-transformed nuclei. There-fore, we investigated single spore isolates from trans-formants TpSetp1 and TpSetp2. The immunoblotrevealed that supernatants of single spore isolates fromTpSetp1 show even stronger signals than the primarytransformant TpSetp1 (Fig. 6).

In conclusion, we successfully constructed three vec-tors, designated pSM1, pSM2 and pIG1783, for theexpression of the egfp gene in fungi. All vectors con-tained the hph gene, encoding hygromycin B phospho-transferase, a commonly used selectable marker fortransformation of fungal protoplasts. The applicabilityof the vectors following transformation into two non-related ascomycetes was demonstrated. Our resultsindicate that vector pSM1, which utilizes the strong gpdpromoter of Aspergillus nidulans, allows high levels ofexpression of the egfp gene in the cytoplasm of fungalhyphae. By means of vector pSM2, the egfp gene can beused as a reporter gene to analyze the strength of fungalpromoters and to visualize spatial and temporal ex-pression patterns of fungal genes. Vector pIG1783 caneasily be used for N-terminal GFP-translational fusions.It is consequently a valuable tool for tagging fungalproteins and analyzing their subcellular localization. The

vectors were primarily constructed for S. macrosporaand Acremonium chrysogenum. However, these shouldprove very effective in a wide variety of fungal species inwhich transformation is based on hygromycin B resis-tance and are expected to facilitate the use of the EGFPpolypeptide for visualization of cell-specific gene ex-pression and subcellular protein localization in fungi.

Acknowledgements The authors wish to thank Silke Giessmann,Ingeborg Godehardt, Swenja Ellßel and Gisela Isowitz for theirexcellent technical assistance, Gabriele Frenßen-Schenkel for helpin the artwork and Drs. Theiß and Mannherz for help and supportwith confocal laser microscopy. We thank Biochemie AG (Kundl)and RheinBiotech (Hilden) for support throughout this work. U.K.thanks Dr. Inglis for providing plasmid pEGFP/gpd/tel. This workwas funded by a grant from the Landesforderung NRW (PTJ-FKZ001-9910-0v08) and a grant from the Deutsche Forschungsgeme-inschaft (SFB 480) to S.P. and U.K.

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