Introduction

Fungi are widely used as model organisms to study the control of cell cycle, organelle movement, protein secretion, fungus-host interactions, etc. In addition, the biology of fungi is intensely studied to unravel the principles of fungal growth, adaptation to environmental conditions, metabolic capacities and the regulation or development of reproductive structures, etc. Gene function analyses comprise mainly the study of loss-of-function or gain-of-function mutations and the monitoring of expression levels or subcellular localisation of proteins. The advent of the Aequoria victoria green fluorescent protein (GFP) had a great impact on fungal molecular biology (Cormack 1998). After the initial application of this technology in Escherichia coli (Chalfie et al. 1994) and Saccharomyces cerevisiae (Niedenthal et al. 1996), GFP has been used in a variety of fungi, such as Ustilago maydis (Spellig et al. 1996), Aspergillus nidulans (Fernandez-Abalos et al. 1998; Suelmann et al. 1997), Schizophyllum commune (Lugones et al. 1999) and Neurospora crassa (Fuchs et al. 2002). Within the past 10 years, a variety of GFP variants has been developed, which show increased sensitivity, faster folding of the protein or altered spectroscopic properties (variants with yellow fluorescence or blue fluorescence; Lippincott-Schwartz and Patterson 2003). The proteins with altered spectral properties are especially useful for co-localisation of two given proteins. In addition, new fluorescent proteins have been characterised and introduced into different organisms. However, in the filamentous fungi, mainly GFP and its derivatives have been used so far (Cormack 1998; Pöggeler et al. 2003). Recently, the Discosoma red fluorescent protein, DsRed, was applied in Penicillium paxilli, Trichoderma species (Mikkelsen et al. 2003) and A. nidulans (Dou et al. 2003). However, DsRed requires tetramer formation for the development of fluorescence (Baird et al. 2000). This may hamper the application of DsRed for protein fusions, because forced tetramerisation of the corresponding fusion proteins is likely to disturb the cellular function of the original polypeptide. In addition, the original DsRed isolate required several days for maturation of the fluorescent properties, which is inappropriate for many applications (Baird et al. 2000). To improve the folding properties, several derivatives were engineered, one of which was DsRed (T4). This protein displays a half-time for maturation of 0.71 h and still has a relative brightness of 0.38, in comparison with the slow-folding version (Bevis and Glick 2002). The additional problem of tetramer formation in DsRed was also solved recently with the monomeric red fluorescent protein derivative (mRFP1; Campbell et al. 2002). mRFP1 functions as a monomer and matures quickly. In addition, the excitation and emission peaks, 584 nm and 607 nm, are about 25 nm red-shifted in comparison with the engineered and improved red fluorescent proteins DsRed (T4) and mRFP1 and the blue fluorescent protein (BFP) in the filamentous fungus A. nidulans. All three proteins were used for organelle-labelling. In addition, we introduce a series of vectors for the efficient cloning of tagged expression constructs.

Materials and methods

Strains, plasmids and culture conditions

Supplemented minimal and complete media for A. nidulans were prepared as described by (Käfer 1977) and standard strain construction procedures were used. Standard laboratory Escherichia coli strains (XL-1 blue, Top 10 F’) were used. The A. nidulans strains used were: RMS011 (pabaA1, yA2; ΔargB::trpCΔB; trpC801, veA1; Stringer et al. 1991), SRF200 (pyrG89; ΔargB::trpCΔB; pyroA4; veA1; Karos and Fischer 1999), SDM1004 (RMS011 transformed with pJH19 and pRS54), SDM25 (RMS011 transformed with pRF280 and pSK700) and SSK90 (RMS011 transformed with pJH19).

Molecular techniques

Standard DNA transformation procedures were used for A. nidulans (Yelton et al. 1984) and E. coli (Sambrook and Russel 1999). For PCR experiments, standard protocols were applied, using a capillary rapid cycler (Idaho Technology, Idaho Falls, USA) for the reaction cycles. DNA sequencing was done commercially (MWG Biotech, Ebersberg). Western blot analysis was performed as described by the supplier of the Hybond membranes and the Western blot kit (Amersham Pharmacia, Freiburg and Roche, Mannheim).

Plasmids

For nuclear labelling, the plasmids used were: pRF280 [gpdA(p)::sgfp::stuA(NLS), argB in pBluescript; a derivative of pRS31; Suelmann et al. 1997], pRF281 [gpdA(p)::sgfp::stuA(NLS), pyr4 in pBluescript], pJW18 [alcA(p)::DsRed (T4)::stuA(NLS), argB], and pJH19 [alcA(p) in pJW18 substituted by the gpd promoter]. For mitochondrial labelling, the plasmids used were: pRS54 [gpdA(p)::citrate synthase N-term::sgfp in pBluescript; Suelmann and Fischer 2000], pSK800 (sgfp in pRS54 substituted by mRFP1) and pSK700 [sgfp in pRS54 substituted by DsRed (T4)].

Destination vectors

For pMT-OvE and pMT-3×HA, the vector pBluescript KS-Δ was used, with argB cloned into NotI and alcA(p) cloned into BamHI. The suicide ccdB box [containing the ccdB gene (Bernard and Couturier 1992), the chloramphenicol cat gene and attR sites] was amplified with pDEST 14 (Invitrogen) as template (primers 5’-CTC GAG ATA GGG AGA CCA CAA CGG-3’, 5’-CTC GAG CAG CTT CCT TTC GGG C-3’) and cloned into XhoI downstream of the alcA promoter. 3×HA was cloned as a KpnI fragment downstream of the ccdB box. For pMT-sGFP, pMT-BFP and pMT-mRFP1, the vector pSNi11 (Schier and Fischer 2002) including argB and alcA(p) was used to insert a commercially available ccdB box-containing fragment (Invitrogen) blunt-ended into a SmaI site, as described by the supplier. The tags were cloned into EcoRI and KpnI. The sGFP gene was amplified with the primer combination 5’-GAA TTC ATG GTG AGC AAG GGC GAG-3’ and 5’-GGT ACC CTA TTT GTA CAG CTC GTC-3’, the BFP gene with the same primers as for sGFP and mRFP1 with the primers 5’-GAA TTC ATG GCC TCC TCC GAG G-3’ and 5’-GGT ACC TTA GGC GCC GGT GGA G-3’. The template for mRFP1 was obtained from Dr. Prastio (University of San Diego, USA) and the template for BFP from Dr. Ram (Leiden University, The Netherlands). For the amplification of all destination vectors, ccdB gene-resistant E. coli cells (Library Efficiency DB3.1 competent cells; Invitrogen) were used. Entry vectors pMT-veA and pMT-stuA(NLS) were based on the vector pENTR/D-TOPO (Invitrogen). The vector pMT-veA included the PCR-amplified veA gene (primers 5’-CAC CGC AAC AAG TCT TCT AGA GC-3’, recombination was performed with the LR clonase enzyme mix (Invitrogen), as described by the supplier. In each reaction, 300 ng destination vector and 300 ng entry vector were used. If more than one destination vector were used in a single reaction, the different destination vectors were used in equal amounts, so that the total amount of destination vectors was always 300 ng per reaction. The same applied for the use of several entry vectors in a single LR reaction. The LR recombination reaction was transformed into E. coli, as described by the supplier.

Fluorescence microscopy

Fluorescent proteins were visualised with appropriate filter combinations (no. 15 for red fluorescence, no. 9 for green fluorescence; Zeiss, Jena, Germany), using an Axiophot microscope (Zeiss). Images were captured with a high-resolution Orca ER camera (Hamamatsu, Munich, Germany). Alternatively, we used a TCSSp2 confocal microscope (Leica).

Results and discussion

Expression of DsRed (T4) and mRFP1 in A. nidulans

In previous work, we fused sGFP to the C-terminal domain containing the nuclear localisation signals (NLS) of the developmental transcription factor StuA and expressed the construct under the control of the constitutive gpd promoter (Suelmann et al. 1997). A similar construct (pJH19) was established with the DsRed (T4) gene instead of the sgfp gene. The construct was introduced into wild-type A. nidulans (RMS011) and stable transformants were analysed for red fluorescence. Microscopic inspection revealed that nuclei were brightly labelled. In previous experiments, we found that the sGFP fusion protein diffuses out of the nucleus during mitosis (Suelmann et al. 1997). This result was surprising, because in fungi the nuclear envelope remains intact during nuclear division. However, small proteins could diffuse through the nuclear pore complex and, since the sGFP-StuA(NLS) fusion protein has a predicted molecular mass of about 45 kDa, it could leak out of the nucleus. In comparison, fluorescent proteins fused to DNA-binding proteins remain in the nucleus during mitosis (Fernandez-Martinez et al. 2003; unpublished data from our laboratory). Since the DsRed protein needs to form a tetramer for fluorescence, the nuclear-targeted protein should have a molecular mass of about 190 kDa. To test whether this fusion protein would remain in the nucleus, we did a time-lapse analysis of mitosis and found that the sGFP-StuA and the DsRed-StuA fusion proteins behaved identically (Fig. 1). These observations suggest that diffusion of the fluorescent proteins out of the nucleus during mitosis is not dependent on the molecular mass of the proteins but rather depends on DNA interaction. Since the StuA(NLS) portion of the StuA protein only comprises the putative NLS but probably not the DNA-interacting domain, it is unlikely that the fusion proteins bind to DNA. Our results could be explained in two ways: (1) the nuclear envelope does not remain intact during mitosis and becomes largely leaky or (2) the nuclear envelope remains intact but the nuclear import machinery is not active.

Fig. 1
figure 1

Localisation of DsRed (T4) in the nuclei of hyphae of A. nidulans before (a), during (b) and after (c) mitosis. Plasmid pJH19 was transformed into RMS011

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To demonstrate that the red fluorescent protein can be used for double-labelling experiments, we combined the red nuclear label [pJW19, DsRed (T4)] with sGFP-labelled mitochondria (pRS54; Fig. 2a). We also expressed a fusion between the mRFP1 targeted to mitochondria (pSK700) in combination with green-labelled nuclei (Fig. 2b).

Fig. 2
figure 2

Double-labelling of nuclei and mitochondria with DsRed (T4) and sGFP. a, c Phase contrast image of germlings. b, d Fluorescence picture of the same germlings as in a, c. a, b Labelling of nuclei with DsRed (T4) and mitochondria with sGFP in strain SDM1004. c, d Labelling of nuclei with sGFP and mitochondria with DsRed (T4) in strain SDM25

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Construction of expression vectors for over-expression and protein tagging with mRFP1, BFP, sGFP or 3×HA

The tagging of proteins by conventional cloning usually involves several time-consuming steps, and sometimes it is hard to achieve because of the lack of unique restriction sites in the vectors and/or the gene of interest. This problem has been solved by the introduction of a vector system based on recombination in vitro (Landy 1989). After cloning of the gene of interest into a vector flanked by attL sites (pENTR/D-TOPO, kanamycin resistance), the gene is transferred to the destination vector by in vitro recombination. Due to the recombination event, the suicide ccdB box (Bernard and Couturier 1992) is replaced by the gene of interest. The obtained expression vectors confer ampicillin resistance to the recipient E. coli strains, which are not resistant to the ccdB box (XL-1 blue, Top 10 F’) and thus only plasmids with successful recombination are able to amplify on ampicillin-containing media. Meanwhile, a great variety of expression vectors are commercially available from Invitrogen and have been adapted for use in plants (Curtis and Grossniklaus 2003; Karimi et al. 2002). However, those vectors are not useful for filamentous fungi, due to the lack of a promoter and a fungus-specific selection marker. Therefore, we designed a number of constructs which allow the tagging of proteins with sGFP, mRFP1, BFP and the haemagglutinin epitope, 3×HA (Fig. 3). To test the functionality of the system, we tagged part of the StuA transcription factor, containing the NLS sequences. The in vitro LR-recombination reaction with the entry vector pMT-stuA(NLS) and destination vectors pMT-BFP and pMT-mRFP1 should result in two different expression vectors: stuA(NLS) tagged with BFP and stuA(NLS) tagged with mRFP1. After transformation of the recombination reaction in E. coli, 24 colonies were analysed. Five of the plasmids contained the stuA(NLS) tagged with mRFP1 and 13 plasmids contained the stuA(NLS) tagged with BFP (Fig. 4). All inserts were in the correct orientation. StuA(NLS) was also tagged with sGFP. The nuclei of the corresponding transformants (SRF200) harboured red, green or blue nuclei, respectively (Fig. 5a). The fluorescence of the BFP is very weak and it is not recommendable for standard use. In another example, we used pMT-veA as the entry vector with three different destination vectors (including pMT-3×HA). Seven from 14 tested E. coli colonies contained the tagged veA gene and three of these were fused to 3×HA. The function of the VeA-3×HA expression vector in A. nidulans (SRF200) was shown by Western blot (Fig. 5b). It was also possible to use several entry vectors with only one destination vector in a single in vitro LR-recombination reaction. Using this strategy, one can transfer several genes of interest into one destination vector (data not shown). Meanwhile, the system was successfully used to label other cellular proteins in our laboratory. Hence, the introduced vectors are very useful tools for quick and efficient protein-tagging in filamentous fungi. High-throughput analyses will be of increasing importance with the increasing number of full fungal genome sequences available.

Fig. 3
figure 3

Scheme of the constructed vectors. The sequences and details of these vectors are available at http://www.uni-marburg.de/mpi/fischer/fischer.html. The web site will be updated by adding new vectors (with relevant information) as they are constructed in our laboratory. Restriction enzyme sites: B BamHI, Bg BglII, E EcoRI, F FspI, K KpnI, N NotI, S SmaI, X XhoI

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Fig. 4
figure 4

Scheme of an example of the recombination reaction between one entry-clone and two destination vectors (a) and the analysis of plasmids obtained after the recombination (b). a The location of the primers for the amplification of the gene of interest is indicated. The forward primer should contain CACC upstream of the ATG and the reverse primer should end just before the stop codon. b Plasmids were digested with FspI and separated on a 1% agarose gel. A total of 13 clones were derived from recombination with pMT-BFP (e.g. lane 1) and five from pMT-mRFP1 (e.g. lane 2). The other six cannot be explained by the recombination event. Lambda DNA digested with Eco1301 was used as a size marker

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Fig. 5 a, b
figure 5

Analysis of SRF200 strains transformed with plasmids created by recombination between pMT-stuA(NLS) and the destination vectors for labelling with fluorescent proteins or the veA gene in the entry vector pMT-veA and the hemagglutinin (HA)-tagging plasmid as destination vector, as described in Fig. 3. a Fluorescence analysis. b Western blot analysis of protein extracts from a control strain expressing the cyclin PclA fused to 3×HA epitopes (left lane; Schier et al. 2001), the wild type without any HA-tagged protein (middle lane) and a strain expressing the transcription factor VeA fused to 3×HA (right lane). The arrows indicate PclA:3×HA and VeA:3×HA. One hundred micrograms of total protein were loaded and epitope-tagged proteins detected with an anti-HA antibody and peroxidase-coupled secondary antibodies

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