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Expression of the red fluorescent protein DsRed-Express in filamentous ascomycete fungi

Lisbeth Mikkelsen, Sabrina Sarrocco, Mette Lübeck, Dan Funck Jensen
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00355-0 135-139 First published online: 1 June 2003


The recently reported red fluorescent protein DsRed from the reef coral Discosoma sp. represents a new marker that has been codon-optimized for high expression in mammalian cells. To facilitate expression of DsRed in ascomycete fungi, we used the clone pDsRed-Express (Clontech) for constructing a plasmid vector, pPgpd-DsRed, containing the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter. This vector was used for co-transformation of Penicillium paxilli, Trichoderma harzianum and Trichoderma virens (syn. Gliocladium virens) together with either pAN7-1 or gGFP, both containing a gene for hygromycin resistance for transformant selection. In addition, gGFP contains a green fluorescent protein (GFP) gene for expression in Ascomycetes. Expression of DsRed-Express was obtained in all three fungi, indicating that DsRed can be used as a highly effective vital marker in Ascomycetes. Dual marked transformants expressed both DsRed-Express and GFP in the same mycelium and were used for non-quantitative comparison of the intensity of the fluorescence using confocal laser scanning microscopy.

  • Trichoderma
  • Penicillium
  • Fungal transformation
  • Reporter gene
  • Fluorescent protein
  • Green fluorescent protein
  • DsRed

1 Introduction

The gfp gene from the jellyfish Aequorea victoria encoding green fluorescent protein (GFP) is widely used as a reporter gene in a large number of organisms. A gene encoding red fluorescent protein, DsRed, has been obtained from a reef coral Discosoma sp. [1]. This protein resembles GFP and the gene may be used as a reporter in a similar way as the gfp gene. GFP has proved an outstanding tool for studying various biological systems. Expression and localization of GFP can easily be detected in living cells without addition of cofactors, substrates or other proteins [2] and the brightly fluorescing molecule allows visualization of events both in situ and in real time. Mutations of the gfp gene have led to the design of a number of improved GFP varieties, which are not only brighter than the wild-type, but also available in different colors (for reviews, see [3,4]). Since Ustilago maydis was successfully transformed with gfp [5], the exploitation of GFP technology has been adapted for use with other filamentous fungi (for reviews, see [6,7]). We have used GFP constitutively expressed in various fungi for studying fungal ecology in relation to biological control of plant diseases [8] and colonization of ryegrass by the endophyte Neotyphodium lolii [9]. However, there is still a need for developing gfp constructs for use in fungi as many of the present gene constructs have been designed for use with other organisms. Detection has been exploited with differently gfp-marked bacteria [10]. In this way different bacteria could be followed simultaneously on plant roots. For further work in the field of fungal ecology and for studying interaction between fungi in vivo we wanted to use a similar approach by transforming different fungi with genes encoding differently fluorescing markers.

The red fluorescent protein DsRed resembles the structure of GFP although it tends to form tetramers [1]. However, due to slow maturation and solubility of the wild-type protein and in order to facilitate expression in mammals, different variants have been developed [11]. The variant DsRed-Express contains nine amino acid substitutions, which improve the maturation and solubility of the protein. Furthermore, these substitutions reduce the level of residual green emission as well as the tendency to aggregate [11]. The DsRed-Express protein has an excitation maximum at 557 nm and an emission maximum at 579 nm. The codon-optimized DsRed has been shown to facilitate expression in plants [12] and the wild-type DsRed was used for dual labelling of Saccharomyces cerevisiae with the yEGFP variant [13]. Expression of DsRed in filamentous Ascomycetes has not previously been reported, probably due to the lack of available constructs.

The experiments reported here were performed to investigate the ability of DsRed to act as a reporter protein in Ascomycetes. The objectives were also to study if dual labelling of filamentous fungi with both DsRed and GFP and labelling of different fungal isolates with either GFP or DsRed could be useful in studies of fungal ecology and in studies of interaction between fungi.

2 Materials and methods

2.1 Strains and plasmids

Cultures of Trichoderma harzianum I252 [14], Trichoderma virens, syn. Gliocladium virens I10 [15] and Penicillium paxilli Bainier, ATCC 26601/IBT22884 (J.C. Frisvad, Technical University of Denmark) were grown on potato dextrose agar (PDA; Scharlau Chemie, Barcelona, Spain) at 20–25°C. Escherichia coli strain MC1061 was used as host for plasmid amplification and cloning [16].

The plasmid pAN7-1 [17] containing the E. coli hygB resistance gene was donated by M. van Montagu (Laboratorium voor Genetica, Gent, Belgium). The plasmid pNOM102 [18] containing the E. coli uidA (GUS) gene was donated by M. Penttilä (VTT, Helsinki, Finland). The plasmid gGFP [19] containing a synthetic version of the gfp gene, sGFP(ser65T), in which Ser65 was replaced by threonine, and the E. coli hygB resistance gene, was donated by A. Sharon (Tel Aviv University, Israel). All genes mentioned contained the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter facilitating expression in many filamentous fungi. The plasmid pDsRed-Express containing an improved DsRed variant was purchased from Clontech Laboratories (Palo Alto, CA, USA).

Plasmid propagation was performed according to [16] while purification of plasmids was carried out using the Qiagen plasmid purification procedure according to the manufacturer's procedure (Qiagen, Chatsworth, CA, USA).

2.2 Plasmid construction

The pPgpd-DsRed construct (Fig. 1) was produced by replacing the β-glucuronidase (uidA) gene in the fungal vector pNOM102 with the DsRed-Express gene sequence, thereby placing the gene under the control of the constitutive gpd promoter and the trpC terminator, both from A. nidulans.

Figure 1

Restriction enzyme map of the 6.4-kb plasmid pPgpd-DsRed. The plasmid was constructed for fungal expression of the DsRed-Express fluorescent reporter protein, an improved variant of DsRed from Discosoma sp. placed under the control of the constitutive A. nidulans glyceraldehyde 3-phosphate promoter (PgpdA) and the A. nidulans trpC transcriptional terminator (TtrpC). The plasmid contains the ampicillin resistance gene (pDraw32 ver. 1, ACALONE Software).

The DsRed-Express gene sequence was excised from pDsRed-Express plasmid with EcoRI, the overhang was filled in, followed by NcoI digestion. The resulting 0.7 kb fragment was inserted into the 5.7-kb BamHI/overhang fill in/NcoI fragment of the pNOM102 vector.

2.3 Transformation methods

Protoplast preparation and transformation of P. paxilli was done as described by [20]. T. harzianum I252 and T. virens I10 protoplasts were prepared according to [21] and transformation of protoplasts was carried out as described by [22]. For transformation of the protoplasts either 4 µg pPgpd-DsRed together with 4 µg of pAN7-1 or 4 µg of pPgpd-DsRed together with 4 µg of gGFP was used. All plates were incubated at room temperature for 1 day and then overlaid with hygromycin B (Calbiochem-Novabiochem, Darmstadt, Germany) in 5 ml molten PDA 0.8% agar to a final concentration of 100, 200 or 150 ppm, respectively.

2.4 Microscopy

The regenerating protoplasts were observed in regeneration plates using the fluorescence stereomicroscope 20 h after transformation.

The Leica MZ FLIII stereomicroscope equipped with a mercury lamp, a Leica DC 300F digital camera and a green fluorescence filter G with excitation 546/10 nm and an emission filter 590 LP (longpass) was used to detect DsRed fluorescence. For detection of GFP fluorescence we used filters GFP2 and GFP3 with excitation filters 480/40 and 470/40 nm, respectively, and barrier filters 510 nm LP and 525/50 nm, respectively (Leica Microsystems, Wetzlar, Germany). Images were handled with the software Leica IM500 ver. 1.20 release 19.

3 Results and discussion

The DsRed-Express coding region from plasmid pDsRed-Express was inserted into pNOM102 replacing the coding region of the uidA (GUS) gene (for details see Section 2) (Fig. 1). The resulting vector, pPgpd-DsRed, was inserted into P. paxilli, T. harzianum and T. virens in co-transformation experiments together with either pAN7-1 or gGFP, both containing a gene encoding hygromycin resistance for transformant selection. The transformants of both Trichoderma strains resulting from the gGFP experiments were, in addition to the DsRed-Express, also marked with the GFP gene.

Expression of DsRed-Express in the transformants was observed under the fluorescence stereomicroscope as red fluorescent mycelium already the day after the transformation (Fig. 2A1). The red fluorescent colonies were easy to detect in the mixture of transient regenerating protoplasts (Fig. 2A2) and no autofluorescence was observed. Some of the Penicillium transformants expressed DsRed-Express in such high amounts that the white mycelium of the colonies turned pink on PDA plates (Fig. 2A3). This was not observed in Trichoderma transformants probably because the mycelium of Penicillium is much denser than Trichoderma on PDA.

Figure 2

Fluorescence stereomicroscope images. A: P. paxilli expressing DsRed-Express (A1) 1 and (A2) 4 days after transformation of protoplasts. A3: Three selected DsRed-Express transformants, grown for 8 days on PDA, are pink compared to the wild-type (lower left). B: Dual-labelled T. harzianum 3 days and C: T. virens 6 days after transformation of protoplasts expressing both DsRed-Express and GFP (B1, C1) observed with the G filter, (B2, C2) with the GFP2 filter and (B3, C3) no fluorescent light. D: Mixed culture of germinated spores of two different P. paxilli transformants expressing either DsRed-Express or GFP. The spores are observed with (D1) the G filter or (D2) the GFP3 filter and (D3) the two images are merged.

Dual marking of fungi with DsRed-Express and GFP also allowed examination of whether both marker genes are expressed within the same cells. Co-expression of both markers was clearly demonstrated as the dually marked Trichoderma transformants co-expressed DsRed-Express and GFP in the same mycelium and was easily detected under the stereomicroscope with the G and the GFP2 filters (Fig. 2B,C). Furthermore, a preliminary comparison of the intensity of the two different fluorescent signals from these dually marked strains using confocal laser scanning microscopy (CLSM) was comparable as judged by CLSM (results not shown). No quantification was made.

Examination of a single-labelled DsRed-Express P. paxilli transformant (Fig. 2D1) co-cultured with a GFP-labelled transformant (from previous experiments) (Fig. 2D2) by fluorescence stereomicroscopy using the two filters G and GFP3 (see Section 2) enabled comparison of the two different fluorescent signals. Fig. 2D3 shows a merge of two images (D1 and D2) acquired with each of the filters. DsRed-Express expression was detected as orange fluorescence with the GFP2 filter (not shown), because this filter allows detection of light above 510 nm and the stereomicroscope is equipped with a separate channel for the excitation light (no beam splitter needed).

The characteristics of DsRed make it an ideal candidate for fluorescence imaging and will be useful for multicolor experiments together with GFP. Thus, when combining different antagonistic fungi for studying their effect, the monitoring of each strain is now possible for differently labelled strains. Simultaneous detection of bacteria marked with different GFP variants has previously been demonstrated [23]. Multichannel detection by CLSM might also be exploited when DsRed- or GFP-marked fungi are to be followed simultaneously on plant surfaces. The GFP variants, emitting blue, cyan and yellow light, have overlapping emission spectra, which means that CLSM and equivalent equipment is necessary for proper discrimination. In contrast, conventional fluorescence microscopy is sufficient to distinguish between co-expressed DsRed-Express and GFP (Fig. 2), because of the considerable difference of the spectral properties of DsRed and GFP. DsRed-Express has excellent properties together with GFP for use in dual marking. The two Trichoderma strains used in this study are under development as biological control agents in a commercial product [24]. Thus, labelling one strain with GFP (unpublished results) and the other with DsRed-Express opens the possibility for studying the performance of each strain simultaneously in situ.

The constitutive A. nidulans gpd promoter was used for expression of DsRed-Express in three different ascomycete fungi. Since the gpd promoter is known to function in a large number of Ascomycetes, we expect that our construct will have general utility for DsRed-Express expression in additional species.


Michael Hansen is gratefully acknowledged for CLSM. Jane Kristine Rohde Hansen, Laila Thuesen Simonsen and Anje Che Lawrance are acknowledged for the GFP-transformed P. paxilli strain. Mari-Anne Newman is gratefully acknowledged for commenting on the manuscript. The University of Pisa and Giovanni Vannacci are acknowledged for their support to S.S. to accomplish her PhD program.


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