Fungal Genetics and Biology 42 (2005) 191–199 www.elsevier.com/locate/yfgbi
Technological advancement
EYcient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns C. Burnsa, K.E. Gregoryb, M. Kirbya, M.K. Cheunga, M. Riquelmec, T.J. Elliottb, M.P. Challenb, A. Baileya, G.D. Fostera,¤ a
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK b Warwick HRI, Wellesbourne, Warwick CV35 9EF, UK c Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK Received 27 November 2003; accepted 12 November 2004 Available online 5 January 2005
Abstract We have developed a “Molecular Toolkit” comprising interchangeable promoters and marker genes to facilitate transformation of homobasidiomycete mushrooms. We describe the evaluation of a range of promoters in the homobasidiomycetes Agaricus bisporus and Coprinus cinereus using green Xuorescent protein (GFP) as a reporter gene; the C. cinereus trp1 promoter and A. bisporus trp2 and gpdII promoters proving successful in driving expression in C. cinereus, with the gpdII promoter also functioning in A. bisporus. Our investigations demonstrate that a prerequisite for GFP expression in C. cinereus and A. bisporus is the presence of an intron. This is the Wrst reported expression of GFP in either C. cinereus or A. bisporus. 2004 Elsevier Inc. All rights reserved.
1. Introduction Green Xuorescent protein (GFP) expression is now a widely used tool in molecular analysis of Wlamentous fungi, as reviewed by Lorang et al. (2001). Although modiWcations of the gfp gene have been necessary for expression, such as base alterations to give preferential codon usage, removal of cryptic intron sites, and a serine to threonine shift at amino acid 65 (Chiu et al., 1996; Cubitt et al., 1995; Yang et al., 1996), few further problems have been experienced. Most of these fungi have been ascomycetes, with new reports of expression being commonplace: recent species to express GFP include Acremonium chrysosporium, Sordaria macrospora, (Pöggeler et al., 2003), Colletotrichum acutatum (Horowitz et al., 2002), and Verticillium fungicola (Amey et al.,
*
Corresponding author. Fax: +44 117 925 7374. E-mail address:
[email protected] (G.D. Foster).
1087-1845/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.11.005
2002). DiVerent coloured Xuorescent proteins are also being developed for use as markers in Wlamentous fungi (Bourett et al., 2002). Despite these advances, to date, GFP has been successfully expressed in only two homobasidiomycetes, Schizophyllum commune (Lugones et al., 1999) and Phanerochaete chrysosporium (Ma et al., 2001). In both these cases, an intron was needed before signiWcant levels of GFP expression were seen; for S. commune the intron was immediately downstream of the 3⬘ end of the gfp coding region, and in P. chrysosporium an intron was embedded between two native exons immediately before the gfp coding region. Transgene expression in basidiomycetes appears to be hampered by a number of phenomena. In the model species Schizophyllum commune, transforming DNA is inactivated by preferential methylation (Mooibroek et al., 1990), ATrich sequences inactivate gene expression (Scholtmeijer et al., 2001; Schuren and Wessels, 1998), and introns are needed for mRNA accumulation to occur (Lugones et al., 1999; Scholtmeijer et al., 2001).
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The homobasidiomycete Agaricus bisporus, or common button mushroom, is the most broadly cultivated mushroom worldwide, with annual production in the region of Wve million tonnes (Kües and Liu, 2000), and an estimated commercial value of £3000 million (Scrase and Elliott, 1998). In the UK alone A. bisporus accounts for around 10% of total horticultural production (Elliott, 1997), with a farm gate value of £140 million in 1999. Application of biotechnology to this commercially signiWcant fungus was hampered until relatively recently by the lack of an eYcient transformation system. Although protoplast based transformation was reported (van de Rhee et al., 1996a,b) this proved relatively ineVective and not readily transferable between laboratories. It was not until Agrobacterium tumefaciens mediated transformation was developed that an eYcient, reproducible method became available (Chen et al., 2000; de Groot et al., 1998; Mikosch et al., 2001). Despite various attempts at development of other selectable markers (Challen and Elliott, 1987), the only reported transgene expression to date has been the Escherichia coli hygromycin phosphotransferase gene (hph), conferring hygromycin B resistance. In A. tumefaciens mediated transformation, Mikosch et al. (2001) used a derivative of vector pAN7-1 (Punt et al., 1987), utilising an Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter to drive expression of hph. In a separate study the A. bisporus gpdII promoter was used with hph, and also with the enhanced gfp gene (eGFP), but although integration of gfp was demonstrated, no expression was detected (Chen et al., 2000). To date, there have been no descriptions of “reporter” gene expression (e.g., luciferase, -glucuronidase, or GFP) in A. bisporus. Coprinus cinereus, commonly known as the inky cap, is a well-studied model homobasidiomycete mush room (Casselton, 1995; Kües, 2000). Transformation of C. cinereus was Wrst reported by Binninger et al. (1987), using a protoplast/PEG mediated complementation of a trp1 auxotrophy with the cloned wild-type trp1 gene. The complementation of trp1 has proved highly eVective and is routinely used with co-transformation to introduce secondary transgenes. Although some other auxotroph based systems (Casselton, 1995; Granado et al., 1997) and dominant selectable markers (Bhattiprolu et al., 1993; Cummings et al., 1999) have been used in Coprinus, hitherto, as with Agaricus, there are no descriptions of reporter gene expression in C. cinereus. As part of our eVort to optimise and improve transformation of A. bisporus we have attempted to develop an “Homobasidiomycetes Molecular Toolkit” to enable rapid interchange of promoter and marker gene elements. In this manuscript, we report the use of this toolkit to assess eYcacy of promoters in C. cinereus, and to demonstrate that intron containing constructs lead to GFP expression in both C. cinereus and A. bisporus.
2. Materials and methods 2.1. Strain and culture maintenance Escherichia coli strain DH5 was used in the construction and transformation of recombinant plasmids. Agrobacterium tumefaciens strains AGL-1 (Hellens et al., 2000) and LBA1126 (Beijersbergen et al., 1992; Bundock and Hooykaas, 1996) were used for A. bisporus transformation. The A. bisporus commercial strain Amycel 2100 was used for transformations. A tryptophan auxotroph strain, LT2 (Binninger et al., 1987) was used for C. cinereus transformations. 2.2. Culture conditions Agaricus bisporus mycelia were maintained at 25 °C on MMP agar plates (Mikosch et al., 2001), and supplemented with hygromycin B to select transformants. Coprinus cinereus mycelia were maintained at 37 °C on YMG agar plates supplemented with 100 g ml¡1 L-tryptophan (Binninger et al., 1987). Transformants were selected and maintained on RA medium (Cummings et al., 1999). 2.3. Transformation of Coprinus cinereus Protoplasts were prepared from oidia of C. cinereus monokaryon LT2 (trp1.1, trp1.6) and co-transformed with ca. 1 g of plasmid pCc1001, which harbours the homologous trp1 gene (Skrzynia et al., 1989), and ca. 1 g of an appropriate secondary plasmid, as previously described (Binninger et al., 1987; Cummings et al., 1999). Trp+ transformants were maintained on RA medium (Cummings et al., 1999) before screening for GFP expression. DNAs for transformation were prepared using Qiagen Midi Prep Kits. 2.4. Transformation of Agaricus bisporus Agaricus bisporus was transformed using A. tumefaciens mediated transfer of DNA as described previously (Challen et al., 2000; Chen et al., 2000; de Groot et al., 1998; Mikosch et al., 2001). Binary vectors were transformed into A. tumefaciens by electroporation (Shen and Forde, 1989). Pieces of A. bisporus gill tissue were vacuum inWltrated with A. tumefaciens cultures (Chen et al., 2000). Agaricus–Agrobacterium co-cultures were incubated for 2–3 days after which the gill pieces were transferred to MMP containing 30 g ml¡1 hygromycin B. Putative resistant transformants were transferred to MMP containing 50 g ml¡1 hygromycin B and screened for GFP expression. 2.5. Screening for GFP expression GFP expression was initially detected in C. cinereus and A. bisporus by microscopic screening using Leica
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UV microscopes (DMKLB or MZFL111) with excitation Wlters at 450–490 nm, dichroic Wlter at 510 nm, and emission Wlter at 515 nm. Further imaging of C. cinereus and A. bisporus samples were obtained on a Zeiss Model LSM510 laser scanning confocal microscope in the Department of Plant Sciences of the University of Oxford, UK, using 488 nm excitation line and a bandpass BP505–550 emission Wlter and Wtted with a 63£ oil plan-apochromat objective (NA 1.4). 2.6. Fungal DNA isolation Mycelia were grown on cellophane discs overlaid on MMP agar plates or YMG agar plates where appropriate. Once the discs were colonised fully, the fungal mycelia were harvested and used for genomic DNA isolation as described previously (Zolan and Pukkila, 1986). Growth times were ca. 10 days for C. cinereus, and ca. 5 weeks for A. bisporus. 2.7. Polymerase chain reaction Presence of the transgenes was conWrmed within transformants by PCR which was carried out using ReddyMix 2£ PCR buVer (AbGene) with a general thermal cycle program of 95 °C for 5 min, (94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min) £ 30, 72 °C for 12 min.
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3. Plasmid construction 3.1. Construction of 5⬘ intron GFP constructs for C. cinereus transformation Novel restriction sites were introduced into an intronegfp -terminator amplicon of pUGiGM3⬘ (Ma et al., 2001) using PCR mutagenesis; a NcoI site was created at the 5⬘ end (primer pug-1: tacttgaccatggcggtcagtaca), and a KpnI site at the 3⬘ end (primer Kpn-utr: ccgggtaccagtccacaatgacag). On restriction with these enzymes, this cassette was used to replace the marker gene-terminator region in a number of toolkit plasmids, providing a range of GFP plasmids with diVerent basidiomycete promoters (Fig. 1 and Table 1). 3.2. Construction of 5⬘ intron GFP constructs for Agaricus bisporus transformation Agrobacterium mediated DNA transfer is used for A. bisporus transformation, requiring use of a binary plasmid. Thus, the transformation cassette from the Homobasidiomycetes Molecular Toolkit was cloned into the binary plasmid pGREENII (Hellens et al., 2000). Plasmid phph004 (see Table 1) and pGREENII were digested with KpnI and SacI, and ligated together to form pGRhph004.
Fig. 1. (A) Homobasidiomycetes Molecular Toolkit. A plasmid toolkit was developed for use in transformation of basidiomycetes, comprising several promoter and marker elements, readily interchangeable with the restriction sites shown. Table 1 lists the range of promoters used within this cassette. (B) Basidiomycete promoters linked to the intron-gfp-UTR region from pUGiGM3⬘. The intron-GFP-UTR region was ampliWed from plasmid pUGiGM3⬘ (Ma et al., 2001) and was cloned into the Homobasidiomycetes Molecular Toolkit, thus linking the gfp section to a range of basidiomycete promoters. These plasmids were used in transformation of C. cinereus. Table 1 details the promoters used. 3⬘UTR from P. chrysosporium. (C) Binary GFP plasmids for transformation of A. bisporus. The hygromycin phosphotransferase gene (hph), under the control of the A. bisporus gpdII promoter was cloned into binary plasmid pGREEN (Hellens et al., 2000) for A. tumefaciens mediated transformation. pUGiGM3⬘ and p004iGM3⬘ were cloned into this binary plasmid to give pGR4GFP and pGR4-4iGM3⬘, respectively. The plasmids are identical except for the promoter controlling the GFP element; in pGR4GFP this is the P. chrysosporium gpd promoter, and in pGR4-4iGM3⬘ this is the A. bisporus gpdII promoter. Details are given in Table 1. 3⬘UTR from P. chrysosporium.
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Table 1 Homobasidiomycete promoters used in the Agaricus Molecular Toolkit Gene description
GenBank Accession No.
Reference(s)
PCR primers used for promoter isolation.
Size (bp)
Construct series code
C. cinereus trp1
Tryptophan synthetase
AH002542
Skrzynia et al. (1989)
cc.p1: cgccgcggccatgatgatgacggtagac cc.p2: ggcgggatccatccatgggagatcaagggtgag
321
p001
A. bisporus cel1
Cellulose growth speciWc protein. Expressed in the presence of cellulose, repressed by other sugars
M86356
Raguz et al. (1992)
cel1.p3: cgcccgcggggtttgatctaccacatgac cel1.p4: ggcgggatccgccaccatggtgagtacttg
500
p002
A. bisporus trp2
Tri-functional protein involved in tryptophan synthesis
AJ298078
Challen et al. (1996); Challen et al. (2002)
trp2.p1: cgccgcggccctgggcttcgttcgccacg trp2.p2: ggcgggatcctgccatggtggatatagagtcg
342
p003
A. bisporus gpdII
Glyceraldehyde-3-phosphate dehydro genase. Constitutively expressed
M81728
Harmsen et al. (1992)
gpd.p2: cgcccgcgggaagaagaattcagaggtccgcaagt gpd.p3: ggcggatccgagagacaaaccatggcgataagc
277
p004
A. bisporus cel3
Cellobiohydrolase. Strongly expressed in fruit body
L24519
Chow et al. (1994)
cel3.p1: cgcccgcggctggctgggtaagtttgggtccgtatgctcg cel3.p2: ggcgggatccgaccatgggttcaagagaaggatggcag
550
p005
A. bisporus gdhA
Glutamate dehydrogenase. Repressed in the presence of ammonium, upregulated with glutamate
X83393
Schaap et al. (1996)
gdh.p1: cgccgcggggaatggaattacgccgctcggg gdh.p2: ggcgggatccgtgaggaaggaccatggtgta
450
p006
A. bisporus hypA
Hydrophobin. Tissue speciWc to cap tissue of the fruit body
X90818
de Groot et al. (1996)
hyp.p1: cgccgcggggcctctagactaagcagagtccc hyp.p2: ggcgggatccggacgcgagagaccatggtg
600
p007
A. bisporus lcc1
Laccase. Expressed in mycelia but downregulated in fruiting body
L10664
Perry et al. (1993)
lcc1.p1: cgcccgcggctacgtcttgacagtatgtc lcc1.p2: ggcgggatccgccccatggttgatcctacc
460
p008
C. cinereus -tub
-Tubulin. Constitutively expressed
AB000116
Matsuo et al. (1999)
-tub1: tccccgcggttcagtctccctggttttgg -tub2M: tcacccatggtgggaacgcgagg
453
p010
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Species/ promoter
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3.3. Construction of pGR4-GFP The GFP cassette, including P. chrysosporium regulatory sequences, was excised from pUGiGM3⬘ using XbaI and EcoRI, and cloned into similarly digested pLitmus28 (New England Biolabs) to form pLitGFP. pGRhph004 and pLitGFP were then digested with XhoI and Kpn I and the pertinent fragments ligated to form pGR4-GFP. 3.4. Construction of pGR4-4iGM3⬘ The promoter-GFP cassette from p004iGM3⬘ was ampliWed by polymerase chain reaction to introduce a HpaI site at the 5⬘ end of the gpdII promoter. The primers used were Kpn-utr, as above, and Hpa-004-long: cccgttaacgaagaagaattcagaggtccg. Restriction with these enzymes allowed the GFP cassette to be cloned alongside the hygromycin B selection cassette to form plasmid pGR4-4iGM3⬘.
4. Results 4.1. Homobasidiomycetes Molecular Toolkit A toolkit was developed whereby a range of promoters from A. bisporus and C. cinereus linked to marker genes could be readily exchanged via conserved restriction sites. All constructs utilise the 3⬘ tryptophan synthetase (trpC) terminator from the ascomycete A. nidulans isolated from the plasmid pAN-7 (Punt et al., 1987), and the cassettes are cloned into pBluescript SK-(Stratagene). Promoters, markers and the terminator were all cloned using conserved restriction sites for ready interchange of transformation cassette elements (Fig. 1). Marker genes initially used in the toolkit were luciferase, sGFP (Sheen et al., 1995), and -glucuronidase, all of which are well-established markers used in other systems. Plasmids developed in the toolkit were labelled systematically, with numbers denoting the promoter used, and letters describing the marker gene as follows: pBUƒ (luciferase), pGUSƒ (uidA), and pGFPƒ (sGFP). Nine promoters were isolated from regions directly upstream of diVerent A. bisporus and C. cinereus genes, some of which are constitutively expressed, with others nutritionally or developmentally regulated (see Table 1). As for the marker genes, the promoters used were number coded as follows; pƒ001 (C. cinereus trp1), pƒ003 (A. bisporus trp2), pƒ004 (A. bisporus gpdII), pƒ008 (A. bisporus lcc1), and pƒ010 (C. cinereus -tub) (full list is provided in Table 1). Transformation of and transient expression in A. bisporus was attempted with the full range of toolkit plasmids, using a range of techniques including particle bombardment, electroporation, PEG/CaCl2 mediated
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transformation, and liposome transformation, but no expression of GUS, LUC or GFP, transient or stable, was observed in any experiments (results not presented). Plasmids were also co-transformed into C. cinereus LT2 with pCc1001, using the basic toolkit and plasmids with a promoter-gene-terminator arrangement, with high numbers of co-transformants recovered for all constructs. Despite extensive screening, no expression of GUS, LUC or GFP could be detected in any of the transformants. In summary, transgene expression was not seen for any of the toolkit plasmids, using any promoter-geneterminator arrangement. 4.2. Transformation of Coprinus cinereus with pUGGM3⬘ and pUGiGM3⬘ As no expression was observed with any of the constructs described above, it was concluded that an additional element such as the presence of an intron might be required to obtain detectable levels of expression in these homobasidiomycetes. Such elements have been shown to be a prerequisite for expression in a number of other systems, including the basidiomycete P. chrysosporium in which Ma et al. (2001) demonstrated that a 5⬘ intron is essential for detectable levels of GFP expression. Coprinus cinereus strain LT2 was co-transformed with pCc1001 and either pUGiGM3⬘ or pUGGM3⬘ (Ma et al., 2001). These plasmids contain the P. chrysosporium gpd promoter, GFP, and a P. chrysosporium 3⬘ UTR. pUGiGM3⬘ has a 5⬘ intron from the P. chrysosporium gpd gene within a small section of gpd coding region directly upstream of the GFP gene, whereas pUGGM3⬘ has no intron. No GFP expressing colonies were seen in transformations with pUGGM3⬘, despite conWrmation of presence of transforming DNA by PCR (results not presented); at least 100 colonies per experiment were screened. However, in a series of experiments, an average of 25% of the trp+ transformants exhibited GFP expression when co-transformed with pUGiGM3⬘; this is in accordance with co-transformation rates seen by others (Casselton, 1995). Transformation of GFP expressing colonies was conWrmed by PCR; however, some additional non-expressing colonies were also screened by PCR (results not presented). This indicates that an intron is necessary for eGFP expression in C. cinereus, but that transformation does not guarantee expression. C. cinereus hyphae and oidia expressing GFP are illustrated in Fig. 2. 4.3. Re-evaluation of toolkit promoters in C. cinereus Promoter eYcacy in C. cinereus was tested using a range of promoters (C. cinereus -tub, trp1 and A. bisporus trp2, gpdII) linked to the intron-GFP-terminator fragment from pUGiGM3⬘. Three of these (C. cinereus
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Fig. 2. (A–J) Expression of GFP in C. cinereus mycelia and oidia expressing green Xuorescent protein from plasmid pUGiGM3⬘. Coprinus cinereus monokaryotic mycelia and oidia were removed from an actively growing colony, suspended in sterile water, and examined microscopically using 40£ objective. (A, C, E, and G) Viewed using phase contrast microscopy; (B, D, F, and H) were visualised using UV light. (A and B) Untransformed mycelia; (C and D) GFP-transformed mycelia; (E and F) untransformed oidia; and (G and H) GFP-transformed oidia. (I and J) Confocal microscope images of C. cinereus transformants expressing GFP. Images clearly show GFP expression in C. cinereus cytoplasm, with no GFP expression in vacuolar spaces (63£ objective).
trp1, A. bisporus trp2, and gpdII) were successful in promoting GFP expression in C. cinereus (see Table 2). As well as the native C. cinereus trp1 promoter, elements from the basidiomycetes P. chrysosporium and A. bisporus were found to be functional in C. cinereus, demonstrating utility and exchange of a wide range of promoters in the homobasidiomycetes. GFP expression was shown to be stable through multiple rounds of subculturing.
The only promoter that proved ineVective was our C. cinereus -tub. This is perturbing, as the -tub promoter region has previously been used to mediate the transformation of C. cinereus to hygromycin B resistance (Cummings et al., 1999): sequence of the -tub promoters are identical with the exception of an additional 5⬘ 60 bp in our toolkit version. LT2 co-transformants with GFP under the control of the constitutive A. bisporus gpdII promoter were mated with compatible mono-
C. Burns et al. / Fungal Genetics and Biology 42 (2005) 191–199
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Fig. 3. Expression of GFP in A. bisporus transformed with pGR4-4iGM3⬘. In (A–D), A. bisporus mycelia were removed from an actively growing colony, suspended in sterile water, and examined microscopically using 40£ objective. (A and C) Viewed using phase contrast microscopy; (B and D) were visualised using UV light. (A and B) Untransformed mycelia; (C and D) mycelia transformed with eGFP plasmid pGR4-4iGM3⬘ and expressing Xuorescence. Although some background Xuorescence was seen in non-transformed A. bisporus mycelia illuminated with UV light, there was a clear diVerence between transformed and non-transformed strains. (E, F, and G) Confocal microscopy of A. bisporus mycelia expressing green Xuorescent protein (63£ objective). (E) Untransformed mycelia; (F and G) mycelia transformed with eGFP plasmid pGR4-4iGM3⬘ and expressing Xuorescence. Although some background Xuorescence was seen in non-transformed Agaricus bisporus mycelia illuminated with UV light, there was a clear diVerence between transformed and non-transformed strains. Background Xuorescence in wild-type Agaricus bisporus was vacuolar (and yellow), and faded quickly when illuminated with UV light. In transformed mycelia, Xuorescence was green and evenly distributed.
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C. Burns et al. / Fungal Genetics and Biology 42 (2005) 191–199
Table 2 Proportion of trp+ C. cinereus transformants which expressed GFP, with GFP under the regulation of a variety of basidiomycete promoters Plasmid name
Promoter
Average % of trp+ transformants expressing GFP
p001iGM3 p003iGM3 p004iGM3 p010iGM3
Coprinus cinereus trp1 Agaricus bisporus trp2 Agaricus bisporus gpdII Coprinus cinereus -tub
21 34 49 0
A total of 190 GFP expressing transformants were isolated.
karyons to form fruiting dikaryons. GFP expression was maintained in dikaryotic mycelia (results not presented). 4.4. GFP expression in A. bisporus Binary plasmids pGR4-GFP and pGR4-4iGM3⬘ were transformed into A. bisporus. These binary plasmids contain a hygromycin B cassette for transformant selection, and either the full GFP cassette from pUGiGM3⬘ driven by the P. chrysosporium gpd promoter, designated pGR4-GFP, or the intron-GFP-UTR section linked to the A. bisporus gpdII promoter, designated pGR4-4iGM3⬘. Hygromycin B resistant colonies were initially screened for transgenes by PCR (results not presented) and subsequently screened microscopically for GFP expression. GFP expression was not detectable in any of the hygromycin resistant pGR4GFP transformants, despite conWrmation of GFP DNA presence by PCR. Conversely, all the pGR4-4iGM3⬘ transformants strongly expressed GFP in mycelia (Fig. 3). Chen et al. (2000) used an almost identical binary construct (pBGgHg), without intron, which produced hygromycin B resistant colonies but no detectable GFP expression, suggesting that an the presence of an intron is required for expression, similar to the observed results for C. cinereus. GFP expression was shown to be stable through multiple rounds of sub-culturing.
5. Discussion This is the Wrst report of GFP expression in the cultivated mushroom A. bisporus and the model ink cap species C. cinereus; previous attempts to express marker genes in these species were not successful (Chen et al., 2000; Gregory, 2001). There are only two previous examples of GFP expression in homobasidiomycetes; Schizophyllum commune (Lugones et al., 1999) and Phanerochaete chrysosporium (Ma et al., 2001) and in both these species introns were required for expression. Using a 5⬘ intron based on the system used by Ma et al. (2001) to transform P. chrysosporium, we have tested intron necessity for GFP expression in A. bisporus
and C. cinereus. In transformation of C. cinereus with plasmids pUGiGM3⬘ (with intron) and pUGGM3⬘ (no intron), only the intron containing plasmid gave any visible GFP expression, showing that an intron is required for GFP expression in this fungus. In A. bisporus, a previous unsuccessful attempt had been made to express GFP (Chen et al., 2000), using the GFP gene under the control of the A bisporus gpdII promoter. By using the same promoter and GFP with a 5⬘ intron, we have shown that A. bisporus gpdII promoter can drive GFP expression in A. bisporus and four diVerent promoters (C. cinereus trp1, A. bisporus trp2 and gpdII, P. chrysosporium gpd) were successful in promoting GFP expression in C. cinereus. We have also used successful GFP expression in C. cinereus to extend the range of available promoters for use in basidiomycete transformation, and in doing so we have shown that promoters from three diVerent basidiomycetes are eVective in C. cinereus. The toolkit we have developed allows easy exchange of promoter, marker gene, and terminator elements and will facilitate further analysis of transformation in A. bisporus and C. cinereus and other basidiomycetes. Acknowledgments We thank Dr. Mike Gold for provision of plasmids pUGiGM3⬘ and pUGGM3⬘. This work was funded by the BBSRC, and through a BBSRC CASE award in collaboration with HRI. References Amey, R.C., Athey-Pollard, A., Burns, C., Mills, P.R., Bailey, A., Foster, G.D., 2002. PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola. Mycol. Res. 106, 4–11. Beijersbergen, A., Dendulkras, A., Schilperoort, R.A., Hooykaas, P.J.J., 1992. Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 256, 1324–1327. Bhattiprolu, G.R., Challen, M.P., Elliott, T.J., 1993. Transformation of the homobasidiomycete Coprinus bilanatus to 5-Xuoroindole resistance using a mutant trp3 gene from Coprinus cinereus. Mycological Research 97, 1281–1286. Binninger, D.M., Skrzynia, C., Pukkila, P.J., Casselton, L.A., 1987. DNA-mediated transformation of the basidiomycete Coprinus cinereus. EMBO J. 6, 835–840. Bourett, T.M., Sweigard, J.A., Czymmek, K.J., Carroll, A., Howard, R.J., 2002. Reef coral Xuorescent proteins for visualizing fungal pathogens. Fung. Genet. Biol. 37, 211–220. Bundock, P., Hooykaas, P.J.J., 1996. Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc. Natl. Acad. Sci. USA 93, 15272–15275. Casselton, L.A., 1995. Genetics of Coprinus. In: Kuck, U. (Ed.), The Mycota II: Genetics and Biotechnology. Springer-Verlag, Berlin, pp. 35–46. Challen, M.P., Elliott, T.J., 1987. Production and evaluation of fungicide resistant mutants in the cultivated mushroom Agaricus bisporus. Trans. Brit. Mycol. Soc. 88, 433–439.
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