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Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.
Key words: fluorescent proteins, fungal promoter, heterologous expression, Pyrenophora tritici-repentis, ToxA, ToxB
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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developed a versatile fungal transformation vector for the expression of the green fluorescent protein (GFP) in filamentous ascomycetes. Vector pCT74 employs the promoter from the Pyrenophora tritici-repentis necrosis-inducing host-selective toxin gene ToxA (Ciuffetti et al 1997) to drive expression of the synthetic GFP gene, sGFP (Chiu et al 1996). The form of sGFP used here possesses human-optimized codon usage, incorporates nucleotide changes that lead to a serine-to-threonine mutation at position 65 of the chromophore, and lacks a cryptic intron donor site that abolished GFP expression in Arabidopsis (Chiu et al 1996). This vector was shown to confer bright cytoplasmic sGFP fluorescence to plant pathogens belonging to eight genera of the Ascomycota where promoters from Aspergillus, Neurospora and Colletotrichum had failed to produce acceptable levels of GFP fluorescence (Lorang et al 2001). Although other fungal GFP expression vectors were also available, none had been evaluated in such a diverse range of fungi. Since then numerous members of the Ascomycota have been successfully transformed with pCT74, including the model fungus Neurospora crassa (Freitag et al 2001), as well as both Trichoderma harzianum, a known biocontrol agent, and Ophiostoma picea, a bluestain fungus of harvested lumber (Xiao et al 2003). Thus, the ToxA promoter has proven very useful for strong expression of GFP in ascomycetes with diverse lifestyles.
Due to the success of ToxA promoter-driven sGFP expression in fungi of the Ascomycota, we sought to use the ToxA promoter to express additional fluorescent protein color variants. Since the isolation of the gfp gene from the jellyfish Aequorea victoria (Prasher et al 1992) and demonstration of it as a fluorescent marker in heterologous organisms (Chalfie et al 1994, Inouye and Tsuji 1994), there has been a continuous effort to develop new GFP variants with altered fluorescent properties (Tsien 1998, Lippincott-Schwartz and Patterson 2003). In addition to improving GFP fluorescence, mutagenesis studies have resulted in color variants with various absorbance and emission spectra. The development of cyan fluorescent protein (CFP) (Heim and Tsien 1996) and yellow fluorescent protein (Y FP) (Ormö et al 1996) made simultaneous visualization of two distinct GFP variants practical (Ellenberg et al 1998, Stuurman et al 2000) and provided an ideal pair for fluorescent energy transfer (FRET) analysis (Tsien 1998, Dye et al 2005). However, the production of a red mutant of GFP from A. victoria for use in multi-spectral imaging has met with little success (Lippincott-Schwartz and Patterson 2003, Czymmek et al 2005).
Fortunately, red fluorescent proteins have been discovered in other marine organisms, though they fluoresce with variable efficacy (Matz et al 1999, Lukyanov et al 2000, Gurskaya et al 2001, Fradkov et al 2002, Labas et al 2002, Wiedenmann et al 2002, Palmer et al 2004). The discovery of the first red fluorescent protein, DsRed, from a coral in the genus Discoma was well received (Matz et al 1999), however its effectiveness was limited by slow maturation times, obligate oligomerization, and emission of fluorescence when excited at wavelengths optimal for GFP (Baird et al 2000). Of the optimized forms of DsRed since developed (Verkhusha et al 2001, Bevis and Glick 2002, Campbell et al 2002, Terskikh et al 2002), mRFP1, a monomeric form developed by Campbell et al (2002), shows promise as the superior partner with GFP for colocalization or FRET analysis and as the preferred candidate for construction of fusion proteins (Campbell et al 2002, Zhang et al 2002). In addition to the red variants, other reef coral fluorescent proteins (RCFPs) also have been added to the palette of available fluorescent proteins, including AmCyan, ZsGreen and ZsYellow (Matz et al 1999, Labas et al 2002, Gurskaya et al 2003, Carter et al 2004, Czymmek et al 2005). However, like DsRed, the other RCFPs are known to form oligomers and high molecular weight aggregates (Czymmek et al 2005). Thus, from the available GFP variants, we chose EYFP, ECFP and mRFP1 for inclusion in ToxA promoter-driven expression vectors. Combined with sGFP, these three colors provide a range of spectral profiles to address challenges with autofluorescence (Tsien 1998), as well as two pairs of proteins with non-overlapping spectra ideal for in vivo colocalization studies and FRET analysis.
The nine copies of ToxB, a chlorosis-inducing host-selective toxin gene from P. tritici-repentis (Martinez et al 2001, 2004), potentially offer multiple promoters for heterologous expression of proteins in fungi. Though the putative promoters of at least five ToxB loci share considerable sequence homology, some variation exists (Martinez et al 2004). The presence or absence of certain sequence elements could contribute to differences in expression by each promoter (Gold et al 2001, Martinez et al 2004). To initially assess heterologous expression from the putative ToxB promoter, we chose the ToxB1 promoter to express sGFP in a heterologous fungus, Verticillium dahliae.
We report a new set of fluorescent protein expression vectors and an additional promoter for general use in filamentous ascomycetes. In addition to sGFP (Lorang et al 2001), the ToxA promoter drives strong expression of EY FP, ECFP and mRFP1 in both P. tritici-repentis and V. dahliae. In V. dahliae, the ToxB promoter drives comparable sGFP expression to that of the ToxA promoter, demonstrating its capacity for heterologous gene expression. We anticipate these vectors will be useful for the study of fungal biology and fungi-host interactions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Martinez et al 2001) and a V. dahliae isolate from potato. P. tritici-repentis was grown on solid V8 agar (20% v/v V8, 0.3% w/v CaCO3, 2% w/v Bacto agar) in constant darkness, 25 C. V. dahliae was grown on solid, half-strength potato dextrose agar (PDA; BD Difco, Sparks, Maryland) amended with 1 ppm streptomycin (Fisher Scientific, Pittsburgh, Pennsylvania) in constant illumination at 25 C. V. dahliae sporulates under these conditions. However, P. tritici-repentis requires induction of conidiation by flooding a culture with sterile water and manually flattening the mycelia followed by 18–24 h of light at room temperature and 18–24 h of dark at 16 C (Lamari and Bernier 1989). Hygromycin-resistant transformants of P. tritici-repentis were transferred onto PDA with 15 µg/mL hygromycin B (In-vitrogen, Carlsbad, California); V. dahliae transformants were transferred onto half-strength PDA with 1 ppm streptomycin and 50 µg/mL hygromycin B.
Fluorescent protein expression vectors.— –
Vector pCT73, which contains sGFP under the control of the ToxA promoter and nos terminator (Lorang et al 2001), was digested with NcoI and SmaI to remove sGFP-nos. To make pCA42 and pCA43, sGFP-nos was replaced by ECFP-nos and EYFP-nos fragments from pAN94 and pAN95, respectively, which were generated via digestion with SalI, treatment to fill-in 5'-protruding ends, and digestion with NcoI. Both vectors then were digested with SalI, followed by ligation to an ~1.6 kb SalI hygromycin resistance cassette fragment (Carroll et al 1994) from pCT48 (Ciuffetti et al 1997) to produce pCA49 and pCA45. The orientation of the hygromycin B phosphotransferase gene, hph, was confirmed by sequence analysis (Central Services Lab, Center for Genome Research and Biocomputing, Oregon State University, Corvallis). The hph gene is in the opposite direction of CFP in pCA49 and YFP in pCA45. The final constructs are ~5.8 kb.
To produce pCA51, we used the following primers to engineer restriction enzymes sites on either side of mRFP1 (Campbell et al 2002) from pCB302Hsp70h-mRFP (Prokhnevsky et al 2005) via polymerase chain reaction (PCR). The forward primer mRFP1 (5'-CATATCCCGGGATGGCCTCCTCCGAGGACGTCATC-3') introduced a 5' SmaI site, and the reverse primer mRFP2 (5'-CATATGGATCCAAGCTTTTAGGCGCCGGTGGAGTG-3') introduced sites for HindIII and BamHI 3' of mRFP1. PCR was performed on ~10 ng plasmid DNA in a standard 50 µL reaction under standard PCR reaction conditions with a 62 C annealing temperature. The amplification product was ligated with pGEM-T Easy (Promega, Madison, Wisconsin) and a clone chosen by sequence analysis. Construct pCA51 was digested with SmaI and BamHI to release mRFP1, which was then inserted into pCT73 vector DNA that had been digested with NcoI, treated to fill-in 5'-protruding ends, digested with BamHI, and gel purified away from sGFP-nos. The resultant construct, pCA53, was digested with NotI for the insertion of ~0.4 kb NotI nos-terminator fragment from pCT73 to form pCA54. The Sal I hygromycin resistance cassette fragment was inserted into SalI-digested pCA54 to create pCA56. The resultant construct, pCA56, is ~5.9 kb.
For pCM56, a portion of the ToxB1 locus (GenBank accession AY425480
[GenBank]
) was amplified from genomic DNA of P. tritici-repentis isolate DW7 with primers TB21 and TB29 (Martinez et al 2004). The PCR product was ligated with pGEM-T Easy to form pCM29. The putative ToxB promoter was PCR amplified from pCM29 with primers TB67 (5'-ATAATCGATTGTTGGAAGGCCTTGTAC-3') and TB68 (5'-GGCGCCATGGTCTAACAAGGGAT-3'). The PCR product was digested with ClaI and NcoI and ligated with pCT73 that had been digested with ClaI and NcoI and purified to remove the ToxA promoter. The resulting plasmid, pCM50, was then digested with XhoI and ClaI and ligated to an XhoI/ClaI hygromycin resistance cassette fragment (Carroll et al 1994) to form pCM56.
All PCR products and gel fragments were purified with QIAquick Spin Kits (Qiagen, Valencia, California). A QIAfilter Midi Plasmid Kit was used to purify high concentrations of each construct. For transformation of P. tritici-repentis, constructs were linearized with SacI and concentrated via standard ethanol precipitation.
Pyrenophora tritici-repentis transformation.— –
For P. tritici-repentis, transformation was via the protoplast method of Turgeon and colleagues (1985) with some modifications. Conidia were collected in water + 0.015% tween-20, inoculated into 100 mL quarter-strength potato dextrose broth (PDB; BD Difco, Sparks, Maryland) and grown at 25 C, 175 rpm, 16 h. After 16 h fungal tissue was ground, inoculated into fresh media and incubated for 2 h. Approximately 10 g of tissue was incubated in 100 mL enzyme-osmoticum (0.7 M NaCl, 3% ß-D-glucanase [Interspex, San Mateo, California] and 1% driselase [Sigma, St. Louis, Missouri]) for ~3 h at 30 C, 80 rpm. Protoplasts were collected through 100 µM pore-size nitex (Tetko Inc., DePew, New York). For each transformation reaction, 20 µg linear, plasmid DNA diluted 2-fold with STC (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5) were added to 1 x 107 total protoplasts in 200 µL. Transformation reactions were aliquoted equally between four plates of solid regeneration media (RM; 1.2 M sucrose, 0.1% yeast extract, 0.1% casein, 1.5% Bacto agar) at 2.5 x 106 protoplasts per plate and spread with a glass rod. After ~24 to 36 h at 25 C, the plates were overlaid with 15 mL 1% water agar (pH 5.9) amended with 60 µg/mL hygromycin B for a final hygromycin B concentration of ~30 µg/mL. Hygromycin-resistant transformants were evident after one week.
Verticillium dahliae transformation.— –
For V. dahliae, transformation occurred via the protoplast method of Dobinson (1995) with some modifications. Four flasks of 50 mL PDB + 0.001% thiamine were inoculated with V. dahliae spore solution to a final concentration of 6 x 106 conidia/mL. PDB was prepared by boiling 200 g potatoes in 1 L e-pure water and filtering the suspension through cheesecloth. 20 g dextrose were added to the filtrate and the volume adjusted to 1 L with e-pure water. Stationary cultures of V. dahliae were aerated 8 h, followed by centrifugation at 1900 x g, 10 min at 4 C. Pellets were resuspended in 1 mL mycelia buffer (10 mM Na2HPO4, pH 7.5, 10 mM EDTA, pH 8.0) for a 30 min, room-temperature incubation, followed by overnight digestion in enzyme-osmoticum (1 mg/mL Novozyme 234 in 1.2 M MgSO4·7H2O, 10 mM NaPO4, pH 5.8) at 25 C, 65 rpm. Protoplasts were collected and transformed as described by Ciuffetti and colleaques (1997). For each transformation reaction, 15 µg circular plasmid DNA diluted twofold with STC were added to a total of 1 x 107 protoplasts in 200 µL. Protoplasts were plated at 2 x 106 protoplasts per plate. Hygromycin-resistant and colleagues colonies were evident after one week.
Fungal slide mounts.— –
Three slide mounting techniques were used: (i) a square of media was placed on a slide, squashed with a cover slip and sealed with clear nail polish; (ii) 20 µL of a conidial suspension were added to a slide, cover-slipped and sealed; and (iii) the touch tape method developed by Harris (2000) was modified as follows. A wood applicator was attached to one end of a piece of clear adhesive tape. The free end of the tape was horizontally cut to allow for ease of detachment from the applicator. The tape was touched to a fungal mat and inverted on a microscope slide sticky side up. A cover slip was then placed over the sample and sealed for microscopic analysis. The media mount was used for both species for both confocal and fluorescence microscopy. The conidia mount was used in confocal microscopy of P. tritici-repentis and the tape mount was used in fluorescence microscopy of V. dahliae.
Microscopy.— – A Leica DMRB epifluorescence microscope (Leica Microsystems, Wetzler, Germany) or a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Jena GmbH, Jena, Germany) was used to observe slides of fluorescent transformants. The epifluorescence microscope was fitted with a mercury lamp and the appropriate filter sets (Chroma Technology Corp., Rockingham, Vermont) for visualization of sGFP (Endow GFP Bandpass Emission Set: HQ470/40x exciter, Q495LP dichroic, HQ525/50m emitter), EY FP (Yellow GFP BP [10C/Topaz] Set: HQ500/20x, exciter, Q515LP dichroic, HQ535/30m emitter), and mRFP1 (TRITC [Rhodamine]/DiI Red Shifted Emission Set: HQ535/ 50x exciter, Q565LP dichroic, HQ620/60m emitter). A Leica MZFLIII stereomicroscope equipped with a mercury lamp and the GFP Plant fluorescence filter set (470/40 nm excitation filter, 525/50 nm barrier filter; Chroma) was used to image the hyphae of V. dahliae transformed with ToxB promoter-driven sGFP. Image-Pro PLUS in conjunction with a CoolSNAP-Pro digital camera (MediaCybernetics, Silver Spring, Maryland) was used to capture images from both the epifluorescence microscope and stereomicroscope.
For confocal microscopy, sGFP, EY FP, and ECFP were excited with an argon laser at 10%, 10% and 17%, respectively, whereas a HeNe laser was used at 80% for mRFP1. The following beam splitter and filter configurations were used: (i) sGFP: HFT488, LP505; (ii) EYFP: HFT405/514, NFT490, BP530-600; (iii) mRFP1: HFT488/ 543, NFT545, LP560; and (iv) ECFP: HFT458, NFT515, BP470-500. The pinhole values for sGFP, EY FP, mRFP1 and ECFP were 114 µm, 114 µm, 127 µm, and 431 µm, respectively. Images were reconstructed in Zeiss LSM 510 software. For both fluorescence and confocal microscopy, slides of wildtype fungi were viewed under all settings as a check for background autofluorescence (data not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), which relies on antibiotic resistance conferred by a modified form of the E. coli hygromycin B phosphotransferase (hph) gene (Carroll et al 1994) for selection, was used as a template for construction of transformation vectors with the ToxA promoter driving expression of an additional set of fluorescent proteins. Genes for EY FP, ECFP, and mRFP1 were cloned downstream of the ToxA promoter to produce the constructs pCA45, pCA49 and pCA56, respectively. Sequence analysis confirmed successful vector construction.
To test the ability of the ToxA promoter (Ciuffetti et al 1997) to drive expression of these fluorescent proteins, the resultant constructs were transformed into two fungal species known to successfully express sGFP from the ToxA promoter (Lorang et al 2001). Positive transformants of P. tritici-repentis, the source of the ToxA promoter, and V. dahliae, an ascomycete distantly related to P. tritici-repentis (Lutzoni et al 2004), were first chosen for their ability to grow under hygromycin selection, followed by confirmation of EY FP and mRFP1 fluorescence via fluorescence or confocal microscopy, and ECFP fluorescence via confocal microscopy. For hygromycin-resistant transformants of both P. tritici-repentis and V. dahliae, wildtype colony morphology and conidiation were confirmed (data not shown). Confirmation of fluorescence by each type of transformant revealed a range of intensities, though only a few lacked any fluorescence (data not shown). The brightest individual of each was chosen for subsequent analysis.
As shown by fluorescence and confocal microscopy, the ToxA promoter successfully drove expression of all three fluorescent proteins, EY FP, ECFP and mRFP1, in the cytoplasm of both P. tritici-repentis and V. dahliae (FIGS. 1–8). Confocal imaging revealed bright EY FP (FIG. 2) and mRFP1 (FIG. 4) fluorescence in conidia and conidiophores of P. tritici-repentis, commensurate with the sGFP fluorescence of the original pCT74 transformant (FIG. 1) (Lorang et al 2001). ECFP fluorescence also occurred in conidia (FIG. 3) and conidiophores (data not shown), but required maximized settings to obtain a signal as intense as the other three fluorescent proteins (FIGS. 1, 2, 4). EY FP, ECFP, and mRFP1 fluorescence also was present in P. tritici-repentis hyphae (data not shown). V. dahliae revealed a similar pattern of fluorescence as P. tritici-repentis (FIGS. 5–8). Fluorescence microscopy showed that EY FP and mRFP1 expression in conidia, conidiophores, and hyphae of V. dahliae is similar to ToxA promoter-driven sGFP expression (Sawyer et al 1998), whereas confocal imaging of the same set of structures again required maximized settings to acquire a readily detectable ECFP signal (FIGS. 5–8). Additionally, V. dahliae sclerotia possess sGFP (Sawyer et al 1998), EY FP, and mRFP1 fluorescence to varying degrees (data not shown). However, ECFP fluorescence was not detectable (data not shown) possibly because it was masked by melanization of sclerotia cell walls. Finally, in contrast to P. tritici-repentis, which had a near uniform distribution of fluorescence among conidia (FIGS. 1–4), V. dahliae conidia showed a heterogeneous range of intensities for all four proteins (FIGS. 5–8).
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). Transformation of V. dahliae with pCM56 produced hygromycin-resistant colonies that grew and sporulated similarly to wild-type. Fluorescence microscopy revealed that, like the ToxA promoter, the ToxB promoter drives strong expression of sGFP in conidia, conidiophores and hyphae of V. dahliae (FIGS. 10, 11). Sclerotia also displayed ToxB promoter-driven sGFP fluorescence (data not shown). Additionally, pCM56-transformed V. dahliae spores exhibited an array of sGFP intensities similar to that observed for ToxA promoter-driven sGFP expression (FIG. 11).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Mikkelsen et al 2003, Pöggeler et al 2003, Toews et al 2004). As recently reviewed by Czymmek et al (2005), live imaging using fluorescent proteins has advanced the study of fungal cell biology, phytopathology and fungal ecology via a number of applications, including as reporters of gene expression or protein distribution, as tags of subcellular compartments or whole fungi, and as tools to study protein kinetics or to measure protein-protein interactions via FRET. To provide an arsenal of colors useful for a variety of applications, we chose the fluorescent proteins EY FP, ECFP and mRFP1 to join sGFP for inclusion in ToxA promoter-driven fluorescent vectors for fungal transformation. Successful expression of these three additional fluorescent proteins by the ToxA promoter in the cytoplasm of both its fungus of origin, P. tritici-repentis (FIGS. 1–4
), and a distantly-related ascomycete, V. dahliae (FIGS. 5–8), suggests these constructs will have as wide ranging applications as the original ToxA promoter-driven sGFP construct, pCT74 (Lorang et al 2001). In addition, a new sGFP expression vector, pCM56, with sGFP under the control of the ToxB promoter (FIG. 9), was assembled. Strong sGFP expression in V. dahliae by the ToxB promoter (FIGS. 10, 11) revealed the potential of this promoter for heterologous expression of proteins in filamentous ascomycetes.
Confocal parameters used in this study revealed that fluorescence due to EY FP and mRFP1 is of similar intensity to that of sGFP, whereas ECFP fluorescence is considerably less intense. The maximized settings required to obtain intensities of ECFP similar to the other fluorescent proteins may be explained by its intrinsic brightness, which is the product of the extinction coefficient, a measure of light absorption ability, and quantum yield, the amount of absorbed light energy that is released as fluorescence (Tsien 1998). Based on this calculation, the intensity of ECFP is expected to be approximately 40% that of EGFP (Tsien 1998, Cubitt et al 1999, Patterson et al 2001), congruent with our observations. In contrast, mRFP1 did not require maximized confocal parameters even though its intrinsic brightness is calculated to be 30% of EGFP (Campbell et al 2002), a value comparable to that for ECFP. A microenvironment favorable to mRFP1 fluorescence, but not to ECFP fluorescence, may explain why mRFP1 appeared more intense than ECFP despite similar calculated values of intrinsic brightness. Indeed, multiple environmental factors, such as temperature and pH, are known to differentially influence brightness over the range of available fluorophores (Patterson et al 1997, Tsien 1998, Cubitt et al 1999). An alternate explanation might be that the extinction coefficient and quantum yield contribute unequally to the overall brightness of a particular fluorophore. The fluorescence of mRFP1, which relies on the strength of its extinction coefficient as the principal contributor to brightness, is more intense than that of ECFP, which attributes its intensity primarily to its quantum yield (Tsien 1998, Cubitt et al 1999, Patterson et al 2001). When comparing these two fluorescent proteins, it appears that the extinction coefficient rather than the quantum yield contributes more to overall brightness. Similarly, Patterson et al (1997) found that mutant forms of GFP with increased extinction coefficients are approximately six-fold brighter than wildtype GFP in spite of corresponding decreases in quantum yields. Cerulean (Rizzo et al 2004), an improved CFP variant, could provide a brighter alternative to ECFP.
From the fungi successfully transformed with ToxA-driven GFP fluorescence (Lorang et al 2001), we chose V. dahliae for transformation with the ToxB promoter-driven sGFP construct, pCM56, due to its taxonomic and pathogenic divergence from P. tritici-repentis. Though V. dahliae and P. tritici-repentis are in the same subphylum, the Pezizomycotina, they diverge at the level of class and thus show considerable phylogenetic distance (Lutzoni et al 2004). Additionally, V. dahliae primarily infects dicotyledenous plant hosts (Pegg and Brady 2002), whereas P. tritici-repentis infects monocotyledonous grass species (Strelkov and Lamari 2003). Thus, we reasoned that if the ToxB promoter expresses GFP in V. dahliae, we might expect the ToxB promoter to have as wide ranging applicability for heterologous expression as the ToxA promoter. That the ToxB promoter rivals the ToxA promoter in driving stable, constitutive GFP expression in V. dahliae (FIGS. 5, 10, 11) gives us confidence that the ToxB promoter will be a useful tool for heterologous expression, at least across the Ascomycota. Furthermore, it will be advantageous to eventually analyze expression from each distinct promoter configuration of the multi-copy ToxB gene. In light of variable levels of expression in fungi from heterologous promoters (Churchill et al 1990, Van Wert and Yoder 1994, Spellig et al 1996, Gold et al 2001, Lorang et al 2001) and that fungal gene expression systems often simultaneously use multiple promoters with one fused to a gene of interest and another fused to a selectable marker (Gold et al 2001, Lorang et al 2001), the advent of new fungal promoters with the potential for widespread application should prove advantageous.
A number of other new constructs for transformation of filamentous fungi are available that use various combinations of promoters and fluorescent protein variants. The greatest diversity of new fungal fluorescent marker constructs has been provided by in planta investigations of plant pathogens. For example, the A. nidulans gpd promoter successfully expressed DsRed2 (Nahalkova and Fatehi 2003) and sGFP (Aboul-Soud et al 2004) in Fusarium oxysporum f. sp. lycopersici; the homologous ribosomal protein 27 promoter expressed four GFP variants in a barley-infecting isolate of Magnaporthe grisea (Czymmek et al 2002); and various promoters were used to express a suite of RCFPs in the plant pathogens M. grisea and Fusarium verticillioides (Bourett et al 2002) as well as Colletotrichum graminicola and F. oxysporum (Czymek et al 2005).
In addition to those generated for in planta observations of fungi, fluorescent expression vectors have been developed to answer basic questions about fungal cell biology. The cytoplasmic ZsGreen constructs developed by Bourett et al (2002) were used to monitor subcellular organelle dynamics and changes in nuclear envelope permeability in M. grisea and F. verticillioides via fluorescent protein exclusion from organelles. Czymmek et al (2005) found the use of the RCFPs ZsGreen and AmCyan for localization to organelles to be fraught with difficulties, however, they successfully localized EGFP and AcGFP, a GFP variant from A. coerulescens (Gurskaya et al 2003), to mitochondria of M. grisea. They also used non-native promoters to express ß-tubulin-EY FP fusion proteins for the visualization of microtubules during mitosis. In contrast, to study mitosis in the model organisms N. crassa and A. nidulans, homologous promoters were used to drive expression of fluorescent protein fusions to tubulin or histone proteins for the analysis of in situ microtubule or nuclear dynamics, respectively. For N. crassa, both histone H1 and ß-tubulin were fused to sGFP and analyzed in separate individuals (Freitag et al 2004), whereas, for A. nidulans, histone H2A was fused to CFP and 
-tubulin was fused to GFP for simultaneous imaging in one individual (Su et al 2004). Finally, to study protein-protein interactions of transcription factors in Acremonium chrysogenum, Hoff and Kück (2005) used EY FP for biomolecular fluorescence complementation. The heterologous glyceraldehyde-3-phosphate (g pd) promoter from A. nidulans was used to drive expression of the N- and C-termini of EY FP fused to two different transcription factors suspected to interact. Interaction of the two transcription factors in the nucleus brought the two domains of EY FP into proximity of each other and restored EY FP fluorescence.
The tremendous success of the green fluorescent protein as an in vivo reporter has revolutionized our understanding of fungal biology and fungi-host interactions (Lorang et al 2001, Jensen and Schulz 2004, Czymmek et al 2005). Advances in fluorescent protein technology allowed for the expansion of the color palette of fluorescent transformation vectors available for use in filamentous fungi. This study describes a new set of fluorescent protein expression vectors that we anticipate will be useful for applications in fungal-related research areas. Though the vectors presented here result in the labeling of whole fungi, we predict that the ToxA and ToxB promoters also can be used to drive expression of fluorescent protein fusions to determine protein distribution or to label cell components or organelles. Additionally, these promoters show promise for heterologous expression in ascomycetes of any protein of choice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006. 
2 Corresponding author. E-mail: ciuffetL{at}science.oregonstate.edu
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LITERATURE CITED |
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