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Appl Environ Microbiol. 2009 November; 75(22): 7196–7204.
Published online 2009 September 18. doi:  10.1128/AEM.01151-09
PMCID: PMC2786515

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing Fungus Clitopilus passeckerianus[down-pointing small open triangle]

Abstract

We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus, Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycin-resistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. passeckerianus, but only when constructs contained a 5′ intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

The basidiomycete Clitopilus passeckerianus (Pleurotus passeckerianus) produces a biologically active compound, pleuromutilin, with strong antimicrobial activity (25). This natural antibiotic is also reported to be produced by Drosophila subatrata, Clitopilus scyphoides (Pleurotus mutilus), and several other species of the genus Clitopilus (17, 24). Pleuromutilin is a tricyclic diterpene product, and its activity is primarily against gram-positive bacteria, such as Staphylococcus aureus, Streptococcus haemolyticus, and Bacillus subtilis (25).

Derivatives of pleuromutilin, namely tiamulin and valnemulin, are used in veterinary medicine to treat swine dysentery and enzootic pneumonia (45). A novel semisynthetic pleuromutilin, retapamulin {mutilin 14-(exo-8-methyl-8-azabicyclo [3.2.1]oct-3-yl-sulfanyl)-acetate}, is active against Staphylococcus aureus and Streptococcus pyogenes (39, 47) and is the first pleuromutilin derivative which has been formulated as a topical antibacterial agent for treating human skin infections (14, 38, 41). In 2007, the Food and Drug Administration and the European Commission approved a new topical retapamulin ointment for the treatment of impetigo and infected small lacerations, abrasions, or sutured wounds. Retapamulin is the first pleuromutilin antibacterial to receive regulatory approval for use in humans, and this class of antibiotics has not been shown to have cross-resistance with other antibiotics (57, 58). Biochemical and crystallization studies suggested that pleuromutilin and its derivatives block the peptide bond formation directly by interfering with substrate binding at both the acceptor and donor sites of the ribosome's peptidyl-transferase center (11, 12, 20, 21, 45). These studies also suggested that, due to a unique mode of action, pleuromutilins may encounter little cross-resistance with other agents.

The dikaryotic nature of the pleuromutilin production strains of Clitopilus passeckerianus makes strain improvement by conventional mutagenesis and screening difficult; any recessive mutations would be sheltered by the second nucleus, and only dominant effects would be observed. These limitations might be alleviated by the use of a molecular biology approach, hence the need to develop the tools for modification of gene expression in this species.

Vectors containing the Escherichia coli hph gene (for hygromycin resistance) coupled with PEG-mediated and/or Agrobacterium-mediated transformation systems have previously been developed for a few basidiomycete species, mainly model systems such as Coprinopsis cinerea (10) and Schizophyllum commune (35, 51), or edible fungi, including Agaricus bisporus (55), Lentinula edodes (50), and Pleurotus ostreatus (42). Phleomycin resistance vectors were also employed to transform C. cinerea (27), S. commune (52), and Trametes versicolor (1). Another marker occasionally used in fungi has been carboxin resistance, based on expression of a resistant version of the iron-sulfur protein subunit of the succinate dehydrogenase gene. This has proven very successful in the hemibasidiomycete Ustilago maydis (3) and has recently been shown to be effective in C. cinerea (27).

The green fluorescent protein (GFP) gene gfp is widely used as reporter gene in filamentous ascomycetes (31). However, to date, successful GFP expression has been reported in only a few basidiomycete species, i.e., A. bisporus (4, 19), C. cinerea (13, 19), S. commune (32), and Phanerochaete chrysosporium (33). In all these fungi, an intron is a prerequisite for expression or maximizes the level of fluorescence. Nevertheless, recent reports of GFP expression in Hebeloma cylindrosporum (36) and Pisolithus tinctorius (48), without the presence of introns, point out some degree of inconsistency among different basidiomycete species.

In this study, we evaluated polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for C. passeckerianus. Concurrently, we investigated the possible use of visual marker genes, namely GFP and DsRed. We also evaluated RNA interference (RNAi)-mediated gene silencing. Although targeted gene disruption is widely used for functional analysis of genes in the yeast Saccharomyces cerevisiae and in many filamentous ascomycetes, it has rarely been used in basidiomycete species due to a low frequency of homologous insertions (2, 53). Many wild isolates of basidiomycetes are dikaryotic and this also reduces the utility of such a method, given the likely difficulty of disrupting the desired target in both nuclear types. RNAi-mediated gene silencing is an alternative mechanism to targeted gene disruption and has already been established in species such as C. cinerea, A. bisporus, and S. commune, with the advantage that targeted insertion is not necessary and that it is likely to deplete specific transcripts irrespective of nuclear origin (8, 9, 18, 37, 56) so it would be phenotypically dominant.

MATERIALS AND METHODS

Strains and culture conditions.

C. passeckerianus strain ATCC 34646 was routinely maintained on potato dextrose agar (PDA) at 25°C. Mature fruiting bodies of C. passeckerianus were obtained following cultivation on MMP medium (1% malt extract, 0.5% mycological peptone, 1.5% agar) (55) at 25°C for 20 to 22 days. The levels of antibiotic resistance were assessed by cultivation of mycelial inocula on PDA plates containing different antibiotics at 25°C for 5 days as previously described for Coprinopsis cinerea (27).

Escherichia coli strain DH5α was used for subcloning plasmids. Agrobacterium tumefaciens strains AGL-1 and LBA1126 were routinely grown in LB medium at 25°C as described previously (5). All DNA manipulations followed standard techniques as previously described (49).

Construction and screening of a C. passeckerianus genomic library.

Fifty micrograms of DNA was partially digested with Sau3AI to generate the maximum yield of fragments in the size range of 9 to 23 kb. The DNA was then size fractionated and cloned in BamHI-linearized vector Lambda GEM-11 (Promega) following the vector's instruction manual. In vitro packaging was performed using the Packagene Lambda DNA system (Promega). Propagation and amplification of the genomic library were performed by infecting E. coli strain KW251. Aliquots of the amplified library were stored in 7% dimethyl sulfoxide at −80°C. The library was screened by plaque hybridization with C. passeckerianus with actin and tubulin probes using standard methods (49). The actin probe of 175 bp was generated using primers ActinF1 and ActinR3, and the tubulin probe of 611 bp was generated using primers Tubf2 and Tubr4 (Table (Table1).1). Sequencing upstream and downstream of the probe regions identified full-length genes.

TABLE 1.
Primers used in this study

Vectors for transformation of C. passeckerianus.

The plasmids pPHT1, phph004, phphi004, pMhph004, pMhphi004, pble004, pblei004, pcbx004, pcbxi004, pGFPi004, and pGFPAnti004 (4, 5, 10, 18, 27) were used for PEG-mediated transformation of C. passeckerianus. A description of each plasmid is summarized (see Table Table3).3). GFP expression vectors driven by endogenous promoters were constructed by replacing the A. bisporus gpdII promoter in p004iGM3 (4) with 1,000-bp and 890-bp SacII-NcoI fragments of C. passeckerianus actin and tubulin promoters, resulting in plasmids p0011iGM3 and p0012iGM3, respectively.

TABLE 3.
Frequencies of GFP-positive clones in PEG-mediated cotransformation experiments with C. passeckerianus as determined by microscopy

A. tumefaciens strains AGL-1 and LBA1126 carrying plasmids pBGgHg based on pCAMBIA (6), pGR4-4iGM3, pGR4GFP, and pGRhph004 all based on pGreen (4, 5), and pBIN7-1 (4-6) based on pBIN19 were used for Agrobacterium-mediated transformation of C. passeckerianus. In all the plasmids described above, hph was under the control of the 277-bp A. bisporus gpdII promoter, except pBIN7-1, which was under the control of the Aspergillus nidulans gpdA promoter (5).

Preparation of protoplasts from C. passeckerianus mycelium.

Small blocks of mycelium were inoculated into general-purpose growth medium (54) and allowed to grow for 5 days at 25°C with shaking at 230 rpm. Mycelia were harvested by centrifugation, washed twice with 0.7 M NaCl, and treated with enzyme solution (50 mg/ml lysing enzymes from Trichoderma harzianum [Sigma-Aldrich] in 1 M MgSO4 and 0.6 M phosphate buffer, pH 6.0) at 25°C for 2.0 to 2.5 h. After incubation, protoplasts were separated from hyphal debris by filtration through a sterile Miracloth and collected by centrifugation at 3,000 × g for 10 min. Protoplasts were washed twice with 1 M sorbitol, and the protoplast density was adjusted to 108/ml with the same.

PEG-mediated transformation.

Fifty microliters of protoplasts (108/ml) was mixed with 10 μg of each plasmid DNA and 12.5 μl of PEG solution (40% PEG 4000, 10 mM Tris-HCl, pH 8.0, 25 mM CaCl2; filter sterilized), and protoplasts were incubated on ice for 20 min. Five hundred microliters of PEG solution was added, gently mixed, and incubated for 5 min at room temperature. One milliliter of ice-cold STC buffer (1 M sorbitol, 10 mM Tris-HCl, pH 8.0, 25 mM CaCl2) was added, and the mixture was then spread on plates containing 20 ml PDAS regeneration agar medium (PDA plus 0.6 M sucrose, pH 6.5). Plates were incubated at 25°C for 48 h, and then 5 ml of PDAS medium containing 600 μg/ml hygromycin B (Duchefa, The Netherlands), 600 μg/ml phleomycin (Invitrogen), or 60 μg/ml carboxin (Duchefa, The Netherlands) was added as an overlay, and plates were further incubated at 25°C until the transformants appeared (5 to 7 days). Transformants were individually subcultured onto fresh PDA plates containing 50 μg/ml hygromycin, 50 μg/ml phleomycin, or 5 μg/ml carboxin.

Agrobacterium-mediated transformation.

A. tumefaciens strains AGL-1 and LBA1126 (for a full list, see Table Table2)2) were grown for 24 h in LB medium supplemented with appropriate antibiotics (5). Bacterial cultures were subsequently diluted to an optical density at 660 nm of 0.15 with Agrobacterium induction medium (AIM) (23) in the presence of 200 μM acetosyringone and grown for an additional 5 to 6 h. Concurrently, 5-day-old C. passeckerianus mycelia obtained from general-purpose growth medium (54) were homogenized using an Ultra-Turrax homogenizer, and hyphal fragments were transferred to fresh general-purpose growth medium and grown for 24 h to give a uniform mycelial slurry.

TABLE 2.
Total number of PEG-mediated C. passeckerianus transformants obtained with various plasmids containing dominant selectable marker genesa

A 100-μl mycelial suspension was mixed with 100 μl of bacterial culture and then spread on cellophane discs, overlaid on AIM agar plates, and incubated at 25°C for 48 h. At least five plates were inoculated in this manner for each transformation experiment. After cocultivation, cellophane discs were transferred to PDA medium containing 200 μg/ml cefotoxime to kill residual Agrobacterium cells and 100 μg/ml hygromycin to select fungal transformants. These were incubated at 25°C until the hygromycin-resistant colonies appeared. Individual colonies were subsequently transferred to PDA medium containing 50 μg/ml hygromycin.

Agrobacterium-mediated transformation of fruiting body gill tissue was carried out as previously described for A. bisporus (6). Mature fruiting bodies were excised from MMP plates using a scalpel and diced into small sections. Fruiting body gill tissue pieces were mixed with induced A. tumefaciens culture and vacuum infiltrated as described previously for A. bisporus (6) until no more air bubbles emerged. The infiltrated gill pieces were transferred to cellulose discs overlaid on AIM agar plates. Cocultivation and selection of transformants were carried out as described above.

Molecular analysis of C. passeckerianus transformants.

PCR was used to confirm the insertions of genes hph, ble, cbx, gfp, and DsRed within the genome of C. passeckerianus transformants, while Southern blot hybridizations were performed to determine the copy number and randomness of integrations. A minimum of 30 transformants were analyzed by using PCR for each plasmid, with at least 8 further characterized by Southern blot analysis. Fungal genomic DNA was extracted (30, 44) from 4-day-old mycelia grown on PDA medium for PCR analysis, and 5-day-old mycelia were grown in general-purpose growth medium for Southern blot hybridizations. Transgenes were amplified using appropriate primers (Table (Table11).

For Southern blot hybridizations of transformants, 5 μg of SacI-digested genomic DNA was separated on a 0.8% agarose gel and vacuum transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech). hph and gfp probes were generated by PCR analysis using hph_f and hph_r, and gfp_f and gfp_r, respectively (Table (Table1).1). The Ready-To-Go labeling kit (Amersham Pharmacia Biotech) was used for probe labeling with [α-32P]dCTP, and hybridizations were performed at 60°C overnight according to the manufacturer's instructions.

For Northern blot analysis, total RNA was extracted from 5-day-old mycelia grown in general-purpose growth medium by using the RNeasy mini kit (Qiagen, United Kingdom) according to the manufacturer's instructions. One microgram of RNA was denatured for each transformant, electrophoresed, and blotted onto a Hybond N+ membrane (Amersham Pharmacia Biotech). Probe labeling and hybridizations were performed as described above using a 600-bp gfp probe, and autoradiographs were developed after an appropriate time period.

Gene silencing.

A strongly expressing GFP transformant, G1, was selected by cotransformation with pblei004 (27) and pGFPi004 (18). This strain was used as a recipient for cotransformations with pMhph004 (27), and the GFP antisense plasmid pGFPanti004 and transformants were screened for GFP levels as previously described (18).

Microscopic analysis for GFP detection.

Microscopic observations for GFP expression were performed as previously described (4, 18).

RESULTS

Selection systems for C. passeckerianus.

To establish a genetic transformation method for C. passeckerianus, dominant selection systems based on antibiotic resistance selection are a prerequisite due to the lack of suitable auxotrophic mutants and the mainly dikaryotic nature of this species. In order to determine the resistance levels of C. passeckerianus mycelia to different antibiotics, mycelial cultures were inoculated on PDA with different concentrations of antibiotics and the growth rates were monitored. Of the eight different antibiotics tested, basta, kanamycin, and nourseothricin showed no appreciable inhibitory action even at 200, 200, and 20 μg/ml, respectively. Zeocin and neomycin showed only intermediate inhibition at 200 μg/ml. Carboxin and hygromycin showed complete growth inhibition at 5 and 50 μg/ml, respectively, with G418 and phleomycin showing complete inhibition at 100 μg/ml.

Agrobacterium-mediated transformation of C. passeckerianus.

Cocultivation of C. passeckerianus mycelium with the A. tumefaciens strain LBA1126 containing pBGgHg allowed the selection of hygromycin-resistant colonies approximately 8 to 10 days after transfer to selection medium. When fruiting body gill tissue pieces were used, hygromycin-resistant growth appeared at the borders of the tissue after 10 to 12 days of cultivation on selection medium. The transformation experiments were performed two to five times. While transformation rates were variable between the repeats, homogenized mycelia consistently generated more transformants (an average of 73 transformants/experiment) than fruiting body gill tissue pieces (16 transformants/experiment). Regardless of the C. passeckerianus tissue used, hygromycin-resistant transformants were never obtained with the A. tumefaciens strain AGL-1 carrying plasmid pBGgHg, indicating the importance of the A. tumefaciens strain to generate C. passeckerianus transformants. Cocultivation of C. passeckerianus mycelium and fruiting body gill tissue pieces with A. tumefaciens strains AGL-1 and LBA1126 carrying the plasmids pGR4iGM3, pGR4-GFP, pGreenhph004, and pBIN7-1 all failed to yield any hygromycin-resistant colonies. These plasmids all contain the truncated hph gene under the control of the A. bisporus gpdII promoter, or in the case of pBIN7-1, the A. nidulans gpdA promoter.

In order to investigate the fate of transforming DNA, 14 different randomly selected pBGgHg transformants originating from homogenized mycelia and fruiting body gill tissue pieces were analyzed for T-DNA integration by Southern blot analysis using a 600-bp hph fragment as the probe. The single hybridizing band in each lane demonstrated that the introduced sequence was integrated into the genome in an apparently random fashion with a single copy in all of the tested transformants (Fig. (Fig.11).

FIG. 1.
Southern blot analysis of Agrobacterium-mediated C. passeckerianus pBGgHg transformants. (A) Lanes 1 to 7 were obtained with homogenized mycelium. (B) Lanes 9 to 15 were obtained with fruiting body gill tissue pieces. SacI-digested total genomic DNA from ...

PEG-mediated transformation of C. passeckerianus protoplasts.

Liquid-grown cultures were successfully protoplasted using Trichoderma sp. lysing enzyme, generating in excess of 108 protoplasts from a single flask culture. Protoplasts transformed with plasmids containing either the full-length hph gene (pPHT1, pMhph004, and pMhphi004) or the truncated hph gene (pAN7-1, phph004, and phphi004) in the presence of PEG-CaCl2 were screened for the hygromycin-resistant colonies on PDAS plates overlaid with hygromycin. After incubation at 25°C for 96 to 120 h, the protoplasts treated with full-length hph gene containing plasmids pPHT1, pMhph004 (without intron), and pMhphi004 (with intron) gave rise to hygromycin-resistant transformants (Table (Table2).2). Despite numerous attempts, no hygromycin-resistant colonies were obtained with the truncated hph gene containing plasmids pAN7-1, phph004 (without intron), and phphi004 (with intron) (Table (Table2).2). There was no significant difference in the number of putative hygromycin-resistant transformants obtained with plasmids pMhphi004 and pMhph004, with and without 5′ intron (Table (Table2),2), indicating that an intron is not required for hph expression in C. passeckerianus. Furthermore, no difference in the growth rate was observed among the transformants obtained with pMhph004 and pMhphi004.

Other dominant selectable markers for C. passeckerianus.

Although hygromycin selection was clearly suitable for C. passeckerianus transformations, in order to carry out multiple transformations of the same strain we developed two other dominant selectable markers, ble and cbx, based on phleomycin and carboxin selection, respectively. C. passeckerianus protoplasts were transformed with intronless and intron-containing ble or cbx plasmids (pble004 and pblei004, and pcbx004 and pcbxi004) and screened for the presence of phleomycin- and carboxin-resistant colonies on PDAS medium overlaid with phleomycin and carboxin, respectively.

While no phleomycin-resistant colonies were obtained with pble004, 32 phleomycin-resistant transformants were obtained with the intron-containing version pblei004 (Table (Table2),2), showing the requirement for an intron for efficient expression. For carboxin-based selection, 23 carboxin-resistant transformants were obtained with pcbx004 and 20 were obtained using cbxi004 (Table (Table2).2). This suggests that the extra intron at the 5′ end of the carboxin gene (three putative introns occur within the gene) is not required for expression in C. passeckerianus. Based on the total number of transformants obtained on selection media, hph was the most efficient dominant selectable marker for transformation of C. passeckerianus, followed by ble and cbx.

Reporter gene expression in C. passeckerianus.

To investigate any intron prerequisite for GFP expression in C. passeckerianus, transformations were carried out with plasmids pGFP004 and pGFPi004. Both plasmids contain the gfp gene under the control of A. bisporus gpdII promoter and A. nidulans trpC terminator, but GFPi004 has an intron in the 5′ terminus of the gene. PEG-mediated cotransformations of pGFP004 and pGFPi004 with a full-length hph gene containing plasmid pMhph004 led to many hygromycin-resistant transformants, and 100 transformants of each plasmid were microscopically examined for fluorescence. However, no fluorescence was detected in pGFP004-pMhph004 cotransformants, while 30 out of 100 pGFPi004-pMhph004 cotransformants exhibited green fluorescence (Fig. (Fig.2)2) with a range of intensities, which suggested that a 5′ intron was essential for GFP expression in C. passeckerianus. Fluorescence was also observed in the hyphal knots and fruiting body gill tissue of a transformant (Fig. (Fig.2).2). Southern blot analysis showed that there were multiple copies of both the hph cassette and gfp cassette within these transformants (Fig. (Fig.3),3), but expression levels were not always correlated to copy number. The same requirement for intron presence was also investigated for DsRed expression (Fig. (Fig.4),4), again showing that expression could be detected only when a 5′ intron was present in the expression cassette.

FIG. 2.
Expression of GFP in C. passeckerianus. A GFP-expressing C. passeckerianus transformant (pGFPi004) was cultivated on MMP agar medium at 25°C and examined microscopically. Mycelia (A and B [bars, 10 μm]) and fruiting body gill tissue (E ...
FIG. 3.
Southern blot analysis of DNA from PEG-mediated C. passeckerianus transformants obtained by cotransformation with pMhph004 (hygromycin resistance) and p004iGM3 (encoding GFP) (lanes 1 to 7) and untransformed wild-type strain (lane 8). SacI-digested total ...
FIG. 4.
Expression of DsRed in C. passeckerianus. Fluorescence analysis of wild-type C. passeckerianus (A and B), intronless DsRed transformant (C and D), and intron-containing DsRed transformant (E and F) were examined directly on PDA plates. Samples shown in ...

GFP expression in C. passeckerianus with endogenous promoters.

A series of GFP reporter plasmids was designed to compare promoter strengths, using the native β-tublin and actin genes from C. passeckerianus. Cotransformations were performed by using plasmids p004iGM3, p0011iGM3, and p0012iGM3, all of which contain a gfp gene under the control of A. bisporus gpdII and C. passeckerianus actin and β-tubulin promoters, respectively, in conjunction with the hph-containing plasmid pMhph004. One hundred hygromycin-resistant transformants for each construct were randomly screened for microscopic analysis (Table (Table3).3). Fluorescence was successfully detected with all the three promoters tested. Forty-two percent of A. bisporus gpdII promoter-driven transformants, 37% of C. passeckerianus actin promoter-driven transformants, and 52% of C. passeckerianus tubulin promoter-driven transformants exhibited fluorescence (Table (Table3).3). Regardless of the promoter used to drive gfp, the majority of the transformants showed fluorescence with intermediate intensity. Nevertheless, 26% of A. bisporus gpdII-driven transformants and 23% of C. passeckerianus tubulin-driven transformants exhibited fluorescence with high intensity, while only 3% of C. passeckerianus actin-driven transformants did (Table (Table3),3), which suggests that the A. bisporus gpdII and C. passeckerianus tubulin promoters were more efficient in C. passeckerianus than the C. passeckerianus actin promoter.

Gene silencing in C. passeckerianus.

To investigate the possibility of triggering RNAi-mediated gene silencing in C. passeckerianus, gfp was used as a selectively neutral reporter gene. As a first step, a strongly expressing GFP transformant (G1) was obtained using phleomycin selection and was subsequently cotransformed with the gfp antisense plasmid pGFPanti004 (17), along with pMhph004, and hygromycin-resistant colonies were selected.

In total, 42 hygromycin-resistant transformants (GA1 to GA42) were obtained. PCR analysis confirmed the presence of the intact gfp sense gene in all 42 transformants, with 27 transformants also indicating the presence of the gfp antisense cassette. The 27 PCR-positive gfp antisense gene transformants were examined for any alteration in fluorescence intensity by microscopy. Six (22%) of the transformants showed similar levels of fluorescence to the GFP host strain (control transformant), suggesting that silencing had not been triggered in these transformants. However, eight (30%) of the transformants showed moderate reduction in fluorescence, five (18%) showed extreme reduction in fluorescence, and eight (30%) showed no detectable fluorescence, indicative of various levels of gene silencing within this fungus (Fig. (Fig.5).5). To investigate whether the reduction in fluorescence was in fact due to the depletion of gfp transcript levels we would expect from gene silencing, Northern blot hybridizations were performed. Total RNA was extracted from the GFP-expressing host strain G1 and from 14 different gfp antisense transformants, which exhibited a range of fluorescent intensities, and was then subjected to Northern blotting using a GFP-derived probe. Hybridization intensities were normalized against the rRNA for an accurate assessment of silencing. For the majority of the transformants analyzed, reduced fluorescence intensities correlated closely with the reduction in gfp transcript levels (Fig. (Fig.66).

FIG. 5.
GFP expression in antisense gfp transformants of C. passeckerianus mycelia. Shown are mycelia obtained from the wild-type C. passeckerianus strain (A and B), the fluorescent host strain G1 (C and D), and the antisense gfp (pGFPanti004) transformants GA13 ...
FIG. 6.
Northern blot analysis of antisense gfp transformants of C. passeckerianus. Total RNA extracted from the fluorescence host strain G1 (lane 1) and antisense gfp transformants (lanes 2 to 15). Transformant numbers are indicated, and fluorescence intensities ...

DISCUSSION

In this study, we succeeded in establishing efficient and reproducible Agrobacterium-mediated and PEG-mediated transformation systems of C. passeckerianus using three different selectable markers. Simultaneously, we attained successful GFP and DsRed expression from a range of native and heterologous promoters and demonstrated efficient gene silencing in C. passeckerianus. The molecular tools which we developed in this study may aid studies identifying and manipulating the genes of this pharmaceutically important fungus.

The availability of genetic transformation systems for basidiomycete fungi is still a limiting factor for exploiting species of interest for biotechnological applications. Our aim was to establish a transformation system for a pharmaceutically important species, C. passeckerianus, which produces pleuromutilin (17, 24), an antibiotic that has a long history of veterinary use and recently has been developed as a human therapeutic (58). Approval of retapamulin, a tricyclic pleuromutilin derivative for the treatment of uncomplicated superficial infections caused by Staphylococcus aureus and Streptococcus pyogenes (57, 58), opens new possibilities for exploitation of basidiomycete species for production of novel antimicrobial agents.

As the dominant selectable marker gene hph has previously been successfully employed to develop hygromycin resistance-based transformation systems for basidiomycete species, such as the edible mushrooms A. bisporus and P. ostreatus and the model systems C. cinerea and S. commune (10, 27, 35, 42, 55), we used the same gene to establish transformation systems of C. passeckerianus. We were successful in generating hygromycin-resistant transformants by using both Agrobacterium- and PEG-mediated methods. As observed using other basidiomycete species (6, 22, 26, 52, 55), Southern blot analysis of Agrobacterium-mediated transformants of C. passeckerianus showed the single-copy insertions, while PEG-mediated transformants showed the multiple-copy insertions within the genomic DNA. This information is of importance if it is desired to express heterologous proteins in C. passeckerianus as appropriate methods of transformation may be adopted depending on the desired levels of gene expression.

Several of the plasmids commonly used in fungal transformation were not able to generate hygromycin-resistant transformants of C. passeckerianus. We have recently reported a similar phenomenon in C. cinerea where the same plasmids (pAN7-1, phph004, and phphi004, all of which contain a truncated hph gene with a deletion of two lysine residues) (27) failed to give hygromycin resistance. Plasmids pPHT1, pBGgHg, pMhph004, and pMhphi004, which contain the full-length hph gene, were able to generate hygromycin-resistant transformants of C. passeckerianus, indicating the importance of these two lysine residues that were deleted in the other nonfunctioning plasmids. These results also explain the unsuccessful attempts at Agrobacterium-mediated transformations of C. passeckerianus with pGR4iGM3, pGR4-GFP, pGreenhph004, and pBIN7-1, all of which contain this same truncated hph gene. In contrast, truncated hph-containing plasmids were able to generate the hygromycin-resistant transformants of A. bisporus, H. cylindrosporum, P. ostreatus, S. commune, and T. versicolor (29, 34, 35, 43, 55), pointing out the variations among different basidiomycete species.

Although ble-containing plasmids have been reported to generate phleomycin-resistant transformants of H. cylindrosporum (40), Paxillus involutus (40), P. chrysosporium (15), S. commune (52), Suillus bovinus (40), and T. versicolor (1), the same ble gene was not able to give rise to phleomycin-resistant transformants of C. passeckerianus. Insertion of an intron at the 5′ end of the gene led to the production of phleomycin-resistant transformants of C. passeckerianus. Previous studies reported the intron requirement for GFP expression in S. commune (32), P. chrysosporium (33), C. cinerea (4), and A. bisporus (4). However, to our knowledge this is the first report of a basidiomycete species that requires an intron for ble expression. Thus, while a 5′ intron is dispensable for hph expression, a 5′ intron is necessary for ble, gfp, and DsRed expression in C. passeckerianus. Although an extra 5′ intron is not required for cbx expression, it should be noted that we expressed the genomic clone rather than the cDNA, and this already contained the three introns at positions commonly conserved in other basidiomycete succinate dehydrogenase genes.

C. passeckerianus protoplasts were more efficiently transformed to hygromycin resistance with the bacterial hph gene than to carboxin resistance with the C. cinerea-derived cbx gene. In contrast, C. cinerea protoplasts were more efficiently transformed to carboxin resistance than to hygromycin resistance (27). This may suggest that the protein-protein interactions required for correct functioning of the carboxin-resistant succinate dehydrogenase protein may not be as efficient in heterologous hosts. This shows that one needs to pay attention to the careful selection of dominant selectable markers to achieve successful and high-efficiency transformation systems for individual species.

As we expected, the A. bisporus gpdII promoter was efficient at driving expression of all the five genes (hph, ble, cbx, gfp, and DsRed) tested in C. passeckerianus. Fluorescence intensities demonstrate that the A. bisporus gpdII promoter is functional in vegetative mycelium, hyphal knots, and fruiting body gill tissues of C. passeckerianus. The 277-bp A. bisporus gpdII promoter has also been successfully used for expression studies in different basidiomycete species, i.e., C. cinerea, H. cylindrosporum, S. bovinus, and Laccaria bicolor (4, 7, 16, 26, 27), and was shown to be the most efficient promoter in C. cinerea (28). This tempted us to use this promoter for expression of heterologous genes in C. passeckerianus.

It has been observed that targeted gene disruption can be inefficient in several basidiomycete species due to the low frequency of homologous recombination (2, 53), and in any case, targeted gene disruption is of restricted utility in fungi with dikaryotic mycelia, such as the antibiotic-producing isolates of C. passeckerianus. We therefore evaluated RNAi-mediated gene silencing as a means of knocking down expression of specific genes in C. passeckerianus. When an antisense gfp cassette was introduced into a fluorescence transformant, approximately 30% of the transformants showed no discernible green fluorescence, showing that significant silencing of expression had occurred. PCR analysis confirmed the presence of the intact gfp sense transgene, so these reductions were not due to gene disruption. An additional 48% showed some reduction in fluorescence, as expected from gene silencing. This was confirmed by Northern blot analysis where fluorescence broadly correlated with transcript levels, showing that this was indeed due to mRNA depletion, as would be expected from RNAi-mediated gene silencing.

Acknowledgments

We thank GlaxoSmithKline for financial support of this study.

We particularly thank Dave Spence and Karen O'Dwyer for helpful discussions on the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 September 2009.

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