Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing Fungus Clitopilus passeckerianus

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 1). Sequencing upstream and downstream of the probe regions identified full-length genes.

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 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.

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 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.

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 1).

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 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).

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. 1).

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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 14 different fungal transformants (lanes 1 to 7 and 9 to 15) and the untransformed wild-type strain (lanes 8 and 16) were probed with a 600-bp hph probe. Hybridization patterns show a single random insertion event within the genome of each transformant.

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 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 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 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 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 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. 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. 2). Southern blot analysis showed that there were multiple copies of both the hph cassette and gfp cassette within these transformants (Fig. 3), but expression levels were not always correlated to copy number. The same requirement for intron presence was also investigated for DsRed expression (Fig. 4), again showing that expression could be detected only when a 5′ intron was present in the expression cassette.

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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 and F [bars, 2 μm] and G and H [bars, 1 μm]) were removed from actively growing colonies for visualization, and hyphal knots (C and D [bars, 5 μm]) were examined directly on MMP agar. Samples shown in panels A, C, E, and G were examined using bright-field microscopy; those shown in panels B, D, F, and H were viewed using epifluorescence. No background fluorescence was observed with the C. passeckerianus wild-type strain (data not shown).

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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 genomic DNA was probed with 600-bp hph probe (A) and 600-bp gfp probe (B), respectively. Hybridization signals indicate multicopy insertion of the plasmids in each transformant.

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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 panels A, C, and E were examined using bright-field microscopy; those shown in panels B, D, and F were viewed using epifluorescence. No background fluorescence was observed with wild-type C. passeckerianus or intronless DsRed transformant (B and D). Bars, 20 μm.

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 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 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 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. 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. 6).

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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 (E and F), GA42 (G and H), and GA6 (I and J). Mycelia were removed from actively growing colonies, suspended in sterile water, and viewed microscopically. Samples shown in panels A, C, E, G, and I were examined using phase-contrast microscopy; those shown in panels B, D, F, H, and J were viewed using UV light. The image in panel H shows the reduction in fluorescence in transformant GA42, and that shown in panel J shows the lack of fluorescence in transformant GA6. Bars, 10 μm.

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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 are indicated by − (no fluorescence), + (low), ++ (medium) and +++ (high), respectively. (A) Probed with 600-bp gfp probe. (B) Total RNA electrophoresed through a 1.2% agarose gel. (C) Normalized densitometry values expressed as a ratio of signal A to B. (D) Relative percentage of gfp transcript levels detected in antisense transformants in comparison to fluorescent (control) transformant.