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Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus
Journal of Microbiological Methods 70 (2007) 519 – 527 www.elsevier.com/locate/jmicmeth
Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus G. Marchand a,1 , E. Fortier a,1 , B. Neveu a , S. Bolduc a , F. Belzile b , R.R. Bélanger a,⁎ a
Département de Phytologie, Centre de Recherche en Horticulture, Pavillon de l'Envirotron, Université Laval, Québec, Québec, Canada G1K 7P4 b Département de Phytologie, Pavillon C.E.-Marchand, Université Laval, Québec, Québec, Canada G1K 7P4 Received 28 March 2007; received in revised form 12 June 2007; accepted 13 June 2007 Available online 3 July 2007
Abstract Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl2-mediated transformation of protoplasts. Electroporation yielded 100–200 transformants per μg of DNA per 108 cells after 3 days on selective medium. For its part, ATMT yielded 60–160 transformants per 106 input cfu after 5–10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl2-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium. © 2007 Elsevier B.V. All rights reserved. Keywords: Agrobacterium tumefaciens; Electroporation; Genetic transformation; Pseudozyma antarctica
1. Introduction Basidiomycetous yeasts belonging to the genus Pseudozyma (Boekhout, 1995) possess several intrinsic characteristics making them attractive systems for different biological applications. For instance, P. flocculosa, and to a lesser extent P. rugulosa have been reported for their biocontrol activity against several powdery mildew fungi (Bélanger and Avis, 2002). In addition, most Pseudozyma spp. along with the closely related model fungus Ustilago maydis were found to produce mannosylerythritol lipids (MELs), a glycolipid biosurfactant molecule with potential pharmaceutical applications in the treatment of schizophrenia and some cancers (Vertesy et al., 2002). Finally, because of their higher position in the fungal kingdom (Bruns, 2006), and their yeast-like morphology, Avis et al. (2005) showed evidence that Pseudozyma spp. ⁎ Corresponding author. Tel.: +1 418 656 2758; fax: +1 418 656 7856. E-mail address: [email protected] (R.R. Bélanger). 1 Both authors contributed equally to this publication. 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.06.014
represented a very versatile and efficient host for the production of complex recombinant proteins. Low proteolytic activity in culture conditions and glycosylation patterns characteristic of higher fungi and animal cells have been documented, along with the expression of green fluorescent protein and hen egg white lysozyme in both P. flocculosa and P. antarctica (Avis et al., 2005). In the latter study, a genetic transformation protocol developed for P. flocculosa and based on polyethylene glycol and calcium chloride — mediated transformation of protoplasts (Cheng and Bélanger, 2000; Cheng et al., 2001) was used. This method has two major drawbacks: the preparation of protoplasts is delicate and time-consuming and protoplasts must be prepared fresh before each transformation experiment as they have poor viability when stored frozen at − 80 °C (Cheng, 2005). The availability and cost of suitable lytic enzymes required to obtain protoplasts can also be a concern. For example, the Novozym 234 enzyme used in the original protocol is no longer commercially available. New methodologies for genetic transformation of filamentous fungi have been proposed recently to manipulate fungi
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refractory to protoplast generation. For instance, techniques previously associated with other organisms, such as electroporation of bacteria and Agrobacterium tumefaciens-mediated transformation (ATMT) of plants, are now being successfully used to transform yeasts and filamentous fungi. Several studies have reported the success of transformation by electroporation with yeasts (Hashimoto et al., 1985; Karube et al., 1985; Becker and Guarente, 1991) and filamentous fungi (Richey et al., 1989; Chakraborty et al., 1991, Sánchez and Aguirre, 1996; Weidner et al., 1998). The electroporation technique is based on the reversible permeabilization of the cell membrane induced by a short duration, high amplitude electric field (Chang, 1992). Different types of fungal cells can be made competent for plasmid DNA uptake by electroporation: cells treated with a weakening agent (Chakraborty et al., 1991), protoplasts (Saito et al., 2001) and untreated germinated conidia (Chakraborty and Kapoor, 1990; Sánchez and Aguirre, 1996). The possibility of directly using untreated conidia, with a simple transformation protocol giving high yields of transformants would make electroporation a clearly advantageous system for transformation of Pseudozyma spp. ATMT of fungal species was first demonstrated in the yeast Saccharomyces cerevisiae (Bundock et al., 1995) followed by different species of filamentous fungi (de Groot et al., 1998). Some 60 fungal species have thus far been successfully transformed by A. tumefaciens (reviewed by Michielse et al., 2005). A. tumefaciens transfers part of its DNA, namely the transfer DNA (T-DNA), into the host cell. The T-DNA is located on a large (ca. 200 kb) plasmid, the tumor-inducing (Ti) plasmid, which also contains several virulence genes that are required for infection of the host and transmission of the genetic material. Two short sequences on the Ti plasmid, called the left and right T-DNA borders, flank the portion of the Ti plasmid that is transferred (see Gelvin, 2003). Attractive features of ATMT include a simple transformation protocol, few T-DNA insertions in the host genome and high yields of transformants. Considering the mounting interest in the properties of Pseudozyma spp. and the limited efforts devoted toward developing transformation protocols for this particular group of basidiomycetous fungi, we aimed to apply both electroporation and ATMT methodologies to P. antarctica and analyze integration events in order to develop versatile, efficient and robust transformation protocols that could eventually be adapted to different Pseudozyma species and other related basidiomycetous yeast-like fungi. 2. Materials and methods
Diagnostics Canada, Laval, Québec, Canada), as well as cefotaxim (200 μM, Sigma-Aldrich Canada Ltd, Mississauga, Ontario) and moxalactam (100 μg ml− 1, Sigma-Aldrich) in the case of ATMT transformants. Unless specified otherwise, liquid cultures were inoculated with three 0.5 cm mycelial plugs and incubated at 25 °C with 150 rpm shaking. All culture media ingredients were supplied by Difco (BD Biosciences, Mississauga, Ontario, Canada). A. tumefaciens strain LBA4404 was provided by D. Michaud (Université Laval, Québec, Canada). For long-term storage, A. tumefaciens strains were maintained in 15% glycerol stocks stored at −80 °C. For short-term storage, strains were maintained on Luria–Bertani (LB) agar containing the antibiotic streptomycin (125 μg ml− 1) at 4 °C. Kanamycin (50 μg ml− 1) was added to the culture medium for strains containing pCAMBIA constructions. 2.2. Plasmid construction For the electroporation experiments, pSceI-hyg or pSPF.GFP were used. pSceI-hyg was provided by Dr. J. Kronstadt (University of British Columbia, Canada) and contains a hygromycin B selection cassette containing the Escherichia coli hygromycin phosphotransferase (hph) gene under the control of the Ustilago maydis HSP70 promoter and terminator sequences. pSPF.GFP (Avis et al., 2005) contains two cassettes in the same orientation, the hygromycin selection cassette from pSceI-hyg and a green fluorescent protein (GFP) expression cassette consisting of the gGFP gene under the control of the U. maydis HSP70 promoter and terminator sequences (Fig. 1). For the ATMT experiments, two constructions were made using the pCAMBIA-0380 binary vector (CAMBIA, Canberra, Australia) that contains a multiple cloning site between the left and right T-DNA borders. pCAMBIA-hyg was created by ligating a 2681 bp AvrII/HindIII fragment containing the hygromycin selection cassette from pSceI-hyg into AvrII/HindIII — digested pCAMBIA-0380. pCAMBIA-hyg-GFP was generated in a similar fashion by ligating a 6726 bp AvrII/XmnI fragment containing both hygromycin selection and GFP expression cassettes from pSPF.GFP into AvrII/XmnI — digested pCAMBIA-0380 (Fig. 1). Both constructions were electroporated into A. tumefaciens strains LBA4404 using a Gene Pulser II with Pulse Controller (Bio-Rad, Hercules, CA) under standard conditions for E. coli (100 Ω resistance, 25 μFD capacitance, 2.5 kV voltage). Plasmid DNA was isolated from E. coli DH5α using the QIAgen MiniPrep kit (QIAgen, Mississauga, Ontario, Canada). DNA restriction and ligation, and agarose gel electrophoresis were carried out as described in Sambrook et al. (1989).
2.1. Strains, growth media and culture conditions 2.3. Transformation protocols Pseudozyma antarctica (Goto, Sugiyama, and Lizuka) Boekhout (CBS 516.83) was maintained on potato-dextroseagar (PDA) at 4 °C. Liquid cultures were performed in 500 ml Erlenmeyer flasks containing 100 ml of yeast extract (3 g l− 1), malt extract (3 g l− 1), peptone water (2.5 g l− 1) and dextrose (5 g l− 1) (YMPD) broth. All transformants were grown in the presence of the antibiotic hygromycin B (300 μg ml− 1, Roche
The electroporation experiments were performed as previously described by Sánchez and Aguirre (1996) and Brown et al. (1998) with some modifications. Cells from 5 ml of 3-day-old P. antarctica broth cultures were collected by centrifugation at 3000 ×g, 4 °C, washed twice in cool sterile water and resuspended in 270 mM sucrose, 5 mM Tris–HCl, 1 mM LiAc, pH 7.5 to
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obtain ca. 1 × 108 cells ml− 1. Fifty microliter aliquots of cell suspension were mixed with XhoI-linearized or circular plasmid DNA in pre-chilled 0.2 cm electroporation cuvettes (VWR International, West Chester, PA) and chilled on ice for 15 min. Electroporation was performed using a Gene Pulser II with Pulse Controller (Bio-Rad). Combinations of voltage (0.65 to 1.90 kV in 250 V increments) and resistance (200, 400, 600 and 800 Ω) were tested. Capacitance was held constant at 25 μF with one pulse. The time constant varied between 4.6 and 14.6 ms. Immediately following the pulse, 1 ml of YMPD broth was added to the transformed cells. The conidia were then transferred to a 15 ml tube (Sarstedt, Montréal, Québec, Canada), chilled on ice for 15 min and then incubated for 45 min at 25 °C with 250 rpm shaking. 100–400 μl aliquots were spread on PDA plates containing 300 μg ml− 1 hygromycin B (Roche). For the ATMT experiments, the transformation protocol was based on that of de Groot et al. (1998), with a few modifications. A. tumefaciens LBA4404 containing pCAMBIA-hyg or pCAMBIA-hyg-GFP was grown in LB broth medium (instead of minimal media) supplemented with appropriate antibiotics at 28 °C with shaking (250 rpm) to an OD600 of at least 0.5.
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Bacteria were collected by centrifugation (15 min at 2000 ×g and 4 °C) and resuspended to an OD600 of 0.15–0.20 in induction medium (IM, Bundock et al., 1995), without antibiotics, with or without 20 μM acetosyringone (AS, SigmaAldrich) and incubated overnight (instead of 6 h). Untransformed A. tumefaciens LBA4404 was used as a negative transformation control. P. antarctica was prepared by centrifuging a 3-day-old liquid culture (containing mostly conidia and some hyphae) for 10 min at 2000 ×g, 4 °C. Cells were resuspended in 0.85% sterile saline solution and a serie of 10fold dilution was prepared. An equal volume of bacteria in IM and fungus in saline solution were mixed by gentle pipetting and 200 μl aliquots were plated on 6-cm Petri dishes containing the co-culture medium (IM containing a reduced glucose concentration of 5 mM) with or without AS overlaid with a sterile disc of cellophane (gel-drying grade, Bio-Rad). After a co-culture period of 24, 48 or 72 h at 25 °C, the cellophane discs overlaid with the co-cultivation mixture were transferred to a selective medium consisting of PDA containing 300 μg ml− 1 hygromycin B (Roche) to select for transformants, and 200 μM cefotaxim (Sigma-Aldrich) and 100 μg ml− 1 moxalactam
Fig. 1. Map of plasmid vectors used for electroporation (A, B) and Agrobacterium tumefaciens-mediated transformation (C, D) of Pseudozyma antarctica. pro: Ustilago maydis heat shock protein 70 promoter, hph: Escherichia coli hygromycin phosphotransferase gene, ter: U. maydis heat shock protein 70 terminator, GFP: green fluorescent protein gene, amp: ampicillin resistance gene, kan: kanamycin resistance gene, LB: left transfer-DNA border repeat, RB: right transfer-DNA border repeat.
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Fig. 2. GFP expression by Pseudozyma antarctica transformants in broth cultures, observed at a magnification of 400× under visible light (A, C, E) and in the dark with GFP excitation at 488 nm (B, D, F). A, B: untransformed P. antarctica (control); C, D: electroporation transformant; E, F: Agrobacterium tumefaciens-mediated transformant.
analyzed with Image Pro Plus software (MediaCybernetics) using an exposition time of 700 ms for GFP images under 488 nm light and 20 ms for images under visible light. Wildtype P. antarctica was used as control. Randomly picked transformants were submitted to PCR analysis using primers 5'-GGAAGTGCTTGACATTGGGG-3' and 5'-CTTCTACACAGCCATCGGTCCAG-3' to amplify a 746-bp fragment of the hph gene, and primers 5'-GGTGAGCAAGGGCGAGGAGC-3' and 5'-CGGTCACGAACTCCAGCAGG-3' to amplify a 677-bp fragment of the GFP gene for pSceI-hyg or pSPF.GFP transformants. Additionally, for A. tumefaciens transformants, primers 5'-TCTACGGGGTCTGACGCTC-3' and 5'-GATGGCGTCCTTTGCTCGG-3' were used to amplify a 584-bp fragment of the kanamycin gene to test for integration of plasmid sequences located outside the T-DNA borders. 10 ng purified genomic DNA was used as DNA template in a 20 μL PCR reaction containing 1 U of HotMaster Taq DNA polymerase (Eppendorf AG, Hamburg, Germany); 1× manufacturer's PCR buffer; 0.2 mM each of dNTPs and 0.5 μM of each primer. Amplification was performed in an Eppendorf Mastercyler programmed as follows: initial denaturation at 94 °C for 2 min, 30 s at 94 °C for denaturing, 30 s at 62 °C for annealing, 1 min at 65 °C for synthesis, repeated for 30 cycles and a final extension step of 10 min at 65 °C. One nanogram of plasmid DNA was used as template for the positive control reaction. Aliquots of 10 μl of amplified product were separated in 1% (w/v) agarose gels in Tris-Acetate-EDTA buffer, stained with ethidium bromide and photographed under UV light. Southern analysis was performed for a subset of transformants using genomic DNA isolated with the QIAgen Genomic Tips 20/G or 100/G kits (QIAgen) and digested with restriction enzymes as well as undigested DNA. DNA was sizefractionated on a 1% agarose gel and transferred onto Hybond N+ nylon membrane (Amersham, GE Healthcare Bio-Sciences Inc., Baie d'Urfé, Québec, Canada) using a VacuGene XL vacuum blotting system (Amersham) according to the manufacturer's instructions. Blots were probed with the PCR products described above for the hygromycin and kanamycin genes labeled with Redivue [ 32 P]dCTP (Amersham) using the
(Sigma-Aldrich), to prevent growth of A. tumefaciens, and incubated at 25 °C. Serial dilutions and plating were used to determine the number of bacterial and fungal input colony forming units (cfu) before they were mixed together. 2.4. Transformant analysis Prior to analysis, transformants obtained by both methods were isolated by transferring fungal tissue from a single isolated colony on the surface of a Petri dish of selective medium to a new Petri dish of selective medium at least three consecutive times. GFP expression was observed as described previously (Avis et al., 2005). Cultures were observed under blue light (488 nm) using an incident fluorescent microscope (Olympus, model BH2-RFCA, Tokyo, Japan) equipped with a BH2-RFLT3 mercury lamp. Images were recorded using a Coolsnap-Pro (MediaCybernetics, Silver Spring, MD) color camera and
Fig. 3. Effect of resistance and voltage conditions for electroporation transformation of Pseudozyma antarctica using 5 μg of XhoI-linearized pSceIhyg. Results are means of two replicates.
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Fig. 4. A) Number of Pseudozyma antarctica transformants obtained by electroporation using increasing quantities of the pSceI-hyg plasmid vector. B) Number of transformants obtained per microgram of input plasmid DNA Results obtained are means of two replicates.
Rediprime random labeling kit (Amersham). Labeled probes were purified using MiniQuick Spin columns (Roche). Hybridizations were performed at 65 °C in 2× SSC, 0.5% SDS, 0.25% skimmed milk hybridization buffer. Hybridized blots were washed three times in 2× SSC, 0.1% SDS and twice in 0.1× SSC, 0.1% SDS at 65 °C and exposed to Kodak Biomax film (Kodak Canada, Toronto, Ontario, Canada). 3. Results and discussion 3.1. Transformation efficiency and stability of transformants Transformants appeared on selective media 3 days after electroporation and 5–10 days after ATMT experiments. Transformation efficiency was in the range of 100–200 transformants/μg of DNA per 108cells for electroporation and 60– 160 per 106 input cfu for ATMT. Transformants obtained by electroporation with pSPF.GFP and ATMT with pCAMBIAhyg-GFP showed strong GFP expression (Fig. 2). Transformants maintained GFP expression and hygromycin B resistance over 6 months of repeated subculturing on selective medium. Five transformants from each protocol were subcultured repeatedly for 10 generations on non-selective medium. All of them maintained hygromycin resistance and GFP fluorescence after being brought back on selective medium. 3.2. Effect of transformation parameters The effect of specific transformation parameters on transformant yield was studied in order to adapt the transformation protocols for P. antarctica. Electroporation experiments are generally conducted under conditions resulting in 50%
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mortality of cells (Brown et al., 1991). For P. antarctica, we determined these conditions in the absence of plasmid DNA to be a voltage of 1.0–1.8 kV with one pulse at 400 Ω resistance. Optimal conditions for transformation were determined first by using a fixed amount (5 μg) of XhoI-linearized plasmid DNA and testing a range of voltage values between 0.65 and 1.90 kV in 250 V increments combined with four resistance values (200, 400, 600 and 800 Ω). As illustrated in Fig. 3, for each fixed resistance value tested except the lowest (200 Ω), transformant yield reached a voltage optimum before decreasing with higher voltage values. At the lowest resistance value (200 Ω), the number of transformants obtained increased with higher voltage values but transformant yield was low compared to those obtained with higher resistance values. The greatest number of transformants was obtained with 1400 V and 400 Ω and with 1150 V and 800 Ω. These optima probably result from equilibrium between DNA uptake/transformation and cell mortality (see Weaver, 1995). The effect of the quantity of input plasmid DNA was then tested by using 0.5 to 17 μg of XhoI-linearized pSceI-hyg with a set number of input cells (ca. 108) and fixed electrical conditions (1400 V and 400 Ω). The number of transformants increased with plasmid DNA quantity up to 13 μg of plasmid DNA (Fig. 4A). At this point, other factors such as cell concentration are probably limiting the transformation process (Suga and Hatakeyama, 2003). When yield was expressed as the number of transformants obtained per μg of plasmid DNA (Fig. 4B), the best values were obtained between 0.5 and 3 μg with a maximum of ca. 200 at 1 μg. This yield is higher or comparable to those reported for other fungi such as protoplasts of Lyophyllum shimeji (Saito et al., 2001), DTT-treated cells of Candida parapsilosis (Gácser et al., 2005) and germinated conidia of Humicola grisea var. thermoidea (Dantas-Barbosa et al., 1998) or Colletotrichum gloeosporioides f. sp. aeschynomene (Robinson and Sharon, 1999). In the ATMT experiments, the effect of AS in the co-culture medium and in the induction medium for the A. tumefaciens culture, the duration of co-cultivation and the number of input P. antarctica cells were investigated. Treatments consisted of a combination of these three factors. AS is a phenolic compound
Table 1 Transformation efficiency of Pseudozyma antarctica by Agrobacterium tumefaciens as influenced by the ratio of bacterial to fungal cells and the duration of the co-cultivation period Transformation efficiency (transformants/106 input colony forming units) a Duration of co-cultivation A. tumefaciens:P. antarctica cells ratio
48 h
72 h
26:1 260:1 2600:1 2.6 × 104:1 2.6 × 105:1 2.6 × 106:1 2.6 × 107:1 2.6 × 108:1
0.68 4.2 21 0 0 0 0 0
7.0 16 63 156 0 0 0 0
a
Results are means of three replicates.
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resembling those emitted by plants at wound sites and is believed to be an inducer of the A. tumefaciens virulence genes (see Gelvin, 2003). AS was used both in induction and co-cultivation medium or withheld from both to test its effect on transformation efficiency. In three initial experiments, transformants were obtained only in the presence of AS; this suggests that AS is essential for ATMT of P. antarctica, as is the case in the majority
of fungal species (Michielse et al., 2005). In the presence of AS in the induction and co-cultivation media, although transformed colonies appeared on selective medium after a co-cultivation of only 24 h in two out of three experiments, a co-cultivation of at least 48 h was required to obtain a significant number of transformed colonies with both plasmids, and increasing the cocultivation period to 72 h further improved transformation
Fig. 5. Southern blot of genomic DNA from Pseudozyma antarctica (lanes Pa) and transformants obtained by electroporation (lanes 1–10) and Agrobacterium tumefaciens-mediated transformation (lanes 11–20) hybridized with a fragment of the hygromycin phosphotransferase gene (A, B) or the kanamycin resistance gene (C) as probe. Restriction enzymes used: S: SacII, P: PvuII, X: XhoI, N: NcoI, U: undigested DNA.
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efficiency. In ATMT, the transformation efficiency is dependent on the ratio of bacteria to fungal cells (e.g. Godio et al., 2004). The transformation efficiency was approximated by co-cultivating decreasing quantities of P. antarctica cfu with a fixed quantity of A. tumefaciens LBA4404 containing binary vector pCAMBIA-hyg in the presence of AS for 48 or 72 h before transferring to selective medium (Table 1). The highest transformation efficiencies obtained were 63 and 156 transformants per 106 input cfus, at bacterial to fungal cell ratios of respectively 2600 and 26 000:1 after a co-cultivation period of 72 h. No transformants were obtained at higher bacterial to fungal cell ratios, probably due to the limiting number of P. antarctica cells. 3.3. Transformant analysis Transformants were tested by PCR and Southern hybridization to characterize the plasmid and T-DNA insertions. All transformants tested yielded a PCR product of the expected size for the hph (all transformants) and GFP genes (pSPF.GFP and pCAMBIA-hyg-GFP transformants) (data not shown). Genomic DNA from randomly selected transformants was examined by Southern hybridization (Fig. 5). Ten transformants obtained by electroporation were analyzed (Fig. 5A), of which four were obtained with XhoI-linearized pSceI-hyg (transformants 1 to 4 in Fig. 5A) and the remaining six (transformants 5 to 10 in Fig. 5A) with circular pSceI-hyg. In transformants 1 to 4, hybridization of the hygromycin probe to undigested genomic DNA confirmed integration into chromosomal DNA whereas no such signal was observed in DNA from untransformed P. antarctica. SacII cuts once in the pSceI-hyg plasmid within the region covered by the hph probe. Thus two bands of a different size (corresponding to junctions between plasmid and genomic DNA) would be expected for each locus of integration. A prominent 5.9 kb band corresponding to the size of the vector was observed in all lanes except for transformant 7 indicating the presence of tandemly inserted copies of the plasmid. The fainter bands likely represent junctions between the tandem plasmid arrays and the flanking host DNA, each integration locus producing a pair of such bands. As for PvuII, it cuts twice within the plasmid, on either side of the hygromycin resistance gene, such that a 3.1 kb fragment should be detected by the probe. In PvuII-digested DNA, a band of the expected size is seen in the first four transformants. An additional band of ∼ 5.9 kb is also commonly found in most if not all of these transformants and may simply be due to partial digestion at one of the two PvuII sites. In addition to these common bands, a unique PvuII junction fragment is expected for each independent locus of insertion. Based on the banding patterns observed in the four electroporation transformants obtained with XhoIlinearized pSceI-hyg analyzed, it is likely that transformants 1 and 2 contain two insertion loci, each of which contains at least two copies of the plasmid in a direct tandem repeat. Transformants 3 and 4 could contain three such loci, and possibly even four in the case of transformant 4, but this is more difficult to interpret because of the more complex banding pattern. Thus, multiple tandem arrays of the plasmid vector
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were found to be the rule when using linearized plasmid for transformation. Similarly, tandem insertions were found to occur in most electroporation transformants in Schizosaccharomyces pombe (Davidson et al., 2004), albeit mostly at single integration sites, while different integration sites and multicopy integration occurs in Humicola grisea var. thermoidea (DantasBarbosa et al., 1998). Simpler integration events were reported for other fungi (e.g. Brown et al., 1998; Gácser et al., 2005), and some P. antarctica transformants electroporated with pSPF. GFP showed evidence of single integration events (data not shown). As pSceI-hyg does not contain any P. antarctica homologous sequences, it is likely that the genomic integration obtained with linearized plasmid is random. In the six electroporation transformants obtained with circular pSceI-hyg, hybridization of the hygromycin probe to undigested genomic DNA is not as evident, and thus integration of the circular plasmid into chromosomal DNA may not have occurred. However, resolution is limiting for high molecular weight genomic DNA on 1% agarose gels and the variation in the banding pattern observed between the different transformants suggests that some of these bands may be junctions with adjoining chromosomal DNA. The 5.9 kb band in PvuII digests and the 3.1 kb band in SacII digests are common to transformants 5, 6, 8, 9, and 10 and may represent autonomously replicating plasmid or two copies chromosomally integrated in a tandem array as discussed above. As pSceIhyg does not contain a eukaryotic autonomously replicating sequence, the mechanism for maintenance of the plasmid is unknown. When using an undigested plasmid containing no homologous sequence or eukaryotic origin of replication to transform the yeast Candida oleophila, Yehuda et al. (2001) obtained no transformants. For A. tumefaciens transformants, 10 transformants obtained with a strain containing the binary vector pCAMBIA-hyg were analyzed. Hybridization of hygromycin (Fig. 5B) and kanamycin (Fig. 5C) probes to undigested genomic DNA indicated that the plasmid was inserted within high molecular weight genomic DNA. The fact that the kanamycin resistance gene was also detected in transformants suggests that transformation was not limited to the T-DNA but also included vector sequences located outside the T-DNA. In agreement with this observation, all transformants shared a common 9.4 kb band upon digestion with XhoI and hybridization with both hygromycin and kanamycin probes. Such an observation is consistent with the presence of at least two copies of the whole pCAMBIA-based plasmid in a direct tandem repeat. Similarly, upon digestion with NcoI, all transformants shared bands of 1.5 and 7.9 kb with the hygromycin probe, and a single band of 7.9 kb with the kanamycin probe. Additional bands are present in transformants 11, 16 and 18 with both probes in digested DNA and could correspond to junctions between a plasmid insertion and adjacent genomic DNA. The presence of transferred vector sequences located beyond the T-DNA borders has been reported in filamentous fungi (Covert et al., 2001) and is believed to occur frequently in plants without being monitored (Martineau et al., 1994). The insertion of the whole vector rather than only the T-DNA has been
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reported before in S. cerevisiae (Bundock et al., 1995), albeit in a small (3 out of 20) number of transformants and in plants (Wenck et al., 1997). As T-DNA transfer normally starts at the right border after single-strand nicking at both borders, it has been hypothesized that transfer of the whole binary vector could result from missing the left T-DNA border during processing of T-DNA from the binary vector or from transfer initiation at the left T-DNA border (Kononov et al., 1997). The 10 transformants analyzed thus show evidence of integration of at least two copies of the whole binary vector, which is atypical as ATMT of fungi generally yields a low number of T-DNA insertions (Michielse et al., 2005). It is also possible, however remote this may seem, that a contamination of the fungal cultures used for DNA purification with the transforming A. tumefaciens is responsible for the presence of the full binary vector. 4. Conclusion Electroporation and ATMT are two new methods suitable for the transformation of P. antarctica with distinct advantages over the previously reported PEG/CaCl2-mediated approach. Electroporation is rapid and simple and allows for the recovery of transformants in only a few days. ATMT does not require the purification of the plasmid prior to transformation or the use of sophisticated equipment once the A. tumefaciens strain carrying the desired construction has been obtained. Both methods allow for the recovery of a high number of transformants and use untreated conidia of P. antarctica as starting material; this greatly simplifies the transformation protocol as lytic enzymes or chemical weakening agents are no longer required, a clear drawback in other fungal transformation systems (Yehuda et al., 2001; Kuo et al., 2004). Analysis of integration events was not definitive but allows insights into the fate of the transforming DNA and is of interest in the context of using P. antarctica for the production of heterologous proteins. The description of these new transformation methods should facilitate further genetic studies and exploitation of P. antarctica and other Pseudozyma species. Acknowledgements We thank D. Michaud for providing A. tumefaciens strain LBA4404, S. Laberge and R. Desgagnés of Agriculture and Agri-Food Canada for providing the hybridization protocol used in this study, C. Labbé for technical assistance with the figures and T.J. Avis for critical revision of the manuscript. Fig. 1 was created using the BVTech Plasmid freeware from BVTech Inc. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Canada Research Chairs Program to R.R. Bélanger. References Avis, T.J., Cheng, Y.L., Zhao, Y.Y., Bolduc, S., Neveu, B., Anguenot, R., Labbé, C., Belzile, F., Bélanger, R.R., 2005. The potential of Pseudozyma yeast-like epiphytes for the production of heterologous recombinant proteins. Appl. Microbiol. Biotechnol. 69, 304–311.
Becker, D.M., Guarente, L., 1991. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 194, 182–187. Bélanger, R.R., Avis, T.J., 2002. Ecological processes and interactions occurring in leaf surface fungi. In: Lindow, S.E., Hecht-Poinar, E.I., Elliott, V.J. (Eds.), Phyllosphere Microbiology. APS Press, St-Paul, MN, pp. 193–207. Boekhout, T., 1995. Pseudozyma Bandoni emend Boekhout, a genus for yeastlike anamorphs of Ustilaginales. J. Gen. Appl. Microbiol. 41, 359–366. Brown, L.E., Sprecher, S.L., Keller, L.R., 1991. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol. Cell. Biol. 11, 2328–2332. Brown, J.S., Aufauvre-Brown, A., Holden, D.W., 1998. Insertional mutagenesis of Aspergillus fumigatus. Mol. Gen. Genet. 259, 327–335. Bruns, T., 2006. Evolutionary biology: a kingdom revised. Nature 443, 758–761. Bundock, P., den Dulk-Ras, A., Beijersbergen, A., Hooykaas, P.J.J., 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14, 3206–3214. Chakraborty, B.N., Kapoor, M., 1990. Transformation of filamentous fungi by electroporation. Nucleic Acids Res. 18, 6737. Chakraborty, B.N., Patterson, N.A., Kapoor, M., 1991. An electroporationbased system for high-efficiency transformation of germinated conidia of filamentous fungi. Can. J. Microbiol. 37, 858–863. Chang, D.C., 1992. Structure and dynamics of electric field-induced membrane pores as revealed by rapid-freezing electron microscopy. In: Chang, D.C., Chassy, B.M., Saunders, J.A., Sowers, A.E. (Eds.), Guide to Electroporation and Electrofusion. Academic Press Inc., San Diego, CA, pp. 9–27. Cheng, Y.L., 2005. (Université Laval.) Personal communication. Cheng, Y.L., Bélanger, R.R., 2000. Protoplast preparation and regeneration from spores of the biocontrol fungus Pseudozyma flocculosa. FEMS Microbiol. Lett. 190, 287–291. Cheng, Y.L., Belzile, F., Tanguay, P., Bernier, L., Bélanger, R.R., 2001. Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi. Mol. Genet. Genomics 266, 96–102. Covert, S.F., Kapoor, P., Lee, M., Briley, A., Nairn, C.J., 2001. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105, 259–264. Dantas-Barbosa, C., Araújo, E.F., Moraes, L.M.P., Vainstein, M.H., Azevedo, M.O., 1998. Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance. FEMS Microbiol. Lett. 169, 185–190. Davidson, M.K., Young, N.P., Glick, G.G., Wahls, W.P., 2004. Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations. Nucleic Acids Res. 32, 4400–4410. de Groot, M.J.A., Bundock, P., Hooykaas, P.J.J., Beijersbergen, A.G., 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol. 6, 839–842. Gácser, A., Salomon, S., Schäfer, W., 2005. Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker. FEMS Microbiol. Lett. 245, 117–121. Gelvin, S.B., 2003. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37. Godio, R.P., Fouces, R., Gudina, E.J., Martin, J.F., 2004. Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium. Curr. Genet. 46, 287–294. Hashimoto, H., Morikawa, H., Yamada, Y., Kimura, A., 1985. A novel method for transformation of intact yeast-cells by electroinjection of plasmid DNA. Appl. Microbiol. Biotechnol. 21, 336–339. Karube, I., Tamiya, E., Matsuoka, H., 1985. Transformation of Saccharomyces cerevisiae spheroplasts by high electric pulse. FEBS Lett. 182, 90–94. Kononov, M.E., Bassuner, B., Gelvin, S.B., 1997. Integration of T-DNA binary vector “backbone” sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J. 11, 945–957. Kuo, C.Y., Chou, S.Y., Huang, C.T., 2004. Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. Appl. Microbiol. Biotechnol. 65, 593–599. Martineau, B., Voelker, T.A., Sanders, R.A., 1994. On defining T-DNA. Plant Cell 6, 1032–1033.
G. Marchand et al. / Journal of Microbiological Methods 70 (2007) 519–527 Michielse, C.B., Hooykaas, P.J.J., van den Hondel, C.A.M.J.J., Ram, A.F.J., 2005. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr. Genet. 48, 1–17. Richey, M.G., Marek, E.T., Schardl, C.L., Smith, D.A., 1989. Transformation of filamentous fungi with plasmid DNA by electroporation. Phytopathology 79, 844–847. Robinson, M., Sharon, A., 1999. Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia. Curr. Genet. 36, 98–104. Saito, T., Tanaka, N., Shinozawa, T., 2001. A transformation system for an ectomycorrhizal basidiomycete, Lyophyllum shimeji. Biosci. Biotechnol. Biochem. 65, 1928–1931. Sambrook, J., Fritsh, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sánchez, O., Aguirre, J., 1996. Efficient transformation of Aspergillus nidulans by electroporation of germinated conidia. Fungal Genet. Newslett. 43, 48–51. Suga, M., Hatakeyama, T., 2003. High-efficiency electroporation by freezing intact yeast cells with addition of calcium. Curr. Genet. 43, 206–211.
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Vertesy, L., Kurz, M., Wink, J., Noelken, G., 2002. Ustilipides, method for the production and the use thereof. US Patent 6,472,158. Weaver, J.C., 1995. Electroporation theory: concepts and mechanisms. In: Nickoloff, J. (Ed.), Electroporation Protocols for Microorganisms. Humana Press, Totowa, NJ, pp. 1–26. Weidner, G., d'Enfert, C., Koch, A., Pieternella, C.M., Brakhage, A.A., 1998. Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase. Curr. Genet. 33, 378–385. Wenck, A., Czako, M., Kanevski, I., Marton, L., 1997. Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacteriummediated transformation. Plant Mol. Biol. 34, 913–922. Yehuda, H., Droby, S., Wisniewski, M., Goldway, M., 2001. A transformation system for the biocontrol yeast, Candida oleophila, based on hygromycin B resistance. Curr. Genet. 40, 282–287.
Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus G. Marchand a,1 , E. Fortier a,1 , B. Neveu a , S. Bolduc a , F. Belzile b , R.R. Bélanger a,⁎ a
Département de Phytologie, Centre de Recherche en Horticulture, Pavillon de l'Envirotron, Université Laval, Québec, Québec, Canada G1K 7P4 b Département de Phytologie, Pavillon C.E.-Marchand, Université Laval, Québec, Québec, Canada G1K 7P4 Received 28 March 2007; received in revised form 12 June 2007; accepted 13 June 2007 Available online 3 July 2007
Abstract Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl2-mediated transformation of protoplasts. Electroporation yielded 100–200 transformants per μg of DNA per 108 cells after 3 days on selective medium. For its part, ATMT yielded 60–160 transformants per 106 input cfu after 5–10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl2-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium. © 2007 Elsevier B.V. All rights reserved. Keywords: Agrobacterium tumefaciens; Electroporation; Genetic transformation; Pseudozyma antarctica
1. Introduction Basidiomycetous yeasts belonging to the genus Pseudozyma (Boekhout, 1995) possess several intrinsic characteristics making them attractive systems for different biological applications. For instance, P. flocculosa, and to a lesser extent P. rugulosa have been reported for their biocontrol activity against several powdery mildew fungi (Bélanger and Avis, 2002). In addition, most Pseudozyma spp. along with the closely related model fungus Ustilago maydis were found to produce mannosylerythritol lipids (MELs), a glycolipid biosurfactant molecule with potential pharmaceutical applications in the treatment of schizophrenia and some cancers (Vertesy et al., 2002). Finally, because of their higher position in the fungal kingdom (Bruns, 2006), and their yeast-like morphology, Avis et al. (2005) showed evidence that Pseudozyma spp. ⁎ Corresponding author. Tel.: +1 418 656 2758; fax: +1 418 656 7856. E-mail address: [email protected] (R.R. Bélanger). 1 Both authors contributed equally to this publication. 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.06.014
represented a very versatile and efficient host for the production of complex recombinant proteins. Low proteolytic activity in culture conditions and glycosylation patterns characteristic of higher fungi and animal cells have been documented, along with the expression of green fluorescent protein and hen egg white lysozyme in both P. flocculosa and P. antarctica (Avis et al., 2005). In the latter study, a genetic transformation protocol developed for P. flocculosa and based on polyethylene glycol and calcium chloride — mediated transformation of protoplasts (Cheng and Bélanger, 2000; Cheng et al., 2001) was used. This method has two major drawbacks: the preparation of protoplasts is delicate and time-consuming and protoplasts must be prepared fresh before each transformation experiment as they have poor viability when stored frozen at − 80 °C (Cheng, 2005). The availability and cost of suitable lytic enzymes required to obtain protoplasts can also be a concern. For example, the Novozym 234 enzyme used in the original protocol is no longer commercially available. New methodologies for genetic transformation of filamentous fungi have been proposed recently to manipulate fungi
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refractory to protoplast generation. For instance, techniques previously associated with other organisms, such as electroporation of bacteria and Agrobacterium tumefaciens-mediated transformation (ATMT) of plants, are now being successfully used to transform yeasts and filamentous fungi. Several studies have reported the success of transformation by electroporation with yeasts (Hashimoto et al., 1985; Karube et al., 1985; Becker and Guarente, 1991) and filamentous fungi (Richey et al., 1989; Chakraborty et al., 1991, Sánchez and Aguirre, 1996; Weidner et al., 1998). The electroporation technique is based on the reversible permeabilization of the cell membrane induced by a short duration, high amplitude electric field (Chang, 1992). Different types of fungal cells can be made competent for plasmid DNA uptake by electroporation: cells treated with a weakening agent (Chakraborty et al., 1991), protoplasts (Saito et al., 2001) and untreated germinated conidia (Chakraborty and Kapoor, 1990; Sánchez and Aguirre, 1996). The possibility of directly using untreated conidia, with a simple transformation protocol giving high yields of transformants would make electroporation a clearly advantageous system for transformation of Pseudozyma spp. ATMT of fungal species was first demonstrated in the yeast Saccharomyces cerevisiae (Bundock et al., 1995) followed by different species of filamentous fungi (de Groot et al., 1998). Some 60 fungal species have thus far been successfully transformed by A. tumefaciens (reviewed by Michielse et al., 2005). A. tumefaciens transfers part of its DNA, namely the transfer DNA (T-DNA), into the host cell. The T-DNA is located on a large (ca. 200 kb) plasmid, the tumor-inducing (Ti) plasmid, which also contains several virulence genes that are required for infection of the host and transmission of the genetic material. Two short sequences on the Ti plasmid, called the left and right T-DNA borders, flank the portion of the Ti plasmid that is transferred (see Gelvin, 2003). Attractive features of ATMT include a simple transformation protocol, few T-DNA insertions in the host genome and high yields of transformants. Considering the mounting interest in the properties of Pseudozyma spp. and the limited efforts devoted toward developing transformation protocols for this particular group of basidiomycetous fungi, we aimed to apply both electroporation and ATMT methodologies to P. antarctica and analyze integration events in order to develop versatile, efficient and robust transformation protocols that could eventually be adapted to different Pseudozyma species and other related basidiomycetous yeast-like fungi. 2. Materials and methods
Diagnostics Canada, Laval, Québec, Canada), as well as cefotaxim (200 μM, Sigma-Aldrich Canada Ltd, Mississauga, Ontario) and moxalactam (100 μg ml− 1, Sigma-Aldrich) in the case of ATMT transformants. Unless specified otherwise, liquid cultures were inoculated with three 0.5 cm mycelial plugs and incubated at 25 °C with 150 rpm shaking. All culture media ingredients were supplied by Difco (BD Biosciences, Mississauga, Ontario, Canada). A. tumefaciens strain LBA4404 was provided by D. Michaud (Université Laval, Québec, Canada). For long-term storage, A. tumefaciens strains were maintained in 15% glycerol stocks stored at −80 °C. For short-term storage, strains were maintained on Luria–Bertani (LB) agar containing the antibiotic streptomycin (125 μg ml− 1) at 4 °C. Kanamycin (50 μg ml− 1) was added to the culture medium for strains containing pCAMBIA constructions. 2.2. Plasmid construction For the electroporation experiments, pSceI-hyg or pSPF.GFP were used. pSceI-hyg was provided by Dr. J. Kronstadt (University of British Columbia, Canada) and contains a hygromycin B selection cassette containing the Escherichia coli hygromycin phosphotransferase (hph) gene under the control of the Ustilago maydis HSP70 promoter and terminator sequences. pSPF.GFP (Avis et al., 2005) contains two cassettes in the same orientation, the hygromycin selection cassette from pSceI-hyg and a green fluorescent protein (GFP) expression cassette consisting of the gGFP gene under the control of the U. maydis HSP70 promoter and terminator sequences (Fig. 1). For the ATMT experiments, two constructions were made using the pCAMBIA-0380 binary vector (CAMBIA, Canberra, Australia) that contains a multiple cloning site between the left and right T-DNA borders. pCAMBIA-hyg was created by ligating a 2681 bp AvrII/HindIII fragment containing the hygromycin selection cassette from pSceI-hyg into AvrII/HindIII — digested pCAMBIA-0380. pCAMBIA-hyg-GFP was generated in a similar fashion by ligating a 6726 bp AvrII/XmnI fragment containing both hygromycin selection and GFP expression cassettes from pSPF.GFP into AvrII/XmnI — digested pCAMBIA-0380 (Fig. 1). Both constructions were electroporated into A. tumefaciens strains LBA4404 using a Gene Pulser II with Pulse Controller (Bio-Rad, Hercules, CA) under standard conditions for E. coli (100 Ω resistance, 25 μFD capacitance, 2.5 kV voltage). Plasmid DNA was isolated from E. coli DH5α using the QIAgen MiniPrep kit (QIAgen, Mississauga, Ontario, Canada). DNA restriction and ligation, and agarose gel electrophoresis were carried out as described in Sambrook et al. (1989).
2.1. Strains, growth media and culture conditions 2.3. Transformation protocols Pseudozyma antarctica (Goto, Sugiyama, and Lizuka) Boekhout (CBS 516.83) was maintained on potato-dextroseagar (PDA) at 4 °C. Liquid cultures were performed in 500 ml Erlenmeyer flasks containing 100 ml of yeast extract (3 g l− 1), malt extract (3 g l− 1), peptone water (2.5 g l− 1) and dextrose (5 g l− 1) (YMPD) broth. All transformants were grown in the presence of the antibiotic hygromycin B (300 μg ml− 1, Roche
The electroporation experiments were performed as previously described by Sánchez and Aguirre (1996) and Brown et al. (1998) with some modifications. Cells from 5 ml of 3-day-old P. antarctica broth cultures were collected by centrifugation at 3000 ×g, 4 °C, washed twice in cool sterile water and resuspended in 270 mM sucrose, 5 mM Tris–HCl, 1 mM LiAc, pH 7.5 to
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obtain ca. 1 × 108 cells ml− 1. Fifty microliter aliquots of cell suspension were mixed with XhoI-linearized or circular plasmid DNA in pre-chilled 0.2 cm electroporation cuvettes (VWR International, West Chester, PA) and chilled on ice for 15 min. Electroporation was performed using a Gene Pulser II with Pulse Controller (Bio-Rad). Combinations of voltage (0.65 to 1.90 kV in 250 V increments) and resistance (200, 400, 600 and 800 Ω) were tested. Capacitance was held constant at 25 μF with one pulse. The time constant varied between 4.6 and 14.6 ms. Immediately following the pulse, 1 ml of YMPD broth was added to the transformed cells. The conidia were then transferred to a 15 ml tube (Sarstedt, Montréal, Québec, Canada), chilled on ice for 15 min and then incubated for 45 min at 25 °C with 250 rpm shaking. 100–400 μl aliquots were spread on PDA plates containing 300 μg ml− 1 hygromycin B (Roche). For the ATMT experiments, the transformation protocol was based on that of de Groot et al. (1998), with a few modifications. A. tumefaciens LBA4404 containing pCAMBIA-hyg or pCAMBIA-hyg-GFP was grown in LB broth medium (instead of minimal media) supplemented with appropriate antibiotics at 28 °C with shaking (250 rpm) to an OD600 of at least 0.5.
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Bacteria were collected by centrifugation (15 min at 2000 ×g and 4 °C) and resuspended to an OD600 of 0.15–0.20 in induction medium (IM, Bundock et al., 1995), without antibiotics, with or without 20 μM acetosyringone (AS, SigmaAldrich) and incubated overnight (instead of 6 h). Untransformed A. tumefaciens LBA4404 was used as a negative transformation control. P. antarctica was prepared by centrifuging a 3-day-old liquid culture (containing mostly conidia and some hyphae) for 10 min at 2000 ×g, 4 °C. Cells were resuspended in 0.85% sterile saline solution and a serie of 10fold dilution was prepared. An equal volume of bacteria in IM and fungus in saline solution were mixed by gentle pipetting and 200 μl aliquots were plated on 6-cm Petri dishes containing the co-culture medium (IM containing a reduced glucose concentration of 5 mM) with or without AS overlaid with a sterile disc of cellophane (gel-drying grade, Bio-Rad). After a co-culture period of 24, 48 or 72 h at 25 °C, the cellophane discs overlaid with the co-cultivation mixture were transferred to a selective medium consisting of PDA containing 300 μg ml− 1 hygromycin B (Roche) to select for transformants, and 200 μM cefotaxim (Sigma-Aldrich) and 100 μg ml− 1 moxalactam
Fig. 1. Map of plasmid vectors used for electroporation (A, B) and Agrobacterium tumefaciens-mediated transformation (C, D) of Pseudozyma antarctica. pro: Ustilago maydis heat shock protein 70 promoter, hph: Escherichia coli hygromycin phosphotransferase gene, ter: U. maydis heat shock protein 70 terminator, GFP: green fluorescent protein gene, amp: ampicillin resistance gene, kan: kanamycin resistance gene, LB: left transfer-DNA border repeat, RB: right transfer-DNA border repeat.
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Fig. 2. GFP expression by Pseudozyma antarctica transformants in broth cultures, observed at a magnification of 400× under visible light (A, C, E) and in the dark with GFP excitation at 488 nm (B, D, F). A, B: untransformed P. antarctica (control); C, D: electroporation transformant; E, F: Agrobacterium tumefaciens-mediated transformant.
analyzed with Image Pro Plus software (MediaCybernetics) using an exposition time of 700 ms for GFP images under 488 nm light and 20 ms for images under visible light. Wildtype P. antarctica was used as control. Randomly picked transformants were submitted to PCR analysis using primers 5'-GGAAGTGCTTGACATTGGGG-3' and 5'-CTTCTACACAGCCATCGGTCCAG-3' to amplify a 746-bp fragment of the hph gene, and primers 5'-GGTGAGCAAGGGCGAGGAGC-3' and 5'-CGGTCACGAACTCCAGCAGG-3' to amplify a 677-bp fragment of the GFP gene for pSceI-hyg or pSPF.GFP transformants. Additionally, for A. tumefaciens transformants, primers 5'-TCTACGGGGTCTGACGCTC-3' and 5'-GATGGCGTCCTTTGCTCGG-3' were used to amplify a 584-bp fragment of the kanamycin gene to test for integration of plasmid sequences located outside the T-DNA borders. 10 ng purified genomic DNA was used as DNA template in a 20 μL PCR reaction containing 1 U of HotMaster Taq DNA polymerase (Eppendorf AG, Hamburg, Germany); 1× manufacturer's PCR buffer; 0.2 mM each of dNTPs and 0.5 μM of each primer. Amplification was performed in an Eppendorf Mastercyler programmed as follows: initial denaturation at 94 °C for 2 min, 30 s at 94 °C for denaturing, 30 s at 62 °C for annealing, 1 min at 65 °C for synthesis, repeated for 30 cycles and a final extension step of 10 min at 65 °C. One nanogram of plasmid DNA was used as template for the positive control reaction. Aliquots of 10 μl of amplified product were separated in 1% (w/v) agarose gels in Tris-Acetate-EDTA buffer, stained with ethidium bromide and photographed under UV light. Southern analysis was performed for a subset of transformants using genomic DNA isolated with the QIAgen Genomic Tips 20/G or 100/G kits (QIAgen) and digested with restriction enzymes as well as undigested DNA. DNA was sizefractionated on a 1% agarose gel and transferred onto Hybond N+ nylon membrane (Amersham, GE Healthcare Bio-Sciences Inc., Baie d'Urfé, Québec, Canada) using a VacuGene XL vacuum blotting system (Amersham) according to the manufacturer's instructions. Blots were probed with the PCR products described above for the hygromycin and kanamycin genes labeled with Redivue [ 32 P]dCTP (Amersham) using the
(Sigma-Aldrich), to prevent growth of A. tumefaciens, and incubated at 25 °C. Serial dilutions and plating were used to determine the number of bacterial and fungal input colony forming units (cfu) before they were mixed together. 2.4. Transformant analysis Prior to analysis, transformants obtained by both methods were isolated by transferring fungal tissue from a single isolated colony on the surface of a Petri dish of selective medium to a new Petri dish of selective medium at least three consecutive times. GFP expression was observed as described previously (Avis et al., 2005). Cultures were observed under blue light (488 nm) using an incident fluorescent microscope (Olympus, model BH2-RFCA, Tokyo, Japan) equipped with a BH2-RFLT3 mercury lamp. Images were recorded using a Coolsnap-Pro (MediaCybernetics, Silver Spring, MD) color camera and
Fig. 3. Effect of resistance and voltage conditions for electroporation transformation of Pseudozyma antarctica using 5 μg of XhoI-linearized pSceIhyg. Results are means of two replicates.
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Fig. 4. A) Number of Pseudozyma antarctica transformants obtained by electroporation using increasing quantities of the pSceI-hyg plasmid vector. B) Number of transformants obtained per microgram of input plasmid DNA Results obtained are means of two replicates.
Rediprime random labeling kit (Amersham). Labeled probes were purified using MiniQuick Spin columns (Roche). Hybridizations were performed at 65 °C in 2× SSC, 0.5% SDS, 0.25% skimmed milk hybridization buffer. Hybridized blots were washed three times in 2× SSC, 0.1% SDS and twice in 0.1× SSC, 0.1% SDS at 65 °C and exposed to Kodak Biomax film (Kodak Canada, Toronto, Ontario, Canada). 3. Results and discussion 3.1. Transformation efficiency and stability of transformants Transformants appeared on selective media 3 days after electroporation and 5–10 days after ATMT experiments. Transformation efficiency was in the range of 100–200 transformants/μg of DNA per 108cells for electroporation and 60– 160 per 106 input cfu for ATMT. Transformants obtained by electroporation with pSPF.GFP and ATMT with pCAMBIAhyg-GFP showed strong GFP expression (Fig. 2). Transformants maintained GFP expression and hygromycin B resistance over 6 months of repeated subculturing on selective medium. Five transformants from each protocol were subcultured repeatedly for 10 generations on non-selective medium. All of them maintained hygromycin resistance and GFP fluorescence after being brought back on selective medium. 3.2. Effect of transformation parameters The effect of specific transformation parameters on transformant yield was studied in order to adapt the transformation protocols for P. antarctica. Electroporation experiments are generally conducted under conditions resulting in 50%
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mortality of cells (Brown et al., 1991). For P. antarctica, we determined these conditions in the absence of plasmid DNA to be a voltage of 1.0–1.8 kV with one pulse at 400 Ω resistance. Optimal conditions for transformation were determined first by using a fixed amount (5 μg) of XhoI-linearized plasmid DNA and testing a range of voltage values between 0.65 and 1.90 kV in 250 V increments combined with four resistance values (200, 400, 600 and 800 Ω). As illustrated in Fig. 3, for each fixed resistance value tested except the lowest (200 Ω), transformant yield reached a voltage optimum before decreasing with higher voltage values. At the lowest resistance value (200 Ω), the number of transformants obtained increased with higher voltage values but transformant yield was low compared to those obtained with higher resistance values. The greatest number of transformants was obtained with 1400 V and 400 Ω and with 1150 V and 800 Ω. These optima probably result from equilibrium between DNA uptake/transformation and cell mortality (see Weaver, 1995). The effect of the quantity of input plasmid DNA was then tested by using 0.5 to 17 μg of XhoI-linearized pSceI-hyg with a set number of input cells (ca. 108) and fixed electrical conditions (1400 V and 400 Ω). The number of transformants increased with plasmid DNA quantity up to 13 μg of plasmid DNA (Fig. 4A). At this point, other factors such as cell concentration are probably limiting the transformation process (Suga and Hatakeyama, 2003). When yield was expressed as the number of transformants obtained per μg of plasmid DNA (Fig. 4B), the best values were obtained between 0.5 and 3 μg with a maximum of ca. 200 at 1 μg. This yield is higher or comparable to those reported for other fungi such as protoplasts of Lyophyllum shimeji (Saito et al., 2001), DTT-treated cells of Candida parapsilosis (Gácser et al., 2005) and germinated conidia of Humicola grisea var. thermoidea (Dantas-Barbosa et al., 1998) or Colletotrichum gloeosporioides f. sp. aeschynomene (Robinson and Sharon, 1999). In the ATMT experiments, the effect of AS in the co-culture medium and in the induction medium for the A. tumefaciens culture, the duration of co-cultivation and the number of input P. antarctica cells were investigated. Treatments consisted of a combination of these three factors. AS is a phenolic compound
Table 1 Transformation efficiency of Pseudozyma antarctica by Agrobacterium tumefaciens as influenced by the ratio of bacterial to fungal cells and the duration of the co-cultivation period Transformation efficiency (transformants/106 input colony forming units) a Duration of co-cultivation A. tumefaciens:P. antarctica cells ratio
48 h
72 h
26:1 260:1 2600:1 2.6 × 104:1 2.6 × 105:1 2.6 × 106:1 2.6 × 107:1 2.6 × 108:1
0.68 4.2 21 0 0 0 0 0
7.0 16 63 156 0 0 0 0
a
Results are means of three replicates.
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resembling those emitted by plants at wound sites and is believed to be an inducer of the A. tumefaciens virulence genes (see Gelvin, 2003). AS was used both in induction and co-cultivation medium or withheld from both to test its effect on transformation efficiency. In three initial experiments, transformants were obtained only in the presence of AS; this suggests that AS is essential for ATMT of P. antarctica, as is the case in the majority
of fungal species (Michielse et al., 2005). In the presence of AS in the induction and co-cultivation media, although transformed colonies appeared on selective medium after a co-cultivation of only 24 h in two out of three experiments, a co-cultivation of at least 48 h was required to obtain a significant number of transformed colonies with both plasmids, and increasing the cocultivation period to 72 h further improved transformation
Fig. 5. Southern blot of genomic DNA from Pseudozyma antarctica (lanes Pa) and transformants obtained by electroporation (lanes 1–10) and Agrobacterium tumefaciens-mediated transformation (lanes 11–20) hybridized with a fragment of the hygromycin phosphotransferase gene (A, B) or the kanamycin resistance gene (C) as probe. Restriction enzymes used: S: SacII, P: PvuII, X: XhoI, N: NcoI, U: undigested DNA.
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efficiency. In ATMT, the transformation efficiency is dependent on the ratio of bacteria to fungal cells (e.g. Godio et al., 2004). The transformation efficiency was approximated by co-cultivating decreasing quantities of P. antarctica cfu with a fixed quantity of A. tumefaciens LBA4404 containing binary vector pCAMBIA-hyg in the presence of AS for 48 or 72 h before transferring to selective medium (Table 1). The highest transformation efficiencies obtained were 63 and 156 transformants per 106 input cfus, at bacterial to fungal cell ratios of respectively 2600 and 26 000:1 after a co-cultivation period of 72 h. No transformants were obtained at higher bacterial to fungal cell ratios, probably due to the limiting number of P. antarctica cells. 3.3. Transformant analysis Transformants were tested by PCR and Southern hybridization to characterize the plasmid and T-DNA insertions. All transformants tested yielded a PCR product of the expected size for the hph (all transformants) and GFP genes (pSPF.GFP and pCAMBIA-hyg-GFP transformants) (data not shown). Genomic DNA from randomly selected transformants was examined by Southern hybridization (Fig. 5). Ten transformants obtained by electroporation were analyzed (Fig. 5A), of which four were obtained with XhoI-linearized pSceI-hyg (transformants 1 to 4 in Fig. 5A) and the remaining six (transformants 5 to 10 in Fig. 5A) with circular pSceI-hyg. In transformants 1 to 4, hybridization of the hygromycin probe to undigested genomic DNA confirmed integration into chromosomal DNA whereas no such signal was observed in DNA from untransformed P. antarctica. SacII cuts once in the pSceI-hyg plasmid within the region covered by the hph probe. Thus two bands of a different size (corresponding to junctions between plasmid and genomic DNA) would be expected for each locus of integration. A prominent 5.9 kb band corresponding to the size of the vector was observed in all lanes except for transformant 7 indicating the presence of tandemly inserted copies of the plasmid. The fainter bands likely represent junctions between the tandem plasmid arrays and the flanking host DNA, each integration locus producing a pair of such bands. As for PvuII, it cuts twice within the plasmid, on either side of the hygromycin resistance gene, such that a 3.1 kb fragment should be detected by the probe. In PvuII-digested DNA, a band of the expected size is seen in the first four transformants. An additional band of ∼ 5.9 kb is also commonly found in most if not all of these transformants and may simply be due to partial digestion at one of the two PvuII sites. In addition to these common bands, a unique PvuII junction fragment is expected for each independent locus of insertion. Based on the banding patterns observed in the four electroporation transformants obtained with XhoIlinearized pSceI-hyg analyzed, it is likely that transformants 1 and 2 contain two insertion loci, each of which contains at least two copies of the plasmid in a direct tandem repeat. Transformants 3 and 4 could contain three such loci, and possibly even four in the case of transformant 4, but this is more difficult to interpret because of the more complex banding pattern. Thus, multiple tandem arrays of the plasmid vector
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were found to be the rule when using linearized plasmid for transformation. Similarly, tandem insertions were found to occur in most electroporation transformants in Schizosaccharomyces pombe (Davidson et al., 2004), albeit mostly at single integration sites, while different integration sites and multicopy integration occurs in Humicola grisea var. thermoidea (DantasBarbosa et al., 1998). Simpler integration events were reported for other fungi (e.g. Brown et al., 1998; Gácser et al., 2005), and some P. antarctica transformants electroporated with pSPF. GFP showed evidence of single integration events (data not shown). As pSceI-hyg does not contain any P. antarctica homologous sequences, it is likely that the genomic integration obtained with linearized plasmid is random. In the six electroporation transformants obtained with circular pSceI-hyg, hybridization of the hygromycin probe to undigested genomic DNA is not as evident, and thus integration of the circular plasmid into chromosomal DNA may not have occurred. However, resolution is limiting for high molecular weight genomic DNA on 1% agarose gels and the variation in the banding pattern observed between the different transformants suggests that some of these bands may be junctions with adjoining chromosomal DNA. The 5.9 kb band in PvuII digests and the 3.1 kb band in SacII digests are common to transformants 5, 6, 8, 9, and 10 and may represent autonomously replicating plasmid or two copies chromosomally integrated in a tandem array as discussed above. As pSceIhyg does not contain a eukaryotic autonomously replicating sequence, the mechanism for maintenance of the plasmid is unknown. When using an undigested plasmid containing no homologous sequence or eukaryotic origin of replication to transform the yeast Candida oleophila, Yehuda et al. (2001) obtained no transformants. For A. tumefaciens transformants, 10 transformants obtained with a strain containing the binary vector pCAMBIA-hyg were analyzed. Hybridization of hygromycin (Fig. 5B) and kanamycin (Fig. 5C) probes to undigested genomic DNA indicated that the plasmid was inserted within high molecular weight genomic DNA. The fact that the kanamycin resistance gene was also detected in transformants suggests that transformation was not limited to the T-DNA but also included vector sequences located outside the T-DNA. In agreement with this observation, all transformants shared a common 9.4 kb band upon digestion with XhoI and hybridization with both hygromycin and kanamycin probes. Such an observation is consistent with the presence of at least two copies of the whole pCAMBIA-based plasmid in a direct tandem repeat. Similarly, upon digestion with NcoI, all transformants shared bands of 1.5 and 7.9 kb with the hygromycin probe, and a single band of 7.9 kb with the kanamycin probe. Additional bands are present in transformants 11, 16 and 18 with both probes in digested DNA and could correspond to junctions between a plasmid insertion and adjacent genomic DNA. The presence of transferred vector sequences located beyond the T-DNA borders has been reported in filamentous fungi (Covert et al., 2001) and is believed to occur frequently in plants without being monitored (Martineau et al., 1994). The insertion of the whole vector rather than only the T-DNA has been
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reported before in S. cerevisiae (Bundock et al., 1995), albeit in a small (3 out of 20) number of transformants and in plants (Wenck et al., 1997). As T-DNA transfer normally starts at the right border after single-strand nicking at both borders, it has been hypothesized that transfer of the whole binary vector could result from missing the left T-DNA border during processing of T-DNA from the binary vector or from transfer initiation at the left T-DNA border (Kononov et al., 1997). The 10 transformants analyzed thus show evidence of integration of at least two copies of the whole binary vector, which is atypical as ATMT of fungi generally yields a low number of T-DNA insertions (Michielse et al., 2005). It is also possible, however remote this may seem, that a contamination of the fungal cultures used for DNA purification with the transforming A. tumefaciens is responsible for the presence of the full binary vector. 4. Conclusion Electroporation and ATMT are two new methods suitable for the transformation of P. antarctica with distinct advantages over the previously reported PEG/CaCl2-mediated approach. Electroporation is rapid and simple and allows for the recovery of transformants in only a few days. ATMT does not require the purification of the plasmid prior to transformation or the use of sophisticated equipment once the A. tumefaciens strain carrying the desired construction has been obtained. Both methods allow for the recovery of a high number of transformants and use untreated conidia of P. antarctica as starting material; this greatly simplifies the transformation protocol as lytic enzymes or chemical weakening agents are no longer required, a clear drawback in other fungal transformation systems (Yehuda et al., 2001; Kuo et al., 2004). Analysis of integration events was not definitive but allows insights into the fate of the transforming DNA and is of interest in the context of using P. antarctica for the production of heterologous proteins. The description of these new transformation methods should facilitate further genetic studies and exploitation of P. antarctica and other Pseudozyma species. Acknowledgements We thank D. Michaud for providing A. tumefaciens strain LBA4404, S. Laberge and R. Desgagnés of Agriculture and Agri-Food Canada for providing the hybridization protocol used in this study, C. Labbé for technical assistance with the figures and T.J. Avis for critical revision of the manuscript. Fig. 1 was created using the BVTech Plasmid freeware from BVTech Inc. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Canada Research Chairs Program to R.R. Bélanger. References Avis, T.J., Cheng, Y.L., Zhao, Y.Y., Bolduc, S., Neveu, B., Anguenot, R., Labbé, C., Belzile, F., Bélanger, R.R., 2005. The potential of Pseudozyma yeast-like epiphytes for the production of heterologous recombinant proteins. Appl. Microbiol. Biotechnol. 69, 304–311.
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