Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation
© Brenna et al.; licensee Springer 2014
Received: 11 April 2014
Accepted: 27 April 2014
Published: 29 May 2014
Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi (“truffles”) with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.
KeywordsTuber spp Truffles Agrobacterium tumefaciens-mediated transformation T-DNA binary plasmid Green fluorescent protein Hygromycin phosphotransferase TAIL-PCR
The ascomycete Tuber borchii is a hypogeous fungus (genus, Tuber; family Tuberaceae; order Pezizales) that establishes a beneficial mutualistic interaction (‘ectomycorrhiza’) with the roots of many tree species. T. borchii has also a pronounced saprobiotic capacity and can be grown in vitro (albeit quite slowly) as vegetative free-living mycelium in the absence of a plant host. This mixed symbiotic/saprophytic lifestyle (Hebe et al. ), together with the lack of asexual spores amenable to in vitro culture has hampered the genetic manipulation of this fungus and obscured our understanding of its complex life cycle. Tuber fruitbodies (‘truffles’) lack an active system for the dispersal of spores, which are disseminated by the action of mycophagous animals (Pegler et al. ). Another peculiarity of certain Tuber spp. is the high commercial value of their fruitbodies which are prized as gourmet food. Tuber melanosporum and T. borchii are the two most studied truffle species. Sequencing and annotation of the T. melanosporum genome, the first symbiotic Ascomycete to be sequenced, revealed many aspects of truffles’ biology and genetic organization, including their massive content of repeated transposable elements and their heterotallic mode of conjugation and ability to outcross through the action of two distinct mating type loci (Martin et al. ). T. borchii is the truffle that can be more easily handled under laboratory conditions.
With the acquisition of new genome sequence data, functional genetic studies are all the more necessary to decipher gene function. Reverse genetics in Tuber has been hindered, however, by the absence of a well-established stable transformation system. An effective approach in this direction is represented by Agrobacterium tumefaciens-mediated (ATM) transformation of intact hyphae. This technique was first successfully applied to fungi 19 years ago (Bundock et al. ) by exploiting the natural ability of A. tumefaciens to transfer a portion of its DNA to a foreign infected organism, most notably dicotyledonous plants. To date, ATM transformation is applied to the study of a variety of fungal species, including Ascomycetes, Basidiomycetes and Zygomycetes. The transfer DNA (T-DNA), located on a >200 kb tumor-inducing (Ti) plasmid, is flanked by two 25 bp direct imperfect repeats, known as left border (LB) and right border (RB) sequences, which also include the vir genes encoding the genetic factors required for transfer (Zupan et al. ). Recent improvements of ATM transformation include the optimization of temperature and co-cultivation conditions, and the development of new selection markers (Michielse et al. ). We previously described an ATM transformation procedure for T. borchii (Grimaldi et al. ), which due to the insertion of transgenic DNA driven by the T-DNA, and consequent lack of stability, could not be effectively exploited for functional genomics studies. Building upon these earlier attempts, in the present work we improved transformation conditions through the development of two new vectors and also gained insight into the fate of the transferred T-DNA, which was found to be randomly integrated, with a preference for repeat element-containing genome sites. These results represent an important technical advancement for the molecular biological investigation of truffles, which will be instrumental to the construction of mutant strain collections.
Materials and methods
Strains and media
T. borchii Vittad. mycelia (isolate ATCC 95640) were grown and propagated in the dark at 24°C on potato-dextrose medium with agar 39 g/l (PDA: 0.2% peptone, 0.2% yeast extract, 1.8% glucose, 0.5% potato starch, 1.5% agar; Liofilchem), as described (Ambra et al. ). For easy medium replacement, mycelial cultures were usually grown on sterile dialysis membranes. For liquid culture replacement, mycelial cultures were inoculated in potato-dextrose liquid medium (PDB, without agar). A. tumefaciens was grown and propagated in Luria- Bertani (LB) (1% tryptone, 0.5% yeast extract, 1.5% agar) solid or liquid (w/o agar) medium at 28°C in the presence of appropriate marker selection supplements. After co-cultivation, mycelia were transferred onto Evans minimal medium (1x Vogel’s salts, 1% glucose, 2 mg/l vitamin B1, 1.5% agar). The hypervirulent Agrobacterium strain AGL-1 [(C58 pTiBo542) recA::bla, T-region deleted Mop (+) Cb (R)] was kindly provided by Peter Romaine (Department of Plant Pathology, Pennsylvania State University, University Park, PA, USA) (Lazo et al. ). Carbenicillin (Sigma-Aldrich, St Louis, MO) 100 μg/ml was used for AGL-1 propagation. The GV3101 strain (background C58; Ti-plasmid cured) was kindly provided by Paolo Costantino (Department of Biology and Biotechnology “La Sapienza” University, Rome, Italy). Rifampicin (25 μg/ml) and gentamicin (25 μg/ml), both provided by Duchefa Biochemie, were used for GV3101 propagation.
T. borchii mycelia were pre-grown in the dark at 24°C for 7 days on cellophane-overlaid PDA plates and then transferred to PDA medium containing 200 μM acetosyringone (AS) to stimulate Agrobacterium virulence (outlined in Additional file 1: Figure S1). Three days before the start of co-cultivation, pre-transformed Agrobacterium cells were grown at 28°C for 2 days in the presence of the appropriate selection marker antibiotics (100 μg/ml carbenicillin for AGL-1; 25 μg/ml each of rifampicin and gentamycin for GV3101; 30 μg/ml kanamycin for both vectors). On the day of co-cultivation, a fresh overnight bacterial culture with an optical density at 600 nm (OD600nm) of 1.2-1.5 was diluted to an OD600nm of 0.075 with antibiotic-supplemented medium also containing 200 μM AS and grown for 4 h to an OD600nm of 0.3. After reaching this cell density, 50 μl of bacteria in LB (1.5 × 103 cells/μl) were overlaid onto T. borchii mycelia and co-cultivation was continued for 3 days at 22-24°C. Following co-cultivation, mycelia were washed with sterile ddH2O and 400 μM cefotaxime, and mycelial samples were sliced and examined by confocal microscopy. The remaining mycelia were transferred to new plates of Evans minimal medium containing 400 μM cefotaxime (to inhibit bacterial growth) and 15 μg/ml hygromycin (to aid in transformant hyphae isolation). Additional mycelial samples were analyzed after 6 days of co-cultivation, and the remaining mycelia were monitored over time for hygromycin B resistance and GFP expression.
Fluorescence and confocal microscopy
Sections (3 × 3 mm) of either control, untransformed T. borchii mycelia or mycelia co-cultivated with A. tumefaciens for 3 days were washed 5 times with distilled water and cefotaxime (400 μM) prior to microscopic analysis. An Axioskop 2 microscope (Carl Zeiss International, Oberkochen, Germany) equipped with a 100X oil immersion objective (Plan Neofluar), a Zeiss filter set (green excitation filter 450–490 HB) and a Zeiss AttoArc2 HB-100 W mercury lamp were used for fluorescence microscopy analysis. Images were captured with a Microcolor camera (RGB-MS-C; CRI, Boston, MA) and processed with Diffraction Micro CCD software and Adobe Photoshop (Adobe System, San Jose, CA). Typical exposure times were 30–60 ms for the SGFP and Nomarski images. Confocal laser scanning microscopy was performed with a Leica microscope equipped with a TCS SP2 Laser (Ar/Kr, Gre/Ne, He/Ne; standard phase contrast plus Normaski contrast). SGFP was excited with a 488-nm laser line and detected at 515-530 nm.
DNA extraction and PCR analysis
Mycelia to be analyzed by confocal microscopy were washed 5 times with ddH2O and 400 μM cefotaxime and then disrupted by freezing in liquid nitrogen. DNA was extracted from frozen mycelia using the DNeasy Plant Minikit (QIAGEN N. V., Hilden, Germany) as per manufacturer’s instructions. The amplification protocols described in (Grimaldi et al. ) were employed for PCR analysis of the aminoglycoside 3'-phosphotransferase (kanR) and sgfp genes, using the oligonucleotide primers listed below.
kanR primers (to amplify a region comprised within the binary plasmid vector):
kanR_Fw: 5′-GGTCATGCATTCTAGGTACT-3′; kanR_Rw: 5′-AATGGCTAAAATGAGAATAT-3′.
Sgfp primers (to amplify a region comprised within the T-DNA):
sgfp_Fw: 5′- CACATGAAGCAGCACGACTT-3′; sgfp_Rw: 5′-TGCTCAGGTAGTGGTTGTCG-3′;
ToxA promoter region (also comprised within the T-DNA):
DNA blot analysis
Genomic DNA from transformed and untransformed (negative control) T. borchii mycelia (10 μg each) plus the same amount of pABr1 plasmid DNA (positive control) were digested with Nco I. A separate sample of pABr1 was digested in parallel in order to generate a pair of DNA fragments to be used as probes. Four fragments were obtained from Nco I-Xho I digestion, including an 840 bp fragment that was used as probe. This fragment comprises a region (located around the T-DNA junction site) that encompasses the site of recombination with genomic DNA. The second probe was obtained by Nco I digestion, which produced three different fragments. The fragment that was utilized as probe was 954 bp in length (from the middle of the hygromycin phosphotransferase cassette to the ToxA promoter). This probe matches the region of the T-DNA that is transferred to T. borchii hyphae. Radioactive labeling with approximately 20 ng of purified templates, blotting, hybridization and washing were performed as described (Green and Sambrook ; Montanini et al. ).
TAIL-PCR amplification, cloning and sequencing of T-DNA integration sites
Genomic DNA sequences flanking the T-DNA were amplified by Thermal Asymmetric Interlaced PCR (TAIL-PCR) as described in (Wang and Li ). The following oligonucleotides were used as primers to amplify the Left Border (LB) and the Right Border (RB) regions, in combination with the degenerate, genome-sequence targeting primer AD1.
LB1 5′-GGGTTCCTATAGGGTTTCGCTCATG-3′ LB2 5′-CATGTGTTGAGCATATAAGAAACCCT-3′ LB3 5′-GAATTAATTCGGCGTTAATTCAGT-3′
RB1 5′-GGCATGGCCGTCGTTTTACAAC-3′ RB2 5′-AACGTCGTGACTGGGAAAACCCT-3′ RB3 5′-CCCTTCCCAACAGTTGCGCA-3′
DNA amplified by TAIL-PCR was cloned into the pDrive A/T vector (QIAGEN) according to the manufacturer’s instructions, followed by DNA sequence analysis of plasmids isolated from transformant colonies.
Mycelial sections (3 × 3 mm) containing ~4500 hyphal apexes, as estimated by counting with a Burker chamber, were used for transformation efficiency determinations. The fraction (%) of transformed hyphae was obtained by dividing the number of fluorescent hyphae (average of multiple determinations) by 4500; data were calculated as the mean ± standard error of the mean (s.e.m.) of at least five independent replicates.
The Vector NTI® Software (Life Technologies) was used for drawing plasmid maps. BLAST searches were conducted against the T. melanosporum genome sequence at the MycorWeb site (http://mycor.nancy.inra.fr).
ATM transformation of T. borchii mycelia using a Pyrenophora ToxA promoter-bearing binary vector
We previously documented the production of transformed, GFP-expressing T. borchii mycelia with the use of ATM and the pBGgHg plasmid (Grimaldi et al. ). However, transformation efficiency was rather low (0.2-0.5%) with a fairly weak GFP signal that was lost after a few weeks, possibly due to the lack of stable integration. In order to improve transformation efficiency and to achieve higher GFP expression levels as well as stable transformants, a new series of cloning vectors for ATM transformation was constructed. We first created a binary vector, named pABr1 (Figure 1a), with the sgfp gene under control of the ToxA promoter from the ascomycete P. tritici-repentis (Ciuffetti et al. ), inserted within the T-DNA region, which also contains the hygromycin phosphotransferase (hph) gene utilized for positive selection of transformants. The sgfp gene, which codes for the S65T variant of GFP, is a commonly utilized transformation marker in fungi (Niwa ) and use of the ToxA promoter has previously been shown to enhance the expression of reporter genes in various fungi including Neurospora crassa (Lorang et al. ). Our goal was to couple the enhanced strength of the ToxA promoter with the increased stability of SGFP in order to obtain a stronger fluorescence signal and thus an improved visualization of transformed hyphae. To this end, we performed ATM transformation by treating mycelia with the AGL-1 strain, carrying either pABr1 or pBGgHg, and comparing the transformation efficiency of these two vectors. After 3 days of co-cultivation (outlined in Additional file 1: Figure S1), a subset of mycelia was transferred to new antibiotic-containing plates (ampicillin, cefotaxime and hygromycin B) and samples were taken for hyphal section visualization by confocal microscopy. This allowed the identification of fluorescent, GFP-producing hyphae, by comparison with the background signal associated with control mycelia transformed with the empty AGL-1 strain (Figure 1b). The diffuse fluorescence background present in mock-transformed samples, contrasted with the localized signal observed in the apical hyphae of T. borchii transformed with AGL-1 carrying the binary vector. Transformation efficiency obtained with the pABr1 vector after 3 days of co-cultivation was consistently found to be at least two-fold higher (≥1%) than that obtained with pBGgHg using the same AGL-1 A. tumefaciens strain (Figure 1c). This was calculated as the ratio between fluorescent hyphae and the total number of hyphae per section (~4500). When the analysis was repeated at 6 days, i.e. after 3 additional days since the end of co-cultivation, the hyphae appeared more fluorescent, likely due to SGFP accumulation (Figure 1b), but without any appreciable change in the relative number of fluorescent hyphae (data not shown). Still, the SGFP signal in pABr1-transformed hyphae appeared stronger than that of hyphae transformed with the pBGgHg vector, thus facilitating the identification of transformants grown over time. Transformation was also verified by PCR amplification of the ToxA promoter using genomic-DNA extracted from transformed mycelia as template (Figure 1d). To confirm that ToxA amplification did indeed result from the mobilized T-DNA rather than from residual bacteria, we performed a parallel PCR analysis using a pair of primers annealing to the kanamycin resistance (kanR) gene region that is present in the plasmid outside of the T-DNA. The absence of a kanR amplification signal ruled out a false positive result caused by residual contaminating bacteria. We then used a different Agrobacterium strain (GV3101) to verify that the improved transformation efficiency obtained with pABr1 was a genuine effect of the plasmid vector, rather than an indirect effect of the specific A. tumefaciens strain utilized for transformation. The results obtained with GV3101 confirmed that also in this strain the transformation efficiency yielded by pABr1 was at least two-fold higher than that obtained with pBGgHg (Additional file 1: Figure S2a,b). However, global efficiency with GV3101-mediated transformation was generally lower than that obtained with AGL-1 (Additional file 1: Figure S2c), suggesting that not all A. tumefaciens strains are equally effective for Tuber transformation. Also in this case, the data were confirmed by PCR analysis, which revealed an sgfp amplicon in the case of DNA extracted from transformed mycelia, but not in the case of mock-infected mycelia (Additional file 1: Figure S2d). Furthermore, a stronger GFP signal was systematically observed in pABr1 transformants compared to transformants obtained with the pBGgHg vector utilized in previous studies (Grimaldi et al. ). Altogether the data confirm the superior performance of the newly designed pABr1 vector.
T-DNA transformation occurs by random integration in the Tuber genome
The P1 probe was obtained by using an 840 bp sequence derived from Nco I-Xho I digestion of pABr1 as template (Figure 2d). P1 recognizes the T-DNA region between the left border and the hph sequence. This region presumably recombines with the Tuber genome as described in other fungi (Thomas and Jones ). The other probe (P2) was generated by using as template, a 954 bp region of the T-DNA obtained by Nco I digestion (Figure 2d). This probe recognizes an internal sequence comprising part of the hph gene, the pdgp promoter and the entire ToxA promoter sequence, that is present regardless of the integrated or extra-chromosomal state of the T-DNA. The P2 probe was thus used as a positive control for hybridization experiments, to verify that DNA blotting had been performed correctly. Total genomic DNA from transformed and untransformed mycelia was then subjected to Nco I digestion, which would generate a smear of fragments recognized by the P1 probe only in the case of T-DNA integration. As shown in Figure 2e (left panel), a weak, P1 probe-positive band was only observed in transformed mycelia. The fact that the size of this band (~10,000 bp) was much higher than expected (840 bp) strongly suggests the occurrence of an integration event. In the same sample, a stronger signal resulting from the sum of integrated and extra chromosomal T-DNA fragments of the same size, was observed with the P2-probe, which hybridized with a DNA fragment, located within the T-DNA region, having an expected size of 954 bp (Figure 2e, right panel). Also in this case, the P2-associated signal was only observed in the case of DNA derived from transformed, but not mock-infected, mycelia. Altogether these data suggest the integration of at least some copies of the pABr1-derived T-DNA in the Tuber genome.
Improved transformation efficiency of the shortened pABr3 vector
Since it was previously impossible to obtain portions of Tuber mycelia capable of growing on media containing more than 15 μg/ml hygromycin B, we also analyzed the ability of AGL-1/pABr3-transformed mycelia to grow in the presence of increasing amounts of hygromycin B. To this end, transformed mycelia were transferred to plates containing 15 μg/ml hygromycin B and the outline of the mycelium was marked so to allow an easy visualization of mycelial clumps overgrowth over time. After 60 days (during which mycelia were transferred to fresh antibiotic-containing medium every 10 days), full-bodied mycelial clumps were found to propagate on medium containing 15 μg/ml of hygromycin B (Figure 3c, top panel). Following isolation and transfer of hygromycin-resistant mycelial slices to media containing increasing concentrations of hygromycin B, we found that transformants were able to propagate in the presence of hygromycin B concentrations as high as 300 μg/ml (Figure 3c, bottom panel). Mycelia subjected to this high-stringency selection procedure were used as starting material for further molecular analyses (see below).
Integration site identification by TAIL-PCR
Tuber spp. are ecologically and economically important filamentous Ascomycetes. These fungi, whose fruitbodies are commonly known as truffles (Pegler et al. ), have a subterranean habitat and establish a symbiotic relationship with several trees and shrubs via ectomycorrhiza formation (Hebe et al. ). Crucial for the success of the ectomycorrhizal interaction is the mutualistic exchange of nutrients between the symbiont and the host plant. Indeed, inoculation of plants with Tuber mycelia leads to an increased plant growth, indicating that the fungus is a mutualistic symbiont (Giovannetti and Fontana ). Recently, the genome sequence of a representative Tuber species, the black truffle T. melanosporum, has been determined (Martin et al. ). Although a large fraction of genes has been functionally defined by sequence homology, a consistent number of them (>40%) remains functionally unknown. Reverse genetic analysis will thus be required in order to assign a role to these orphan genes. In Tuber, conventional genetic transformation procedures failed due to inefficient protoplast regeneration (Poma et al. ). We sought to circumvent this problem with the use of A. tumefaciens- mediated transformation, a foreign DNA transfer strategy largely used in plants (Sheng and Citovsky ) and fungi (Michielse et al. ).
Our first attempt toward ATM-transformation in T. borchii was based on the pBGgHg binary vector (Grimaldi et al. ). Although the T-DNA entered host cells, transformation was transient and the organization of T-DNA (extrachromosomal vs. integrated) was not clear. Moreover, transformation efficiency was rather low (0.2-0.5%) and the EGFP fluorescence signal as well as hygromycin resistance were lost quite early, typically after 4–5 weeks of in vitro culture. To overcome these limitations, while trying to achieve integrative transformation, we built two new binary vectors (pABr1 and pABr3). In both pABr1 and pABr3, GFP expression is driven by the strong ToxA promoter from the pathogenic fungus Phirenophora trittici repentis, which has been employed successfully for transgene overexpression in other filamentous fungi (Freitag et al. ; Lorang et al. ). Transformation with AGL-1/pABr1 (Figure 1a) produced stable transformants with a strong and long-lasting SGFP expression (Figure 1b). Also, transformation efficiency increased by about two-fold compared to AGL-1/pBGgHg (Figure 1c). This difference in transformation efficiency was confirmed with GV3101, an Agrobacterium strain less virulent than AGL-1 (Additional file 1: Figure S2).
A further increase in transformation efficiency (~6-fold compared to pBGgHg) was obtained with pABr3 (Figure 3a, b), a shortened derivative of pABr1, lacking the unnecessary plant promoter/GFP/NOS terminator region (Figure 1a). This suggests that a smaller T-DNA size facilitates DNA transfer into host cells. Another important goal in order to strengthen selection efficiency was to maximize the concentration of hygromycin B that could be tolerated by transformed mycelia. Unlike transformation with the other AGL-1/binary vector combinations, AGL-1/pABr3 allowed the amplification of transformed mycelial clumps capable of growing in the presence of hygromycin B concentrations as high as 300 μg/ml (Figure 3c). This enabled the selective isolation and propagation of transformed mycelial regions. This is important, especially considering the lack of conidia-like elements in Tuber spp., the syncytial nature of Tuber hyphae, and thus the fact that transformants are mosaics, likely bearing more than one transformed locus. We noted, however, that transformed hyphae are not randomly distributed within mycelia, but appear to be concentrated at the periphery of mycelial clumps, the only region from which the fungus could be propagated. This suggests that T-DNA may require a metabolically active tissue for activation and efficient functioning.
With pAbr3, the SGFP signal not only increased in intensity and distribution, but most of the analyzed mycelial sections appeared to be enriched in transformed hyphae even after 1 year from the initial transformation. In fact, in transformed mycelia, the GFP signal progressively became more diffuse and increasingly spread to contiguous hyphae (Figure 3a). This can be explained if the T-DNA is integrated in the Tuber genome, so that following duplication of a transformed nucleus the amount of produced/accumulated GFP gradually increases over time.
Integrative transfer was indeed demonstrated by PCR amplification of the ToxA (Figure 1d) and the sgfp (Additional file 1: Figure S2d) sequences, as opposed to the negative amplification results obtained with the kanR gene region, which is located outside of the T-DNA (Figure 1d and Additional file 1: Figure S2d). This was further corroborated by the results of hybridization analyses conducted with region-specific probes. Most notably, the fact that the size of the fragment recognized by a probe hybridizing with the left T-DNA border (i.e., a region that is usually involved in the integration process (Thomas and Jones )) increased from an expected size of 854 bp to ~10,000 bp.
Lastly, we used transformed, hygromycin B-selected mycelia for the identification of the genomic sites of integration by TAIL-PCR (Liu and Whittier ), a widely used approach for the analysis of transformants in systems where homokaryotic colonies are hard to obtain (Poma et al. ). We sequenced T-DNA/Tuber genomic DNA flanking site amplicons obtained from transformed mycelia and found that two of them matched transposon-related elements (Figure 4). In Tuber, repeated elements, including retrotransposons, account for about 60% of the genome (Martin et al. ), suggesting that integrative transformation mediated by heterologous recombination is likely driven by repetitive DNA sequences.
The highly improved genetic transformation procedure described in this work represents a first, but crucial step toward a functional genomic analysis of Tuber, and perhaps other transformation-recalcitrant filamentous fungi as well, with the ultimate goal of producing a comprehensive site-specific mutant collection as in Aspergillus nidulans, N. crassa and Magnaporthe oryzae (http://www.fgsc.net/). Although whole genome sequence data are only available for T. melanosporum so far, we used T. borchii as an experimental organism to set up this improved transformation procedure because of the easier and more efficient propagation of its mycelium under axenic culture conditions. However, ongoing work, including the setting up of more efficient mycelial growth conditions, indicates that the optimized AGL-1/pABr3-protocol here applied to T. borchii also works well in T. melanosporum, with the production of well detectable levels of GFP and resistance to hygromycin concentrations as high as 50 μg/ml (A.B and P.B., unpublished).
The next step will be the disruption of non-homologous end joining (NHEJ) mediated by the Ku70 and Ku80 proteins in order to favor integration at homologous sites. The NHEJ pathway, discovered for the first time in mammals (Jeggo et al. ) and so named because it does not require homologous sequences to join DNA ends, repairs double-strand breaks in the absence of a homologous donor (Moore and Haber ). As shown in N. crassa (Ninomiya et al. ), disruption of the ku70/ku80 genes strongly enhances homologous recombination and thus the production of site-specific mutants. The presence of ku70/ku80 homologs in the T. melanosporum genome (GSTUMT00005220001 and GSTUMT00001928001 gene models; http://mycor.nancy.inra.fr) suggests that such an approach may also be feasible in Tuber.
Enhanced green fluorescent protein:
Hygromycin B phosphotransferase:
Kanamycin resistance gene, aminoglycoside 3'-phosphotransferase:
Synthetic green fluorescent protein:
Thermal Asymmetric Interlaced PCR:
We are grateful to P. Romaine and to L. Ciuffetti for the kind gift of the BGgHg and CT74 plasmids, respectively. We thank R. Gerace for help in maintaining the Tuber strains, G. Buglia for skillful assistance with microscopy analysis and P. Costantino’s group for the GV3101 A. tumefaciens strain. P.B. thanks La Sapienza University, Rome (Fondi di Ateneo) for partial financial support. A.B. was supported by a Teresa Ariaudo fellowship.
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