Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation

Abstract

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.

Keywords: Agrobacterium tumefaciens-mediated transformation; Green fluorescent protein; Hygromycin phosphotransferase; T-DNA; TAIL-PCR; Truffles; Tuber spp; binary plasmid.

Figure 1

ATM-transformation with the pABr1 vector. a) pABr1 (11.4 kb) vector map: Hygromycin (R), hygromycin phosphotransferase gene (hph); Kanamicin resistance gene (kanR); ToxA promoter, promoter sequence from P. tritici-repentis; SGFP, the S65T variant of the green fluorescent protein (GFP); CAMV35S cauliflower mosaic virus promoter; mGFP5, folding-enhanced green fluorescent protein variant; NOS polyA, Nopaline synthase terminator sequence; T-border (R) and T-border (L), right (RB) and left (LB) borders of A. tumefaciens T-DNA. The blue highlighted box is the plasmid region derived from pCT74. b) Images (100x magnification) of T. borchii hyphae transformed with either the pABr1 or the pBGgHg vector, plus an untransformed control (mock), obtained by phase-contrast (Nomarski) and GFP fluorescence microscopy (GFP). The type of treatment, vector and co-cultivation times are indicated on the left; a merge of the Nomarski and GFP images is shown in the rightmost panels. c) Quantification of transformed hyphae obtained with pBGgHg or pABr-1, expressed as percentage with respect to the total number of hyphae (~4500) present in each analyzed section. Data are the mean ± s.e.m. of at least five independent experiments. d) PCR amplification with ToxA-specific primers of total DNA extracted from mock-infected mycelia (lane 1), pABr1-transformed mycelia (lane 2), and pABr1/AGL-1 bacterial cells (lane 3) is shown in the upper panel. The results of PCR amplification of the same samples with kanR-specific primers are shown in the lower panel.

Figure 2

Following the fate of the T-DNA in pABr1-transformed hyphae. a) Sections (3 x 3 mm) of transformed T. borchii mycelia analyzed by confocal microscopy (GFP wavelength) at the indicated times after Agrobacterium infection. b) Histogram representation of cumulative data produced by the experiments in panel a) expressed as percentage of fluorescent hyphae with respect to the total number of hyphae present in each section (~4500). Data are the mean ± s.e.m. of at least five independent experiments. c) PCR amplification with sgfp specific-primers of genomic DNA obtained from mock-transformed mycelia (WT) using 0.1 μg (lane 3) and 1 μg (lane 4) of template DNA, and from AGL-1/pABr1-transformed mycelia (0.1 μg of template DNA; lane5). A PCR negative control (no DNA) and size markers (λ/HIII) are shown in the two leftmost lanes. d) Schematic representation of the probes (P1 and P2) utilized for DNA blot analysis. e) DNA blot analysis of DNA extracted from AGL-1/pABr1-transformed mycelia (“transformant”, lane 1), mock-transformed mycelia (WT, lane 2), and AGL-1/ABr-1 bacterial cells (pABr1, lane 3), hybridized with the P1 probe, which detects T-DNA integration regions. After stripping, the same membrane was hybridized with the P2 control probe.

Figure 3

pABr3 transformation, comparison of transformation efficiency with pABr1, and hygromycin transformant amplification. a) Images (100x magnification) of pABr3-transformed hyphae, at 3 days and 1 year after transformation as indicated, obtained by phase-contrast (Nomarski) and SGFP fluorescence microscopy. A negative, untransformed control (mock) is shown at the top; a merge of the two types of images is shown in the rightmost panels. b) Comparison of the transformation efficiencies obtained with pABr1 and pABr3. Data are the mean ± s.e.m. of at least five independent experiments. c) Transformed mycelia were grown on hygromycin (15 μg/ml)-supplemented medium for 60 days and then transferred to solid medium containing the indicated concentrations of hygromycin B. After 7 days, mycelia were visualized under a stereo-microscope at the indicated magnifications to detect the presence of tufts grown beyond the dotted line.

Figure 4

TAIL-PCR and integration site identification. a) TAIL-PCR amplification of DNA fragments covering pABr3 integration sites. Representative results obtained from three amplification runs (PCR1, PCR2, PCR3) carried out with three nested primers annealing with the left border (LB) of the T-DNA (LB1, LB2 and LB3) and a degenerate primer annealing with the neighboring genome sequence (AD1) (see explanatory scheme on the right and “Materials and Methods” for details). Template genomic DNA was extracted from a pABr3-transformed mycelium (T) and from a mock-transformed mycelium (WT); size markers (M) are in the leftmost lane. b) Amplification of the right border (RB). c) Integration site sequences retrieved from TAIL-PCR analysis of transformants #2, #8, #10, #11 and #12; The underlined and highlighted in bold sequences represent the portion of the T-DNA adjacent to the unknown sequence obtained by PCR.