Construction of Disarmed Ti Plasmids Transferable between Escherichia coli and Agrobacterium Species

Bacterial strains and culture conditions.Bacterial strains used in this study are listed in Table 1. E. coli strains were grown at 37°C in LB medium (1% Bacto tryptone, 0.5% NaCl, 0.5% yeast extract). A. tumefaciens strains were cultured at 28°C in LB medium or IFO medium (1% polypeptone, 0.2% yeast extract, 0.1% MgSO₄). A. rhizogenes strains were cultured at 28°C in IFO medium. Antibiotics were added at the following final concentrations: 50 μg/ml gentamicin, 50 μg/ml kanamycin, 30 μg/ml nalidixic acid, 50 μg/ml rifampin, 50 μg/ml ampicillin, and 50 μg/ml neomycin.

Plant materials used for transformation. Nicotiana tabacum SR-1 and Kalanchoe sp. were used as host plants for infection and DNA transfer experiments. N. tabacum SR-1 was cultured azenically on MS medium solidified with 0.8% agar at 28°C with continuous illumination. Kalanchoe sp. was cultured in a greenhouse. Leaves were surface sterilized with 1% sodium hypochlorite for 15 min and rinsed for 2 min with sterile distilled water four times before azenic experiments were performed.

Plasmid construction.Construction of tool plasmids pLRS-GmsacB and pLRS-Gms2 is described in the supplemental material. The 1.4-kbp left fragment (LL) just outside the left border of T-DNA and the 1.0-kbp right fragment (RR) just outside the right border of T-DNA were derived from pTi-SAKURA (24). The gentamicin resistance (Gm^r) gene was obtained from pUCGm2, the sacB gene and the Km^r gene were obtained from pK18mobsacB (21), IncP type (RK2) oriT was obtained from pJP5603 (18), and low-copy-number pSC101 oriV was obtained from pMW119 (Nippon Gene, Tokyo, Japan).

The binary plasmid pBIN-GI was prepared as follows. A 2.6-kbp HindIII-EcoRI fragment containing the β-glucuronidase (GUS) gene with an intron was obtained from pIG221 (15) and inserted into pBIN19 (2).

DNA preparation and analysis.Plasmid DNA was extracted from bacterial cells by the alkaline sodium dodecyl sulfate method (3). Manipulation of plasmid DNA was performed using standard methods.

Bacterial transformation.Modified shuttle Ti plasmids were extracted from A, tumefaciens strains by the modified alkaline sodium dodecyl sulfate method and purified by ethidium bromide-CsCl gradient ultracentrifugation. Purified shuttle Ti plasmids were introduced into E. coli strains by electroporation as described previously (20, 32).

Plasmids were transferred from E. coli to Agrobacterium strains by conjugal transfer as described elsewhere (28), with some modifications. The E. coli-Agrobacterium cell mixture was spotted onto LB agar for conjugation of A. tumefaciens and onto IFO agar for conjugation of A. rhizogenes. After overnight incubation at 28°C, cells were resuspended and spread onto appropriate selective agar media.

Plant transformation.Transformation of tobacco leaf disks was carried out as described by Clemente (6), with some modifications. Agrobacterium strains transformed with the binary vector pBIN-GI were grown overnight in liquid media supplemented with the appropriate antibiotics. Tobacco leaf disks (diameter, 1 cm) were immersed in the Agrobacterium suspension (optical density at 660 nm, 0.8) for 5 min and cocultivated for 2 days at 22°C with continuous fluorescent light illumination. After cocultivation, the leaf disks were cultivated on MS selective agar with 200 μg/ml claforan and 100 μg/ml kanamycin at 28°C with fluorescent light illumination. Kalanchoe leaf disks were subjected to the same transformation procedure but with different phytohormone and antibiotic concentrations (0.5 mg/liter benzyladenine, 2.0 mg/liter naphthylacetic acid, and 50 μg/ml kanamycin).

Quantitative and histochemical analyses of GUS activity were carried out as described by Jefferson et al. (13).

Construction of disarmed shuttle Ti plasmids.We designed a simple engineering scheme that can make pathogenic Ti plasmids disarmed, stably maintainable in E. coli, and mobilizable between E. coli and Agrobacterium species. As an example, we used the scheme with nopaline-type plasmids. We first constructed pLRS-GmsacB and pLRS-Gms2 (see Fig. S1 in the supplemental material) as tool plasmids to modify nopaline-type Ti plasmids. These tool plasmids are pK18mobsacB containing two fragments, LL and RR, which neighbor to the left of LB and to the right of RB of T-DNA in pTi-SAKURA, respectively, and a cassette containing a gentamicin resistance gene, the low-copy-number type replication origin (oriV) derived from pSC101, and the IncP-type transfer origin (oriT) sandwiched between LL and RR. The pSC101 replication ori should allow the chimeric plasmids to replicate at a very low copy number in E. coli.

Two nopaline-type Ti plasmids, pTiC58 and pTi-SAKURA, were modified using pLRS-GmsacB, as shown in Fig. 1. First, the pLRS-GmsacB plasmid in E. coli was introduced by conjugation into two pathogenic nopaline-type strains belonging to A. tumefaciens (biovar 1). C58rif is a pathogenic strain harboring pTiC58. C58C1 is a Ti-less strain. C58C1 harboring pTi-SAKURA is another pathogenic strain. Because pLRS-GmsacB cannot replicate in Agrobacterium cells, the tool plasmid should integrate into the Ti plasmids by homologous recombination at either LL or RR in the transformants (Fig. 1A). The Agrobacterium transconjugants were resistant to gentamicin and kanamycin and sensitive to sucrose due to the Gm^r, Km^r, and sacB genes on the fusion plasmids.

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FIG. 1.

Conversion of pathogenic Ti plasmids so that they are disarmed and transferable between E. coli and Agrobacterium. The modification of pTiC58 and pTi-SAKURA consists of two steps. (A) pLRS-GmsacB was inserted in vivo into pTiC58 and pTi-SAKURA by homologous recombination at either RR or LL. (B) Cells harboring the fused plasmid DNA were cultivated on LB agar containing sucrose and gentamicin in order to select for the subsequent crossover products. Only the recombinant that did not include the T-DNA portion was selected by cultivation on the medium.

Next, the transconjugants harboring the resulting fusion plasmid were cultured on LB agar supplemented with gentamicin and sucrose. Cultivation in a sucrose-containing medium selects for cells that do not have the sacB gene. Loss of the fusion plasmid can occur at a high frequency. Loss of this plasmid converts cells to Gm^s, Km^s, sucrose-resistant cells. Deletion of the sacB gene from the plasmid can take place at a high frequency through homologous recombination in two ways: recombination between two RR segments, resulting in removal of the pLRS-GmsacB portion, or, alternatively, recombination between two LL segments, resulting in loss of the T-DNA region (Fig. 1B). The former recombination converts cells to Gm^s, whereas the latter maintains Gm^r genes. Thus, colonies on the selective agar plate were expected to have a disarmed type of pTi. To confirm the lack of T-DNA in the derivatives of pTiC58 and pTi-SAKURA, for each Ti plasmid four colonies were randomly chosen from the selective agar culture and analyzed by PCR. T-DNA products were not detected in any of the colonies examined, whereas the virB gene was detected in every colony examined in another PCR experiment (data not shown). These results suggest that there was accurate and frequent removal of the long T-DNA region by replacement using pLRS-GmsacB and the simple selection media. The resultant Ti plasmids were designated pTiC58-S and pTi-SAKURA-S.

Introduction of modified Ti into Agrobacterium species via E. coli.Modified Ti plasmids pTiC58-S and pTi-SAKURA-S were extracted from the Agrobacterium strains. The plasmid DNAs were introduced into two E. coli strains, S17-1λpir and SURE. In order to check the structural integrity of the modified Ti plasmids during maintenance in E. coli, the plasmid DNAs were extracted from the E. coli transformants. The EcoRI fragment ladder profiles suggest that pTi-SAKURA-S was maintained stably in S17-1λpir (Fig. 2A) and that pTiC58-S was also maintained stably in the same E. coli strain (data not shown). Structural alteration was not detectable even after three serial repetitions of the E. coli culture (Fig. 2B). In contrast to the plasmids in S17-1λpir, pTi-SAKURA-S suffered from significant deletions in the other E. coli strain, strain SURE (Fig. 2A).

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FIG. 2.

Stability of the modified Ti plasmids. pTiC58-S and pTi-SAKURA-S were extracted from Agrobacterium cells and then introduced into two E. coli strains, S17-1λpir and SURE. Plasmid DNA was extracted from each E. coli transformant culture and then digested with EcoRI before electrophoretic separation in a 0.8% agarose gel. (A) pTi-SAKURA-S transformant colonies of S17-1λpir and of SURE were cultivated in liquid medium. (B) Cultivation of one S17-1λpir transformant was repeated serially three times. The presence (+Gm) or absence (−Gm) of gentamicin in the medium is indicated.

Because S17-1λpir possesses the IncP-type tra genes in its chromosome, it was expected that the S17-1λpir transformants could mobilize the modified Ti plasmids to various bacteria by conjugation. The Ti plasmid-less Agrobacterium strain C58C1 was cocultivated with the S17-1λpir transformants harboring the modified Ti plasmids. The resulting Rif^r Gm^r transconjugant frequencies were 5 × 10⁻⁵ for pTiC58-S and 4 × 10⁻⁵ for pTi-SAKURA-S. Similarly, the modified Ti plasmids were also introduced successfully by conjugation into another Ti plasmid-less A. tumefaciens strain, strain MNS-1, and into an Ri plasmid-less A. rhizogenes strain, strain A4RL.

Evaluation of reconstructed Agrobacterium strains.We performed plant transformation experiments to confirm the ability of the Agrobacterium transconjugants constructed as described above. For this experiment, the Agrobacterium transconjugants were transformed with an intron-containing GUS reporter plasmid pBIN-GI. The activity of the reconstructed Agrobacterium strains for transformation of tobacco leaf disks was as high as that of the original Agrobacterium strains in which the Ti plasmids were modified (see Fig. S2 in the supplemental material). This result indicates that the modified Ti plasmids have T-DNA transfer ability even after transmission from E. coli to Agrobacterium.

As shown above, pTiC58-S and pTi-SAKURA-S in S17-1λpir were mobilizable into Agrobacterium strains, and this enabled us to easily convert Agrobacterium strains to a disarmed type. We also tried to evaluate the disarmed Ti plasmids, as well as the Ti- and Ri-free strains. As mentioned above, we introduced each of the two disarmed Ti plasmids into two A. tumefaciens strains, C58C1 and MNS-1, and one A. rhizogenes strain, A4RL. The disarmed-plasmid-containing strains were transformed with the GUS reporter binary plasmid pBIN-GI. Then transformation of tobacco and Kalanchoe leaf disks was carried out with these reconstructed Agrobacterium strains. Two weeks after cocultivation with the donor Agrobacterium strains, kanamycin-resistant (Km^r) calluses were observed on the tobacco leaf disks. pTi-SAKURA-S was as effective as pTiC58-S in all strains tested (data not shown). Km^r calluses were induced in tobacco frequently by C58C1 strains containing this plasmid and less frequently by A4RL strains containing the same disarmed plasmid. However, Km^r calluses were rarely induced by MNS-1 strains containing this plasmid. The data for GUS activity in the tobacco leaf disks (Fig. 3A) was comparable to the data for formation of Km^r calluses. Regenerated recombinant tobacco plants were obtained from the Km^r calluses and showed GUS activity in their leaves and roots (see Fig. S3 in the supplemental material). When we treated Kalanchoe leaf disks, however, A4RL strains containing the disarmed plasmid induced higher GUS activity than C58C1 strains containing the same plasmid, as shown in Fig. 3B. The preference for A4RL of Kalanchoe sp. was in contrast to the preference for C58C1 rather than A4RL of tobacco.

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FIG. 3.

Evaluation of the plant transformation efficiencies of reconstructed Agrobacterium strains with different genome backbones. (A) Expression of GUS activity in tobacco leaf disks cocultivated with reconstructed Agrobacterium strains harboring pBIN-GI. (B) Expression of GUS activity in Kalanchoe leaf disks cocultivated with reconstructed Agrobacterium strains harboring pBIN-GI. Cell extracts of the leaf disks were prepared. The filled bars indicate the relative GUS activity of leaf disks transformed with C58C1 harboring pTiC58-S and pBIN-GI. The open bars indicate specific GUS activity. The data averages and with standard deviations of three independent experiments (five leaf disks each). 4MU, 4-methylumbelliferone.