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Applied and Environmental Microbiology, April 2009, p. 1845-1851, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01856-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Kazuya Kiyokawa, Shinji Yamamoto, Kei Sakuma, Katsuyuki Tanaka, Kazuki Moriguchi, and Katsunori Suzuki^*

Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan

Received 11 August 2008/ Accepted 22 January 2009

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Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1pir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

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Agrobacterium-mediated transformation has been considered the most efficient and reliable method for plant biology and biotechnology. This methodology has been established for many plants, but not for others. One of the major factors affecting its applicability is the limited number of donor Agrobacterium strains, because the method depends exclusively on the host ranges of the strains.

Wild-type Agrobacterium strains harboring a tumor-inducing (Ti) plasmid are the causative agent of crown gall tumor disease in dicotyledonous plants (35). The transfer DNA (T-DNA) and virulence gene (vir) regions in the Ti plasmid are essential for tumorigenesis. The vir gene products nick the T-DNA region at its left border (LB) and right border (RB) and then transfer T-DNA into plant cells. T-DNA contains phytohormone synthesis genes, whose expression causes infected plants to suffer from unregulated growth (5, 26). The hairy-root-inducing (Ri) plasmid has a similar T-DNA system.

The binary vector system (11) is widely used for Agrobacterium-mediated transformation. Binary vectors are small plasmids with a cloning site and a selectable marker gene between LB and RB (2). To ensure transformation without tumorigenicity, Agrobacterium strains used for transformation contain a modified Ti plasmid, which lacks T-DNA (disarmed) but retains the entire vir region. Unfortunately, only a small number of Ti plasmids have been disarmed.

Most pathogenic Agrobacterium strains are classified into three species: Agrobacterium tumefaciens (biovar 1, Rhizobium radiobacter), Agrobacterium rhizogenes (biovar 2, Rhizobium rhizogenes), and Agrobacterium vitis (biovar 3, Rhizobium vitis) (33). The genomic organizations of the Agrobacterium species are diverse (25, 27, 29). Pathogenic strains of each species are variable (1), and some of them might be potentially more effective for transformation than the strains used previously. For instance, Agrobacterium strain KAT23 causes tumors in legume plants, including common bean and soybean, very effectively (34). Disarmed Ti or Ri plasmids are either chosen from mutants or created by homologous recombination with a plasmid designed for this purpose (12, 16, 17). Both methods require either extensive screening or knowledge of structural and functional information for the plasmids. However, the large size of Ti and Ri plasmids, approximately 200 kbp, makes structural analysis and modification difficult. Complete nucleotide sequences of several Ti and Ri plasmids (for example, pTi-SAKURA, pTiC58, and pRi1724) have been reported (9, 14, 24, 26, 31). Accumulation of such nucleotide sequence information makes targeted replacement easier than it was previously. However, the large size of T-DNA obstructs the double crossover in the removal process during engineering. In addition to Ti plasmids, chromosomal virulence genes are necessary for plant transformation. It has been pointed out that combining a Ti plasmid with certain chromosomal backgrounds can markedly influence virulence (8). Thus, transfer of large plasmids to various Agrobacterium strains is another important engineering step, which is still not easy for researchers who are not familiar with Agrobacterium biology.

In this study, we describe a simple method and tool plasmids for constructing versatile disarmed nopaline-type Ti plasmids mobilizable from Escherichia coli to Agrobacterium strains, conversion of nopaline-type Agrobacterium strains to disarmed strains using the tool plasmids and simple selection media, and conversion of Ti-less Agrobacterium strains to disarmed strains using the modified Ti plasmids.

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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.

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TABLE 1. Bacterial strains and plasmids used in this study

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).

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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, S17-1pir 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 S17-1pir (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 S17-1pir, 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, S17-1pir 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 S17-1pir and of SURE were cultivated in liquid medium. (B) Cultivation of one S17-1pir transformant was repeated serially three times. The presence (+Gm) or absence (–Gm) of gentamicin in the medium is indicated.

Because S17-1pir possesses the IncP-type tra genes in its chromosome, it was expected that the S17-1pir transformants could mobilize the modified Ti plasmids to various bacteria by conjugation. The Ti plasmid-less Agrobacterium strain C58C1 was cocultivated with the S17-1pir transformants harboring the modified Ti plasmids. The resulting Rif^r Gm^r transconjugant frequencies were 5 x 10^–5 for pTiC58-S and 4 x 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 S17-1pir 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.

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In this study, we described a new disarming scheme and construction of versatile disarmed nopaline-type Ti plasmids mobilizable from E. coli to Agrobacterium strains and then conversion of Ti-less Agrobacterium strains to disarmed strains taking advantage of the modified Ti plasmids. Stable maintenance of Ti plasmids both in E. coli and during the transfer step is a prerequisite for delivering the disarmed plasmids to many strains of Agrobacterium and related genera and subsequent examination of their plant transformation abilities. Several research groups have tried to maintain Ti plasmids in E. coli. Native Ti plasmids cannot replicate in E. coli and therefore require additional replication genes that are functional in E. coli. Sprinzl and Geider (23) added the phage fd ori to a nopaline-type Ti plasmid. However, the modified Ti plasmid was inserted into chromosomal DNA of E. coli. Velikov and Buryanov (30) added ColE1 ori to a nopaline-type Ti plasmid, but the modified Ti plasmid was either inserted into chromosomal DNA or maintained as a much smaller plasmid resulting from large deletions.

In this study, we replaced T-DNA with a cassette containing oriT derived from RK2 and oriV derived from pSC101. This replacement was efficient using the tool plasmid constructed in this study. Two modified Ti plasmids were stably maintained in E. coli strain S17-1pir. Substitution of low-copy-number oriV for high copy-number-number oriV is likely to be effective for stable maintenance in E. coli. On the other hand, the modified Ti plasmids were damaged in another E. coli strain, strain SURE, due to large deletions, even though SURE was developed using a scheme to increase plasmid structural stability by mutating genes related to DNA recombination and repair pathways (10). In any case, it is clear that the E. coli strain used is very important for Ti plasmid maintenance.

It was easy to transfer the modified Ti plasmids from S17-1pir to Agrobacterium strains. Moreover, reconstructed A. tumefaciens and A. rhizogenes strains harboring the modified Ti plasmids successfully transformed plant cells. Therefore, using E. coli strain S17-1pir harboring the shuttle Ti plasmids, various Ti- and Ri-less Agrobacterium strains could be easily converted to disarmed strains useful for plant transformation tests. Plasmid delivery by IncP-type system conjugation does not require addition of any special inducer molecules and enables transfer to wide range of bacteria, while conjugation with the tra regulon on Ti plasmids requires a special inducer, such as agrocinopine (7, 19), which is not available commercially.

Broothaerts et al. (4) mobilized pTiEHA101 derivatives that contain IncP-type oriT using transferable helper plasmid RP4-4 into Sinorhizobium meliloti, Mesorhizobium loti, and a Rhizobium species. They detected T-DNA transfer ability in the transconjugant bacteria. It was necessary to remove the helper plasmid from the transconjugants, because the transconjugants received not only Ti but also the helper plasmid and the latter suppressed the T-DNA transfer ability. E. coli donor strain S17-1pir employed in this study was easy to select against and moreover is convenient in that it does not deliver the helper IncP plasmid to recipient cells.

The C58C1 strains having modified Ti transformed tobacco leaf disks more efficiently than the A4RL strains harboring the same modified Ti did. On the other hand, the latter strains were more effective at transforming Kalanchoe leaf disks. These results suggest that the various genomic backgrounds of the Agrobacterium strains differentially influence the fitness for each plant. There might be strains among pathogenic Agrobacterium strains that are more efficacious than the commonly used Agrobacterium strains. The disarmed Ti plasmids constructed in this study would help screening for such strains.

Complete nucleotide sequences are available for several different types of Ti and Ri plasmids (26). The difference in the auxiliary vir region affects the host range in part. It is worth replacing the LL and RR segments in the tool plasmids with the corresponding segments of various types of plasmids in order to develop disarmed strains of a type other than the nopaline type.

In addition to pLRS-GmsacB, we constructed pLRS-Gms2 (see Fig. S2 in the supplemental material). The latter tool plasmid can also be used to disarm nopaline-type plasmids and is superior to pLRS-GmsacB since it lacks the Ap^r gene in the cassette and therefore does not increase the resistance to β-lactam antibiotics in the disarmed strains. Using a simple and efficient Ti-curing method which we reported previously (32) and the shuttle Ti plasmids constructed in this study, it would be easy to convert many pathogenic Agrobacterium strains to disarmed strains, even for researchers who are not familiar with Agrobacterium biology.

 
This research was supported in part by the Ministry of Education, Science, Sports and Culture (grant-in-aid for scientific research 20570221) and by the Japan Science and Technology Agency.

 
* Corresponding author. Mailing address: Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi Hiroshima 739-8526, Japan. Phone: 81-82-424-7455. Fax: 81-82-424-0734. E-mail: ksuzuki{at}hiroshima-u.ac.jp

Published ahead of print on 30 January 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, April 2009, p. 1845-1851, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01856-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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