Update on Binary Vectors

Current Status of Binary Vectors and Superbinary Vectors

Toshiyuki Komori*, Teruyuki Imayama, Norio Kato, Yuji Ishida, Jun Ueki, and Toshihiko Komari Plant Innovation Center, Japan Tobacco Incorporated, Iwata, Shizuoka 4380802, Japan A binary vector was invented soon after it had been elucidated that crown gall tumorigenesis was caused by genetic transformation of plant cells with a piece of DNA, T-DNA for transferred DNA, from a Ti plasmid (tumor-inducing plasmid) harbored by the soil bacterium Agrobacterium tumefaciens (Fraley et al., 1986). A key nding was that the virulence genes, which are involved in the transfer of T-DNA, could be placed on a replicon separate from the one with T-DNA (Hoekema et al., 1983). Thus, combination of a disarmed strain, which carries a Ti plasmid without the wild-type T-DNA, and an articial T-DNA within a plasmid that can be replicated both in Escherichia coli and A. tumefaciens turned out to be fully functional in plant transformation. The term binary vector literally refers to the entire combination, but the plasmid that carries the articial T-DNA is usually called a binary vector. Now, binary vectors are the most popular tools in the plant science community. One of the binary vectors constructed in the early days was pBin19 (Bevan, 1984), and pBI121 was created shortly after by adding a marker gene to pBin19 (Jefferson, 1987). Soon these vectors and their derivatives were widely distributed among plant scientists. Another popular series of vectors are pPZP vectors (Hajdukiewicz et al., 1994) and pCAMBIA vectors (www.cambia.org), which are modied from pPZP vectors. Recent modications of binary vectors provide a number of user-friendly features, such as wide selection of cloning sites, high copy numbers in E. coli, high cloning capacity, improved compatibility with strains of choice, a wide pool of selectable markers for plants, and a high frequency of plant transformation. Our quick survey of 180 recently published papers, in which transformation of higher plants mediated by A. tumefaciens was described, revealed that derivatives of pBin19 were employed in 40% of the studies and derivatives of pPZP vectors in 30% of them (Table I). Therefore, while recent improvements are very useful, the classic vector conguration appears still to be good enough on many occasions. The nding that some of the virulence genes exhibited gene dosage effects (Jin et al., 1987) led to the development of a superbinary vector, which carried
* Corresponding author; e-mail toshiyuki.komori@ims.jti.co.jp. The author responsible for distribution of materials integral to the ndings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Toshiyuki Komori (toshiyuki.komori@ims.jti.co.jp). www.plantphysiol.org/cgi/doi/10.1104/pp.107.105734

additional virulence genes (Komari, 1990). The superbinary vector has been highly efcient in the transformation of various plants and especially useful in the transformation of recalcitrant plants, such as important cereals (Hiei et al., 1994; Ishida et al., 1996).
BASIC STRUCTURE OF BINARY VECTORS

A binary vector consists of T-DNA and the vector backbone (Fig. 1). T-DNA is the segment delimited by the border sequences, the right border (RB) and the left border (LB), and may contain multiple cloning sites, a selectable marker gene for plants, a reporter gene, and other genes of interest. The vector backbone carries plasmid replication functions for E. coli and A. tumefaciens, selectable marker genes for the bacteria, optionally a function for plasmid mobilization between the bacteria and other accessory components.
T-DNA BORDERS

The RB and the LB are imperfect, direct repeats of 25 bases and said to be the only essential cis-elements for T-DNA transfer (Yadav et al., 1982). The RB and the LB are integrated in binary vectors as DNA fragments cloned from well-known Ti plasmids, either octopine or nopaline type. Because factors that enhance T-DNA transfer have been identied near the borders (Peralta et al., 1986; Wang et al., 1987), a few hundred bases of natural sequences adjacent to the T-DNA are retained by popular vectors, such as pBin19 (Bevan, 1984), pPZP series (Hajdukiewicz et al., 1994), and pSB11 (Komari et al., 1996).
SELECTABLE MARKER GENES FOR PLANTS

Choice of selectable marker genes is a key factor in plant transformation. Genes to give resistance to antibiotics or herbicides, such as kanamycin, hygromycin, phosphinothricin, and glyphosate, are very popular. Kanamycin resistance has been most frequently employed in the transformation of many dicotyledonous plants. Hygromycin resistance is the most effective in rice (Oryza sativa) transformation (Hiei et al., 1994), whereas phosphinothricin resistance is the most effective in maize (Zea mays; Ishida et al., 1996). If the development of herbicide-resistant plants is aimed at, a trait gene could also be a selectable marker gene. Because of concerns over antibiotic resistance genes in commercial transformants, genes to add metabolic
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Table I. Well-known binary and superbinary vectors

Vector Plant Selection Markera Bacterial Selection Markerb Replication Replication Origin for Origin for Mobilization A. tumefaciens E. coli Reference Frequency of Use in Recent Literaturec

pBin19 pBI121 pCAMBIA series pPZP series pGreen series pGA482 pSB11e pSB1e pPCV001 pCLD04541 pBIBAC series pYLTAC series
a

Kan Kan Kan Kan Kan or Hyg Cm or Kan Kan or Gen Cm or Sp Kan, Hyg, Sul, or Bar Kan Kan Tc, Kan None Sp None Tc Kan Ap Kan Tc, Kan Kan or Hyg Kan Hyg or Bar Kan

IncP IncP pVS1 pVS1 IncW IncP None IncP IncP IncP pRi pRi

IncP IncP ColE1 ColE1 pUC ColE1d ColE1 ColE1d ColE1d IncP F factor Phage P1

Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes No

Bevan (1984) Jefferson (1987) www.cambia.org Hajdukiewicz et al. (1994) Hellens et al. (2000) An et al. (1985) Komari et al. (1996) Komari et al. (1996) Koncz and Schell (1986) Tao and Zhang (1998) Hamilton (1997) Liu et al. (1999)

40% 40% 30% 30% 3% 3% 3% 3% 1% 1% 0% 0%

b Kan, Kanamycin; Hyg, hygromycin; Gen, gentamicin; Sul, sulfonylurea; Bar, phosphinothricin. Kan, Kanamycin; Cm, chloramphenicol; Sp, c spectinomycin; Tc, tetracycline; Ap, ampicillin. From issues between 2005 and 2007 of 12 leading plant journals, 180 papers, in which plant d transformation mediated by A. tumefaciens is described, were randomly chosen and surveyed. Although IncP is also active in E. coli, it is likely e that the plasmid is replicated mainly by the ColE1 system. pSB11 and pSB1 are an intermediate vector and an acceptor vector of the superbinary vector system, respectively.

capabilities have been drawing considerable attention. For example, plant cells expressing a phospho-Man isomerase can grow on media with Man as the sole carbon source. Such markers are referred to as positive selection markers (Joersbo et al., 1998). Although more than 20 selectable marker genes have been reported in the transformation of higher plants (Komari et al., 2006) to date, many of them were tested only in a limited number of plant species on a limited scale. Therefore, further studies of marker genes may contribute to improvement of the transformation of certain plant species. Selectable marker genes are usually driven by constitutive promoters. The promoters of the 35S transcript of Cauliower mosaic virus (Odell et al., 1985) and the nopaline synthase of A. tumefaciens (Depicker et al., 1982) are very popular in dicotyledons, and the promoters of the ubiquitin gene of maize (Christensen et al., 1992) and the actin gene of rice are popular in monocotyledons (Zhang et al., 1991). Selectable marker genes are followed by a DNA fragment, the so-called 3# signal. The 3# regions of the cauliower mosaic virus 35S transcript and the nopaline synthase gene in the wild-type T-DNA of A. tumefaciens are frequently used as a 3# signal.

proteins of interest may be employed to examine the subcellular localization of the proteins. Reporter genes that are connected to constitutive promoters may be used to monitor the process of transformation. Expression of the reporter genes soon after the inoculation of plant cells with A. tumefaciens, which is referred to as transient expression, is a good indication of transfer of the T-DNA from the bacteria to the nuclei of plant cells. Expression of the reporter genes later in a cluster of cells growing on selection media is a piece of evidence for integration of the T-DNA in

REPORTER GENES

Reporter genes, whose expression can be easily monitored, are useful in many ways in plant transformation. Strength and temporal, spatial, and other types of regulation of promoters and other elements may be conveniently assayed by connecting these elements to the reporter genes. Genes for GUS ( Jefferson, 1987), luciferase (Ow et al., 1986), and GFP (Pang et al., 1996) are popular examples. Gene fusions of the reporters and
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Figure 1. Typical structure of a binary vector. Key components and their major options are displayed. Plant Physiol. Vol. 145, 2007

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plant chromosomes. A binary vector that carries a constitutive selectable marker and a constitutive reporter is very useful as a control vector both in transformation experiments and in assays of gene expression.

INTRODUCTION OF DNA FRAGMENTS TO T-DNA

Insertion of genes of interest into appropriate locations of a binary vector is traditionally carried out by standard subcloning techniques. Multiple cloning sites, which are similar or identical to those in pUC, pBluescript, and other standard vectors, are still very useful in this regard, but recently constructed vectors are more user friendly. Recognition sites for rare cutters, which are restriction enzymes with long recognition sequences, are very convenient in this respect because the DNA fragments that are to be inserted scarcely have such sites. In some of the recently created vectors termed modular vectors, a series of these rare sites are placed in the T-DNA (Chung et al., 2005). An extensive set of auxiliary plasmids, which have full sets or subsets of these rare sites and other restriction sites, are provided, and some of the plasmids also carry frequently used promoters, marker genes, and/or 3# signals. Various types of expression units may be constructed in auxiliary plasmids, and then the units may be inserted into the modular binary vectors. Thus, several expression cassettes could easily be assembled in a binary vector. The GATEWAY system (Invitrogen), which is a cloning technology based on the site-specic recombination mechanism of phage lambda, provides another user-friendly feature. A DNA fragment anked by a pair of short, specic sequences may easily be replaced with another DNA fragment by the GATEWAY system. Thus, introduction of DNA fragments into a binary vector with the sites for the GATEWAY system is a straightforward step and is useful in many applications. Combination of the modularity based on rare-cutting restriction enzymes and the GATEWAY recombination sites provides an extensively versatile cloning system, which is especially useful in the production of T-DNA with multiple genes (Chen et al., 2006).

mids in bacterial cells. High-copy-number vectors, such as those that carry the origin of replication of ColE1, are very convenient in molecular construction processes. The origin of replication of pUC vectors is a derivative of the origin of ColE1 and gives a much higher copy number due to a mutation (Sambrook and Russell, 2001). On the other hand, low-copy-number vectors, such as ones with the origin of IncP, IncW, or the F factor, are preferable for the transfer of large DNA fragments, e.g. fragments larger than 15 kb. Vectors designated as BIBAC (Hamilton, 1997) and TAC (Liu et al., 1999) were specically designed to clone and transfer extremely large genomic fragments.
BACTERIAL SELECTION MARKER

Antibiotic resistance genes in common cloning vectors, such as genes that can confer resistance to kanamycin, carbenicillin, gentamicin, spectinomycin, chloramphenicol, and tetracycline (Sambrook and Russell, 2001), are also employed in plant transformation vectors. Care must be exercised as some bacterial strains without vector plasmids have certain intrinsic antibiotic resistance, e.g. the kanamycin resistance of A. tumefaciens EHA101 (Hood et al., 1986). In the process of plant transformation, A. tumefaciens should be removed from plant cells by antibiotics after infection (Cheng et al., 2004). When carbenicillin is used for the purpose after infection as previously described (Zhao et al., 2001), it should be noted that the A. tumefaciens strain that carries a vector with an ampicillin resistance gene, which also confers resistance to carbenicillin, cannot be removed by the treatment.
PLASMID MOBILIZATION FUNCTIONS

PLASMID REPLICATION FUNCTIONS

Binary vectors need replication functions active in E. coli and A. tumefaciens. Replication functions active in a wide range of bacteria, such as ones of plasmid incompatibility group P (IncP; Pansegrau et al., 1994) or W (IncW; Okumura and Kado, 1992), may be conveniently employed. Alternatively, replication functions for A. tumefaciens, such as ones for the Ri plasmid ( Jouanin et al., 1985) or pVS1 (Deblaere et al., 1987), and for E. coli, such as ones for the F factor, phage P1, ColE1, or P15A (Sambrook and Russell, 2001), may be combined. The types of replication functions determine the copy number and the stability of the plasPlant Physiol. Vol. 145, 2007

The origin of transfer (OriT) of IncP plasmids (Pansegrau et al., 1994) or the bom function of the ColE1 plasmid (Sambrook and Russell, 2001) is carried by most of the binary vectors. The plasmids with OriT or the bom may be mobilized from E. coli to A. tumefaciens aided by a conjugal helper plasmid, such as pRK2013. This function is not necessary when vectors are introduced into A. tumefaciens by electroporation or freeze-thaw methods, but it is a good idea to have a wider option because the conjugal transfer is a very efcient process.
SUPERBINARY VECTOR

Until the early 1990s, Agrobacterium-mediated transformation had been used mainly in dicotyledons, and it had been difcult to apply the method to cereals. Later, a superbinary vector was developed and successfully used for the transformation of monocotyledons, such as rice and maize (Hiei et al., 1994; Ishida et al., 1996). A superbinary vector is an improved version of a binary vector and carries the 14.8-kb KpnI
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fragment that contains the virB, virG, and virC genes derived from pTiBo542, which is responsible for the supervirulence phenotype of an A. tumefaciens strain, A281 ( Jin et al., 1987; Komari, 1990). Since the total size of vector components is relatively large in the superbinary system, it is not a realistic choice to introduce additional genes of interest into a superbinary vector by ordinary subcloning methods. Therefore, cointegration of an intermediate vector such as pSB11 and an acceptor vector such as pSB1 via homologous recombination between the shared DNA segments in A. tumefaciens is employed in the nal construction step of a superbinary vector (Fig. 2; Komari et al., 1996). The intermediate vector is a small plasmid with a T-DNA and the origin of replication of ColE1 and is replicated only in E. coli. The acceptor vector is an IncP plasmid, which can be replicated in E. coli and A. tumefaciens, and carries the 14.8-kb KpnI fragment. If a gene of interest is to be introduced into plants in tandem with a marker gene, the two genes are rst inserted into an intermediate vector, and then the vector is introduced into a strain of A. tumefaciens that carries an acceptor vector. Note that the resultant A. tumefaciens carries the cointegrated superbinary vector in addition to an intrinsic disarmed plasmid, such as pAL4404, which contains the full set of virulence genes derived from a wild-type A. tumefaciens strain, Ach5 (Hoekema et al., 1983).
VECTOR BACKBONE ISSUE

proposed for the transfer of backbone sequences. In one model, formation of the transfer intermediates of T-DNA, which is initiated at the RB, is not terminated at the LB. In the other model, formation of the intermediates is started at the LB. Sequence contexts around the borders greatly affect the frequency of the backbone transfer in a complex way (Podevin et al., 2006). A method of placing more than one LB repeat was reported, and this simple modication suppressed the transfer of the vector backbone in a nearly perfect fashion (Kuraya et al., 2004). This is a useful indication for the reduction of the transfer of vector backbone, although the effect of such a modication may be different depending on the types of vectors, strains, plant species, and other factors. Another method was to place a killer gene, whose product is harmful to plant cells, in the vector backbone so that the cells that acquired the backbone are eliminated (Hanson et al., 1999).
REMOVAL OF SELECTABLE MARKERS FOLLOWING COTRANSFORMATION

It had been widely believed for a long time that only T-DNA delimited by the border repeats had been transferred to plants. However, many reports have shown for the last 10 years that the transfer of vector backbone sequences is quite common. The ratio of the plants that acquired the backbone sequences in transformants ranged typically between 20% and 50% and was sometimes as high as 75% or more. Two mechanisms were

Figure 2. Final step in the construction of a superbinary vector. An intermediate vector with T-DNA constructed in E. coli is transferred to an A. tumefaciens strain that carries an acceptor vector, and the cointegrate is created via homologous recombination between the shared sequences. Abbreviations: OriV, origin of vegetative replication of the IncP plasmid; O, origin of replication of ColE1; Trf, transacting replication function of the IncP plasmid: OriT, origin of transfer of the IncP plasmid; B, bom site for plasmid transfer of ColE1; C, Cos site of phage lambda. 1158

Although a selectable marker gene is an essential component in plant transformation technology, it is not only an unnecessary DNA piece in established transformants but also a source of public concerns over genetically modied crops. Thus, removal of selective marker genes from commercial transgenic plants is highly desired. A simple approach is to cotransform plant cells with two separate pieces of T-DNA, one with a selective marker gene and the other with genes of interest, and to select marker-free progeny segregated from the cotransformants. To carry out cotransformation in the superbinary vector system, a T-DNA with a selectable marker was located in an acceptor vector. For example, pSB4 and pSB6 were constructed by locating a T-DNA carrying the hygromycin resistance gene and the phosphinothricin resistance gene, respectively, and have been tested in a number of plant species (Komari et al., 1996; Ishida et al., 2004). The frequency of cotransformation, which is the ratio of transformants with the genes of interests among the number of plants with the selective marker gene, has been quite high, ranging typically between 50% and 80%. Marker-free transformants have then been obtained from more than 50% of the cotransformants. Cotransformation may be carried out using other types of vectors. For example, Huang et al. (2004) placed a marker gene in the vector backbone in a regular binary vector and observed that plants were cotransformed with one T-DNA processed from the RB and another T-DNA processed from the LB.

REMOVAL OF MARKER GENES AND OTHER UNNECESSARY SEGMENTS BY RECOMBINATION

Recombinases from phages and yeasts, such as Cre, FLP, and R, which recombine specic sites loxP, FRT,
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and RS, respectively, are powerful tools to remove selectable marker genes (Ow, 2001). A DNA segment placed between two of the specic recombination sites may be excised from the plant chromosome if the corresponding recombinase is somehow expressed in the plant cell. For example, transgenic lines that contained the loxP sites were crossed with lines that expressed the Cre gene (Moore and Srivastava, 2006). Various sophisticated vector congurations and means to express the recombinases were reported to exploit this system (Wang et al., 2005; Jia et al., 2006). The recombinases may be able to cut out not only marker genes but also other unnecessary DNA segments. For example, tandem integration of two or more copies of T-DNA in a single locus has been observed quite frequently (Krizkova and Hrouda, 1998); it is a cumbersome phenomenon because clean, single-copy transformants are generally preferred. If a recombination site is possessed by the T-DNA, a segment between two of the sites in the tandem T-DNA could be deleted so that a clean, single T-DNA integration pattern could be generated.
CHOICE OF TRANSFORMATION VECTORS

good enough in many applications, improved vectors exhibit various user-friendly features. Vectors that are specically designed to resolve certain regulatory issues, such as removal of marker genes and reduction of transfer of the vector backbone, are also available. It is likely that the improvement of vectors will be continued as new technical demands arise in the plant science community. Now, although it is not difcult to nd a vector that can somehow be used in a particular experiment, a further search for vectors better suited to the experimental purpose is often very useful.
ACKNOWLEDGMENT
Assistance by Ms. Kumiko Donovan is highly appreciated. Received July 30, 2007; accepted August 24, 2007; published December 6, 2007.

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A wide range of binary vectors and superbinary vectors is available now. Helpful guidance for selection of the vectors has already been provided in the literature (Hellens et al., 2000; Komari et al., 2006). Unfortunately, there is no vector that is good for all purposes, but, fortunately, many of the vectors currently available are quite versatile. They may be used in various types of experiments, and there is a good chance that vectors you and/or your colleagues are routinely using can be employed in the experiment of interest. If this is not the case or better options are worthwhile searching for, a series of questions need to be asked about the size and nature of the DNA fragments, the strains of A. tumefaciens to be employed, the species of plants to be transformed, and the purposes of the experiments. If the DNA fragments are larger than 15 kb, IncP, BIBAC, and TAC vectors are recommended. Otherwise, high-copy-number plasmids are very convenient, and a wide range of vectors varying in restriction sites, selectable markers, and GATEWAY sites is available. A series of vectors designed for specic purposes, e.g. vectors for suppression of plant genes by RNA interference technology (Miki and Shimamoto, 2004), may also be chosen.
CONCLUSION

The basic framework of binary vectors was developed in the 1980s, and extensive improvements have continuously been conducted to provide a wide selection of cloning sites, high copy numbers in E. coli, high cloning capacity, GATEWAY recombination sites, improved compatibility with the strains of choice, a wide pool of selectable markers, a high frequency of transformation, and so on. While classic vectors are still
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