Mol Gen Genet (1992) 236:1 7 © Springer-Verlag 1992

Design of a novel system for the construction of vectors for Agrobacterium-mediated plant transformation Teresa Mozo and Paul J. J. Hooykaas Institute of Molecular Plant Sciences, Clusius Laboratory, LeidenUniversity,Wassenaarseweg64, NL-2333 AL Leiden, The Netherlands Received March 30, 1992 / Accepted July 10, 1992

Summary. The loxP-Cre site-specific recombination system of phage P1 was used to develop a novel strategy to construct cointegrate vectors for Agrobacteriummediated plant transformation. A pTi disarmed helper plasmid (pALl166) was constructed by replacing the oncogenic T-DNA by a loxP sequence and a spectinomycin resistance marker in the octopine-type pTiB6 plasmid. The cre gene was cloned into an unstable incP plasmid. A third plasmid, which did not replicate in Agrobacterium and contained another loxP sequence together with a kanamycin resistance marker, was used to test the system. Electroporation of this third plasmid into an Agrobacterium strain harbouring both pAL1166 and the Cre-encoding plasmid resulted in kanamycinresistant cells containing a cointegrate between pAL 1166 and the incoming plasmid. Cointegration occurred by Cre-mediated recombination at the loxP sites, and the cointegrate was stabilized in the Agrobacterium cells by the loss of the Cre-encoding plasmid shortly after the recombination event had taken place. Key words: Cointegrates - loxP-Cre - Plant Site-specific recombination

Vectors -

Introduction The gram-negative soil bacterium Agrobacterium tumefaciens causes the neoplastic disease crown gall in dicotyledonous plants by means of the natural system it possesses for transferring oncogenic DNA to plant cells. The transferred DNA (T-DNA) is a segment of the large Ti plasmid (200 kb) harboured by all virulent Agrobacterium strains. Mobilization of the T-DNA to plant cells is mediated by products of genes located in another region of the Ti plasmid, the virulence (vir) region, in a process that resembles bacterial conjugation (for reviews see Melchers and Hooykaas 1987; Zambryski 1988; Correspondence to: P.J.J. Hooykaas

Zambryski et al. 1989). This natural gene transfer system can be modified in order to deliver desired genes into plant cells. The T-DNA is flanked by 24 bp imperfect direct repeats (T-DNA border repeats), which are the only elements essential in cis for the transfer process. In plant vectors, therefore, the genes that are to be transferred to plant cells are cloned between the border repeats along with a plant selectable marker. The second essential element, the vir region, can be provided in trans (Hoekema et al. 1983). This fact has allowed the development of the so-called binary vector system, in which the T-DNA is cloned into a broad-hostrange plasmid while the vir functions are supplied by a Ti plasmid from which the T-DNA has been deleted (disarmed helper plasmid; De Framond et al. 1983; Hoekema et al. 1983; Bevan 1984). The broad-hostrange plasmids used in the binary vector system have disadvantages compared to routinely used Escherichia coli cloning vectors, such as pBR322 and pUC19, due to their large size and low copy number. In addition, these plasmids are usually unstable in Agrobacterium in the absence of selective pressure. As an alternative to using binary vectors, the process of homologous recombination can be used to insert the desired genes into the (disarmed) T-DNA of a Ti plasmid via a single cross-over (Hille et al. 1983 ; Zambryski et al. 1983; Deblaere et al. 1985). A major disadvantage of this procedure is that the resulting cointegrates contain large repeats either within or flanking the T-DNA. This may limit the stability of the cointegrates in Agrobacterium; additionally, the presence of large direct repeats within the T-DNA may affect the expression of the T-DNA genes in plants and cause instability of the T-DNA within the plant cell. Here, we present the design of a novel type of plant vector, which can be maintained in Agrobacterium after site-specific recombination (cointegration) with a resident replicon via the loxP-Cre site-specific recombination system of phage P1 (Sternberg and Hamilton 1981). The Cre recombinase protein recognizes the 34 bp loxP DNA sequence consisting of two 13 bp inverted repeats

separated by an asymmetric region of 8 bp, and promotes its recombination with another loxP sequence. When the two loxP sites are present in different circular DNA molecules, the recombination event causes their cointegration; however, when the two loxP sites are in the same replicon, the DNA lying in between is excised or inverted depending on whether they occur in a direct or inverted repeat (Abremski et al. 1983; Hoess et al. 1986). The system has been used in vitro for the insertion of recombinant DNA into a pseudorabies virus vector (Sauer et al. 1987) and has been shown to function in vivo not only in E. coli but also (after modification) in yeasts (Sauer 1987), mouse cells (Sauer and Henderson 1989), and plant protoplasts (Dale and Ow 1990). Recently, the loxP-Cre system was used for site-directed recombination in the genome of transgenic tobacco plants also (Odell et al. 1990). In this paper we describe the construction of a loxPcontaining disarmed Ti helper plasmid and demonstrate its Cre-mediated cointegration with a loxP-containing E. coli vector in Agrobacterium cells. The stability of the cointegrate is ensured by the loss of the cre gene from Agrobacterium, shortly after the cointegration has taken place. The opportunities offered by the system for the development of new types of plant vectors are discussed.

LC medium (Hooykaas et al. 1977) at 29 ° C. Antibiotic concentrations used were: carbenicillin, 75 gg/ml; kanamycin, 100 gg/ml; spectinomycin, 250 gg/ml; tetracycline, 2 gg/ml; rifampicin, 20 gg/ml; erythromycin, 100 gg/ml.

DNA procedures. DNA isolation from E. coli, restriction, ligation, transformation, nick translation of probes and Southern blotting to nitrocellulose filters were performed according to standard techniques (Sambrook et al. 1989). Plasmid DNA was isolated from Agrobacterium by a modification of the alkaline lysis procedure of Birnboim and Doly (1979), in which an extra denaturing step was included by the addition of a NaOH-phenol solution (2 volumes 0.2 N NaOH: 1 volume TRIS-saturated phenol) to the alkaline lysate (1/10 volume). Hybridization was carried out at 42 ° C in 50% formamide, 5 x SSC (1 x SSC is 0.15 M NaC1, 15 mM sodium citrate) and the hybridized filters were washed at 42 ° C in 2 x SSC, 0.1% SDS. Plasmid transfer to Agrobacterium strains. Plasmids were routinely introduced into the Agrobacterium strains by electroporation, as described previously (Mozo and Hooykaas 1991). Triparental matings according to Ditta et al. (1980) were done when genes had to be inserted into the Agrobacterium genome via double cross-over.

Materials and methods Bacterial strains, plasmids and bacteriophage. E. coli MH1 (Goddard et al. 1983) and JM101 (Yanisch-Perron et al. 1985) strains were used for cloning. A. tumefaciens strains were: LBA1010 (C58 rifampicin resistant, Rift; pTiB6) (Koekman et al. 1982), MOG1010 (containing pMOG1010, which is derived from LBA1010 by substitution of the T-DNA with a spectinomycin resistance (Sp9 marker; kindly provided by A. Hoekema, MOGEN Int. N.V., Leiden, The Netherlands), and UIA143 (C58 erythromycin resistant, Eryr; recA mutant, pTi-cured) (Farrand et al. 1989). Plasmids used in this work were: pUC19 (Yanisch-Perron et al. 1985), pIC20H (Marsh et al. 1984), pHP45 ~ K m (pBR322 derivative containing a kanamycin resistance (Kin r) cassette; Fellay et al. 1987), pDK15 (pBR322 derivative containing two pTiB6 T-DNA flanking fragments) and pDK 16 (pDK 15 with a Sp r gene cloned in between the two pTiB6 fragments; obtained from D. van der Kop and A. Hoekema, MOGEN Int. N.V., Leiden, The Netherlands), pNJ5000 (unstable RP4 derivative with a tetracycline resistance, Tc ~, marker; Grinter 1983), pBIN19 (incP binary vector containing a Km r gene; Bevan 1984), and pRK2013 (Ditta et al. 1980). The P1 phage used was PlclrlO0 Cm, received as a lysogen in E. coli HB101 from J. Engebrecht (The Agouron Institute, La Jolla, California, USA).

PCR amplification of the cre gene. In order to isolate the cre gene, a polymerase chain reaction (PCR) was performed in a Biozym-PREM processor. The DNA isolated from 0.5 ml of an overnight culture of Pl-containing E. coli cells was EcoRI-digested (the cre gene has no EcoRI sites (Sternberg et al. 1986)), and used for PCR in a final volume of 100 ~tl. The reaction mixture contained: 50 mM KC1, 10 mM TRIS-HC1 (pH 8.8), 1.5 mM MgC12, 100 gg/ml bovine serum albumin, 50 gM of each nucleotide (Pharmacia), 0.45 gM of each primer and 5 U of Taq polymerase (Promega). Reactions were overlaid with 75 ~tl of mineral oil. The mixture was heated at 93 ° C for 2 rain to denature the DNA, cooled to 55°C for 1 min to anneal the primers, and heated again to 72 ° C for 1 min to initiate the amplification. The actual amplification took place during 20 cycles of 93 ° C for 1 rain, 55 ° C for 1 min and 72 ° C for 1 rain, followed by a final elongation step of 5 rain at 72 ° C. The mixture was finally extracted twice with chloroform to remove the oil. The primers were designed to amplify the coding sequence of the cre gene flanked by a SphI site at the 5' end and an EcoRI site at the 3' end (Sternberg et al. 1986). The sequence of the upstream primer was 5'G G G C A T G C G G A G T G T T A A A T G T C C - 3 " , and the downstream primer was 5 " - G G G A A T T C A T G G C T A ATCGCCATC-3' (start and stop codons are underlined).

Culture conditions. E. coli cells were grown in LC medium (Hooykaas et al. 1977) at 37 or 30°C (Pl-containing cells). Antibiotics were added to the following concentrations: carbenicillin, 100 ~tg/ml; kanamycin, 25 ~tg/ml; spectinomycin, 50 ~tg/ml; tetracycline, 5 ~tg/ml; chloramphenicol, 50 ~tg/ml. Agrobacterium strains were grown in

loxP sequence. A 41 bp synthetic BamHI-EcoRI loxP sequence was constructed from the oligonucleotides: P 1, 5'-GGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATG-3', and P2, 5'-GAATTCATAACTTCGTATAGCATACATTATACGAAGTTATG-3'.

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Fig. 1. Strategyfor the constructionof cointegrateplant vectors by loxP-Cre-mediated site-specific recombination. LB, left border; RB, right border; M1, M2, M3; antibioticresistance markers; chr, chromosome. Further details are described in the text Results

Strategy for use of the loxP-Cre system in Agrobacterium We have developed a novel approach to integration of any desired T-DNA into disarmed Agrobacterium Ti plasmids by using the loxP-Cre site-specific recombination system of phage P1. The design of the strategy for construction of loxP-Cre-mediated pTi cointegrates is shown in Fig. 1. The T-DNA of a wild-type Ti plasmid is substituted by a loxP sequence and an antibiotic resistance marker (M1), giving rise to a loxP-containing disarmed helper plasmid. The cre gene is cloned into an incP plasmid carrying a second marker (M2). The DNA sequences that are to be integrated into the new pTi-loxP helper plasmid are cloned in a plasmid that cannot replicate in Agrobacterium and that has a loxP sequence and a third marker for selection (M3). In order to obtain the desired cointegrates, the latter plasmid is introduced into an Agrobacterium strain harbouring both the pTi-loxP disarmed helper plasmid and the incP-cre plasmid by electroporation. Strains with cointegrates are selected on medium containing the antibiotics M 1 and M3. Since the cointegrates would be resolved by the action of the Cre enzyme in due time, their stability depends on the rapid loss of the cre-containing plasmid after cointegrate formation. This can be achieved by cloning the cre gene into an unstable plasmid, the loss of which can be assessed by using the M2 marker.

Construction of a cre-containing plasmid unstable in Agrobacterium On the basis of the sequence of the cre gene (Sternberg et al. 1986) a PCR fragment was obtained as described

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Fig. 2. Constructionof a disarmedTi helper plasmidcarryinga loxP site. Cb, carbenicillin resistance marker; Spc, spectinomycinresistance marker; RB, right border; LB, leR border; B, BamHI; E, EcoRI; H, Hin~II. The shaded boxes represent DNA from the octopine Ti plasmid pTiB6. Further details are described in the text

in the Materials and methods. This PCR fragment containing the cre gene was digested with SphI and EcoRI and cloned into the polylinker of plasmid pUC19 under the control of the lac promoter, which is constitutively expressed in Agrobacterium in the absence of a cloned lacIq repressor (Chen and Winans 1991). The resulting pUC19-cre plasmid was subsequently cloned as a whole into the unique EcoRI site of the unstable incP plasmid pNJ5000. The carbenicillin resistance (Cb') marker of the pUC19 plasmid, allowing positive selection for the recombinant clones, made straightforward an otherwise inefficient and difficult cloning step, given the large size (44 kb) and low copy number of plasmid pNJ5000. The stability of the resulting construct (pNJ-cre) was checked in the Agrobacterium strain MOG1010. In these experiments, between 50 and 70% of the population turned out to have lost the pNJ-cre plasmid after 24 h of growth in liquid medium in the absence of antibiotics, whereas the same plasmid was stably maintained in similar cultures that were grown for the same length of time in medium containing carbenicillin and tetracycline (data not shown), Thus, it can be concluded that the stability of

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pNJ5000 had not been increased by the insertion of the

pU C19-cre plasmid. Construction of a pTi-loxP disarmed helper plasmid An EeoRI fragment containing both the loxP sequence and a spectinomycin resistance marker (loxP-Spc cassette) was constructed in two steps in plasmid pIC20H (Fig. 2). This cassette was subsequently cloned into the EcoRI site of plasmid pDK15 resulting in plasmid pDK15-loxP-Spc. This plasmid contains two fragments from the octopine Ti plasmid pTiB6, which flank the T - D N A on either side (Fig. 2) and can, therefore, be used as an intermediate vector for T - D N A elimination or substitution by double homologous recombination in these two pTi fragments. Plasmid pDK 15-loxP-Spc was introduced into Agrobaeterium strain LBA1010 by triparental mating; double recombinants, in which the T - D N A had been replaced by the loxP-Spc cassette,

were selected by resistance to spectinomycin and checked for sensitivity to carbenicillin (indicating loss of the pBR322 vector part of pDK15). The presence of the loxP-Spc cassette in the resulting pTiB6(AT-DNA)::loxP-Spc helper plasmid, designated pALl166, was checked by Southern blotting analysis (not shown).

Cointegration experiments In order to determine whether the two plasmids described above can indeed mediate loxP site-specific recombination, a third plasmid was constructed by cloning a Km' marker into the EcoRI site of plasmid pIC20HloxP (Fig. 3). The resulting plasmid (p!C2OH-loxP-Km) was used to check for cointegrate formation after electroporation into various Agrobacterium strains. Besides the Agrobacterium strain with both the loxP-containing disarmed helper plasmid pAL1166 and the pNJ-cre plasmid, strains containing either pALl166 alone or the pNJ-cre plasmid together with a pTi helper plasmid lacking a loxP sequence were used in these experiments as recipients. In addition, a strain in which pIC2OH-loxPSpc had been integrated into pMOG1010 by single recombination at the Sp r marker gene was included in the experiments in order to compare the efficiency of the loxP-Cre mediated site-specific recombination with that of normal homologous recombination. In a parallel experiment and as a way of assessing that all the Agrobacterium strains used had similar levels of competence for D N A uptake, the same strains were also electroporated with the incP plasmid pBIN19, which has a Km ~marker. The results of these experiments are presented in Table 1A. As can be seen from the table, the strains used did not differ significantly in their competence for DNA uptake of the control plasmid pBIN19. However, when pIC2OH-loxP-Km was electroporated into the strains that did not contain any of the loxP-Cre elements (MOG1010) or only contain the 34 bp loxP sequence (LBA1166), few or no Km' colonies were obtained. This indicates that, as expected, recombination between two loxP sites does not occur in the absence of the Cre protein. In contrast, Km r colonies appeared at a significantly higher frequency not only when the recipient strain contained the complete loxP-Cre system, but also when it contained the pNJ-cre plasmid but had a pTi helper plasmid lacking a IoxP site (pMOG1010). From these data, therefore, it cannot be concluded that the loxP-Cre system is functional in Agrobacterium. It has to be noted that the incoming Km ~ plasmid pIC2OH-loxP-Km and the pNJ-cre plasmid have pUC sequences in common (Fig. 3), which probably serve as substrates for homologous recombination. Normal homologous recombination apparently is rather efficient, as indicated by the large number of Km ~colonies that were obtained when pIC2OH-loxP-Km was electroporated into the strain containing pIC2OH-loxP-Spc integrated into the pTi plasmid pMOG1010 (Table 1). An indirect approach was therefore followed in order to check whether the loxP-Cre site-specific recombination system had indeed been operational in our experiments.

5 Table 1. Frequency of Km r transformants in A RecA + and B RecAelectroporation with pBIN19 or pIC2OH-loxP-KmDNAs Back- Resident ground plasmids

Agrobacterium strains containing the loxP-Cre system after

Incoming plasmid~ pBIN19 Km r

pIC20-loxP-Km

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c

Frequency a

Km r Survivorsc transformants b

transformants b

Frequencya

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RecA + pMOG1010 RecA + pALl166 RecA ÷ pMOG1010: :pIC20H-loxP-Spc RecA + pMOGlOlO+pNJ-ere RecA + pALl166+pNJ-cre

3.1 × 103 3.3x103 1.8 x 103 2.8 x 103 4.4x 103

9.4x 109 1 A x l 0 t° 4.8 × 109 2.4 x 109 2.1 x 109

3.3x 10 -7 2.3x I0 -7 3.7x 10-7 1.2×10 .6 2,1 x 10 -6

0 1.0x 2.4x 2.6x 7.6 x

3.2 x 6.0 x 3.8 x 3.3 x 2.6 x

1.5 x 1.7 x 4.5 x 4.2 x 4.2 x

2.1x10 6 3.5x 10-6 8.4x 10 -7 7.9x 10 -7 6.2x 10 -7

2.0x10 o 1.0x 10° 1.0x 10° 3.8x 10z 4.2x 102

10° 103 103 103

1.3x 2.1 x 7.5x 3.5x 2.1 x

101° 101° 109 109 109