A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens
© Traore and Zhao; licensee BioMed Central Ltd. 2011
Received: 24 August 2011
Accepted: 7 December 2011
Published: 7 December 2011
Cloning genes into plasmid vectors is one of the key steps for studying gene function. Recently, Invitrogen™ developed a convenient Gateway® cloning system based on the site-specific DNA recombination properties of bacteriophage lambda and the cytotoxic protein ccdB, which is lethal to most E. coli strains. The ccdB protein, however, is not toxic to Agrobacterium tumefaciens, an important player often used for studying gene function in planta. This limits the direct application of the Gateway® cloning system in plant transformation-mediated research.
In this study, we constructed a novel Gateway®-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker.
Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway® cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.
The wide-spread availability of genomic sequences from many organisms has raised interest in characterizing the biological functions of newly discovered genes through various high-throughput methodologies . In order to study gene function, a gene-of-interest needs to be cloned into different plasmid vectors. In plant biology research, the gene-of-interest is frequently cloned into binary vectors that can be used for Agrobacterium- mediated transformation .
Gateway® technology was developed as a convenient and fast gene cloning system (Invitrogen™, Carlsbad, CA). This method involves three key steps: (1) amplifying the targeted gene by PCR; (2) directly cloning the PCR product into a TopoEntr/D™ vector without digestion/ligation; and (3) subcloning the targeted gene from the entry vector into any destination vector using the Gateway® LR cloning technique. The Gateway® LR cloning reaction is mediated by the site-specific homologous DNA recombination properties of bacteriophage lambda . This method is more convenient than other methods because it does not involve either DNA digestion nor ligation, two processes that can hinder the cloning process . The Gateway®-compatible destination vector harbors a negative selectable marker, the ccdB gene, which produces a toxic protein that is lethal to most E. coli strains, including DH5α[5, 6]. During the Gateway® LR reaction, the ccdB gene in the destination vector is replaced by the targeted gene from an entry vector through site-specific homologous DNA recombination. The LR reaction mixture containing both recombinant and non-recombinant plasmids are subsequently transferred into an E. coli strain, such as DH5α, that is sensitive to the toxic effect of ccdB. Only recombinant destination plasmids that have lost the ccdB gene are able to survive in the transformed E. coli cells.
Recently, a collection of Gateway®-compatible binary destination vectors, that can be used for Agrobacterium-mediated transformation, have been constructed and are available to the plant research community [7–10]. Targeted genes can be easily cloned into these binary vectors through Gateway® LR cloning. However, the Gateway® LR cloning mixture has to first be transformed and screened in E. coli strains before the recombinant plasmid can be mobilized to Agrobacterium tumefaciens (A. tumefaciens) by either conjugation or electroporation. This process could be simplified if the recombinant binary plasmids from the Gateway® LR reaction could be directly transformed and selected in A. tumefaciens. Unfortunately, A. tumefaciens is insensitive to the toxic effect of ccdB . Thus, the binary vectors that contain the ccdB gene cannot be negatively selected against in A. tumefaciens. To overcome this problem, a new negative selectable marker that is functional in A. tumefaciens must be tested.
The SacB-SacR genes were originally isolated from Bacillus subtilis and encode levansucrase, an enzyme involved in both the hydrolysis of sucrose and the biosynthesis of levan [11, 12]. Levan cannot be metabolized by most gram-negative bacteria, including E. coli and A. tumefaciens, and is therefore toxic to this group of organisms [13, 14]. The SacB-SacR gene cassette, driven by its native promoter, has been used as a negative selectable marker for many gram negative bacteria and works by preventing the transformed bacterial strains from growing on culture medium supplemented with 5% sucrose . Therefore, it will be interesting to test if the ccdB gene in any Gateway®-compatible binary vector can be replaced with the SacB-SacR gene cassette as the negative selectable marker. This would allow for direct transformation and screening of the Gateway® LR cloning product in A. tumefaciens.
In this study, one popular Gateway®-compatible destination binary vector, pEarleyGate101 , was modified by replacing the ccdB gene with a SacB-SacR gene cassette. The novel destination binary vector, pEG101-SacB/R, would allow for the direct selection of Gateway® recombinant clones in A. tumefaciens. To test the efficiency and functionality of pEG101-SacB/R, a luciferase gene (Luc) and a bacterial effector gene Aae2166 were used as reporters and cloned into the pEG101-SacB/R destination vector. The Luc gene encodes the fire fly luciferase protein, an enzyme that catalyzes luciferin and produces a luminescence signal when expressed in plant cells. This makes the Luc gene a convenient reporter gene for this study . The bacterial type III putative effector gene Aae2166, isolated from bacterial pathogen Acidovorax avenae pv. citrulli, encodes a homolog of AvrBsT. Transient expression of Aae2166 in the leaves of Nicotiana benthamiana (N. benthamiana) could trigger a cell death phenotype (Traore S and Zhao B, unpublished results). Therefore, Aae2166 is also a convenient marker for testing the functionality of the newly developed pEG101-SacB/R destination vector. The results of this study demonstrated that both genes were successfully cloned, selected for in A. tumefaciens, and expressed in N. benthamiana leaves. Therefore, the new destination vector is a convenient tool for cloning and expressing genes in A. tumefaciens. The new vector also has potential to be used in high-throughput cloning applications in planta.
Escherichia coli (E. coli) DH5α [F - endA glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(r K - m K + ), λ-] and A. tumefaciens (GV2260) [C58 background, rifampicin resistant with the Ti plasmid (pTiB6s3)]
Preparation of electroporation competent cells
E. coli DH5a and A. tumefaciens GV2260 strains were streaked on Luria Broth (LB) agar media supplemented with appropriate antibiotics and incubated at 37°C and 28°C, respectively.
Single colonies of DH5a and A. tumefaciens (GV2260) were inoculated in 50 ml of LB liquid media, incubated at 37° and 28°C, respectively, and shaken at 200 rpm for 24 hours.
A new 500 ml LB liquid culture was re-inoculated from the initial saturated bacterial culture to produce a final concentration of OD600 nm = 0.1. The liquid cultures were incubated at 18°C and 28°C for DH5a and A. tumefaciens (GV2260), respectively, until the OD at 600 nm reached 0.6.
The bacterial liquid culture was subsequently chilled on ice for 20 minutes.
Bacterial cells were harvested by centrifugation at 6000 × g for 10 minutes at 4°C.
The bacterial cells were washed twice with 250 ml of ice-cold water, and then finally washed with 25 ml of 10% ice-cold glycerol.
The bacterial cells were pelleted by centrifugation and re-suspended in 5 ml of 10% ice-cold glycerol.
A 100 µl aliquot of bacterial cells was transferred to 1.5 ml microtubes and immediately frozen in liquid nitrogen.
The cells were stored at -80°C for at least six months without loss of competency.
N. benthamiana (PI 555478) plants were propagated in a growth chamber programmed for 16 hours light (140 μmol/m2/s cool white fluorescent irradiance) at 28°C and 8 hours dark at 24°C. Agrobacterium-mediated transient assays were conducted on three to four week old plants.
Construction of the destination binary pEG101-SacB/R vector
The SacB-SacR genes were amplified by PCR from plasmid pMsacB  using primers SacB/R Forward: 5'-AATCAGGAAGGGATGGCTGAGG GATATCGGATCGATCCTTTTTAACCCATCAC-3'and SacB/R Reverse: 5'-AGACCGGCACACTGGCCATATCGGTGG GATATCTTATTTGTTAACTGTTAATTGTCCT-3'. The Bbv CI and Bst XI restriction sites appear in the primers as bolded and underlined text, respectively. The nucleotide sequences of the SacB-SacR genes, including their native promoter, can be found in GenBank [GenBank:AB183144].
The iProof ™ high fidelity Taq DNA polymerase (Bio-Rad, Hercules, CA) PCR program consisted of 1 cycle at 98°C (2 min), followed by 30 cycles at 98°C (30 s), 55°C (45 s), and 72°C (1 min), and finished with a 1 cycle extension at 72°C (7 min). The PCR products were digested with Bbv CI and Bst XI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA).
The destination binary plasmid pEarleyGate101  was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH) and digested with Bbv CI and Bst XI to remove the ccdB gene. The gel-purified PCR product of the SacB-SacR gene cassette was then cloned into the Bbv CI and Bst XI restriction sites of pEarleyGate101. The ligation products were transformed into DH5α using a gene pulse electroporation apparatus (2.5 Kv/uM/mSec) (Bio-Rad). Successfully transformed bacteria were selected for on LB medium supplemented with kanamycin (50 mg/ml) and chloramphenicol (25 mg/ml). The derived new destination vector was re-named pEG101-SacB/R.
Plasmid DNA isolation
All plasmid DNA was isolated using an AccuPrep™ Plasmid Extraction Kit (Bioneer).
Negative selection for the SacB-SacR gene
Cloning of the Luc gene and Aae 2166 into the TopoEntry/D vector
The open reading frame (ORF) of the Luc gene was cloned from plasmid pDesA-luc  with primers: Sal_LucFor:5'-caccgtcgacGGCGGTGGCTCATCTGGCGGAGGTatg gaagacgccaaaaac-3' and Pst_LucRev: 5'-ctgcagTTA CAATTTGGACTTTCCGCCCTTCTTGG-3'. The start and stop codons of the Luc gene are bolded and underlined in the forward and reverse primers, respectively. The ORF of the Aae2166 gene was amplified from genomic DNA of Acidovorax avenae subs citrulli strain AAC-001 using primers: Aae2166 For: 5'-caccAGATCTATG AAGAATTTCATGCGATCGAT -3' and Rev: 5'- GTCGACTTCGATAGCTTTTCTGATTTTTCTCA-3'. The start codon of Aae2166 is bolded and underlined. The PCR products of the Luc and Aae2166 genes were gel purified and cloned into the TopoEntr/D™vector (Invitrogen) following the instructions of the user's manual. Plasmid DNA was sequenced at the Core facility of the Virginia Bioinformatics Institute (Blacksburg, VA).
LR reaction and transformation of Agrobacterium tumefaciens by electroporation
The Gateway® LR clonase enzyme mix kit (Invitrogen) was used for the LR recombination reaction. In brief, the LR reaction mixture contained: 1-5 μl (100 ng) of entry clone plasmid DNA, 1 μl (100 ng) of destination vector pEG101-SacB/R plasmid DNA, 2 μl of 5 × LR clonase reaction buffer, and 2 μl of LR clonase enzyme. The reaction mixture was brought to a final volume of 10 μl with TE buffer (pH 8.0) and incubated at room temperature for 1 hour. Following the incubation, a 3-5 μl aliquot was mixed with 100 μl of competent A. tumefaciens (GV2260) cells. A gene pulse apparatus (Bio-Rad), programmed for 2.5 Kv/uM/mSec, was used to transform the cells by electroporation. Following electroporation, the mixture was resuspended in 1 ml of liquid LB and incubated shaking at 28°C/200 rpm for 3 hours. Successfully transformed cells were then selected for on LB agar supplemented with kanamycin (50 mg/ml), rifampicin (100 mg/ml), and 5% sucrose. The LB plates were incubated at 28°C for 2-3 days. After incubation, ten colonies were randomly selected for further analysis. Recombinant plasmids were confirmed by PCR with primers: 35S forward 5'-AAGAAGACGTTCCAACCACGTC-3' and NLuc reverse: 5'-AACTGCAGTCATCCATCCTTGTCAATCAAG-3' to detect the Luc gene (1.4 kb) . Recombinant plasmids were also confirmed by PCR using primers 35S forward plus Aae2166 reverse: 5'- GTCGACTTCGATAGCTTTTCTGATTTTTCTCA-3' to detect the Aae2166 gene (1.1 kb). The PCR fragments were separated on a 0.8% agarose gel, stained with 0.01% ethidium bromide solution, and visualized using the Gel-Document Image System™ under UV light (Bio-Rad).
Agrobacterium-mediated transient assays in N. benthamiana plants
Agrobacterium-mediated transient assays in N. benthamiana plants were performed as described previously . In brief, the Agrobacterium strain was streaked on LB medium supplemented with appropriate antibiotics and incubated at 28°C for 2 days. Bacterial cells were harvested and re-suspended in induction buffer composed of 10 mM MgCl2, 10 mM MES (pH 5.6), and 100 μM acetosyringone and incubated for 3 hours at room temperature. The bacterial inoculums were adjusted to OD600 nm = 0.6 and infiltrated into the stomata of fully expanded N. benthamiana leaves using a 1 ml blunt-end syringe without a needle. The inoculated plants were incubated at room temperature under continuous light for 20-48 hours before the detection of expressed proteins. Transient expression of the Luc gene was detected by applying 1 μM luciferin to the inoculation site . The chemical fluorescent signals were captured with a CCD camera and visualized using the Gel-Document Image System (Bio-Rad). The fluorescent signal of the Aae2166-GFP fusion protein was monitored 20 hours after inoculation by fluorescent microscopy (Zeiss Axio Observer.A1, Carl Zeiss MicroImaging, Inc., Thornwood, NY).
Results and discussion
Constructing a novel Gateway®-compatible binary vector pEG101-SacB/R
Testing the stability of binary vector pEG101-SacB/R
Generating recombinant clones in Agrobacterium tumefaciens through Gateway LR cloning
As shown in Figure 1A, the cloned Luc and Aae2166 genes are driven by the 35S promoter. The ORF of Aae2166 was cloned, without a stop codon, into the pEntry/D vector. After LR cloning, the lack of a stop codon allowed the Aae2166 ORF to be fused in-frame with the N-terminus of the GFP gene in the recombinant pEG101-Aae2166 plasmid. Conversely, a stop codon was added to the C-terminus of the Luc gene; therefore, GFP fusion would not have occurred.
Validating the function of recombinant binary plasmids by Agrobacterium-mediated transient assays
In this report, a novel Gateway®-compatible binary vector pEG101-SacB/R is described that allows direct transformation and selection of recombinant LR-generated clones in A. tumefaciens. The Luc gene, along with a putative bacterial effector gene, Aae2166, were used to demonstrate the efficiency and functionality of this newly developed Gateway®-compatible binary vector. The ability to directly clone and select recombinant genes in A. tumefaciens would save time, labor, and would also minimize potential contamination problems associated with conjugation or transformation manipulation. The new vector pEG101-SacB/R described here has great potential for simplifying and improving the efficiency of cloning genes in A. tumefaciens for plant transformation research. The pEG101-SacB/R vector can also be adapted for high- throughput applications. For example, a gene-of-interest mutant library could be generated by Error-Prone PCR from a TopoEntr/D™ (Invitrogen) plasmid template. The loss-of-function mutants could be identified through Agrobacterium-mediated transient assays in N. benthamiana leaves. Therefore, the new destination binary vector pEG101-SacB/R may be a valuable tool for high-throughput functional analysis of genes in planta.
The authors thank Drs. Richard Veilleux, Gregory E. Welbaum, Ms. Taylor Frazier and Kerri Mills for their critical comments on the manuscript. Financial support was provided by grants from Binational Agricultural Research and Development Fund (BARD) (US-4216-09 to BZ), US NSF (IOS-0845283 to BZ), and the Virginia Agricultural Experiment Station (VA135872).
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