Recombinational Cloning with Plant Gateway Vectors

The study of biological systems relies to a large extent on DNA cloning technologies enabling the analysis of recombinant genes through transgenic research. In this context, the advent of recombinational cloning methods was a significant progress because DNA fragments can now be assembled regardless of their sequence. In particular, the Gateway system was designed to join fragments in a predefined order, orientation, and reading frame. The recent development of transformation vectors and large-scale clone resources amply demonstrate that plant researchers have adopted the Gateway platform and that it will remain an important asset in projects requiring systematic cloning, modular assembly, and expression in various contexts. Agrobacterium tumefaciens binary vectors are widely used for plant transformation. They vary in size, origin of replication, bacterial selectable markers, T-DNA borders, and overall structure. Binary vectors are cumbersome to handle in conventional cloning schemes involving DNA restriction and ligation reactions, and substantial efforts have been invested in the creation of smaller vectors with a choice of unique restriction sites within the T-DNA region (Hajdukiewicz et al., 1994; Hellens et al., 2000a; Goderis et al., 2002; Tzfira et al., 2005; http://www.cambia.org/). But the recent introduction of robust site-specific recombinational cloning methods has greatly facilitated the construction of expression units in a large variety of in vivo and in vitro systems (Marsischky and LaBaer, 2004). In particular, the Gateway technology developed originally by researchers at Life Technologies, Inc. (Hartley et al., 2000), and now commercialized by Invitrogen, has been endorsed by a large community and compatible vectors have been created for most applications requiring the creation of recombinant DNA molecules. This review gives a summary of the site-specific Gateway recombinational cloning system and presents related vectors generated by different plant research laboratories. Schematic representation of att sites and Gateway recombination reactions. A, In a BP clonase reaction, attB sites (in a PCR product or plasmid) recombine with the matching attP sites of a donor vector (pDONR) to yield attL sites in a novel entry vector (pENTR) and attR sites in a byproduct. B, In a LR clonase reaction, attL sites in an entry vector (pENTR) recombine with the matching attR sites of a destination vector (pDEST) to yield attB in a novel expression vector (pEXPR) and attP in a byproduct. C, In a single MultiSite LR clonase reaction, the compatible att sites carried by entry clones originating from independent BP clonase reactions (in this example pENTR L4-fragment1-R1, pENTR L1-fragment2-L2, and pENTR R2-fragment3-L3) and by a MultiSite destination vector (for example pDEST R4-R3) recombine to yield a single contig in which the DNA fragments of interest are separated by short attB sites. Blue boxes, DNA fragments of interest assembled in BP and LR clonase reactions; black box with vertical white stripe, attB sites, also at the core of the attP, attL, and attR sites; yellow box, portion of the attP and attL sites; red box, portion of the attP and attR sites. B1 to B4, attB1 to attB4 sites; L1 to L4, attL1 to attL4; R1 to R4, attR1 to attR4. To enable directional cloning, the inventors of the Gateway system engineered variants of the original attB, attP, attL, and attR sites so that attB1 will react specifically with attP1, but not with attP2, attP3, etc. (Cheo et al., 2004; Sasaki et al., 2004). The standard oriented BP and LR reactions involving the att1 and att2 series (Fig. 1, A and B) always maintain the frame register that is necessary to build translational fusions. The availability of multiple recombination site series is also useful in alternative protocols to assemble simultaneously multiple DNA fragments in a single LR clonase reaction yielding an expression clone with two or more contiguous DNA fragments in predefined order and orientation. This specific technology with more than two att series has been dubbed MultiSite Gateway and has already been adapted for the construction of plant binary destination vectors (Fig. 1C; Karimi et al., 2005). For additional information about the basics of the Gateway technology, the reader is referred to the manuals accessible online (http://www.invitrogen.com/). A given destination vector intended for a particular functional assay can be recombined with any sequence captured in a compatible entry clone. Conversely, the same entry clone (such as an open reading frame [ORF] entry clone) can be recombined with many different destination vectors. The resulting expression clones are the constructs used to test gene functions, for example after transformation into plants. Typically, the desired plasmids are created by in vitro recombination, transformed in Escherichia coli strains, and segregated from other reaction by-products and input vectors through appropriate antibiotic selection and counterselection based on the ccdB (control of cell death) gene (Hartley et al., 2000). Importantly, the backbones of the plant binary T-DNA destination vectors must contain different bacterial antibiotic resistance markers from the donor (and entry) vectors (Table I Gateway plant binary destination vectors The asterisk refers to the DNA selectable markers as indicated in the T-DNA marker column. BSM, Bacterial selectable markers; Cm, chloramphenicol; K, kanamycin; Sm, streptomycin; Sp, spectinomycin. RfA, RfB, or RfC, Gateway reading frames. T-DNA selectable markers: B, Basta; H, hygromycin; K, kanamycin; NM, no marker. References are: 1, Karimi et al. (2002); 2, Curtis and Grossniklaus (2003); 3, Earley et al. (2006); 4, Karimi et al. (2007); 5, Helliwell and Waterhouse (2003); 6, Hilson et al. (2004); 7, http://www.pi.csiro.au/rnai/vectors.htm; 8, Wielopolska et al. (2005); 9, http://www.bio.utk.edu/vonarnim/BRET/BRET-vectors.html; 10, Joubès et al. (2004); 11, De Schutter et al. 2007; 12, Brand et al. (2006); 13, Brown et al. (2006); 14, Rohila et al. (2006); 15, Van Leene et al. (2007); 16, Liu et al. (2002); 17, http://www.psb.ugent.be/gateway/; 18, Karimi et al. (2005); 19, Marjanac et al. (2007). Other useful resources include: http://www.biology.wustl.edu/pikaard/Vectors%20homepage.html; http://www.cambia.org/daisy/cambia/home.html; and http://www.unizh.ch/botinst/Devo_Website/curtisvector/. RLUC, Renilla LUC; hRLUC, humanized LUC. Gateway plant binary destination vectors The asterisk refers to the DNA selectable markers as indicated in the T-DNA marker column. BSM, Bacterial selectable markers; Cm, chloramphenicol; K, kanamycin; Sm, streptomycin; Sp, spectinomycin. RfA, RfB, or RfC, Gateway reading frames. T-DNA selectable markers: B, Basta; H, hygromycin; K, kanamycin; NM, no marker. References are: 1, Karimi et al. (2002); 2, Curtis and Grossniklaus (2003); 3, Earley et al. (2006); 4, Karimi et al. (2007); 5, Helliwell and Waterhouse (2003); 6, Hilson et al. (2004); 7, http://www.pi.csiro.au/rnai/vectors.htm; 8, Wielopolska et al. (2005); 9, http://www.bio.utk.edu/vonarnim/BRET/BRET-vectors.html; 10, Joubès et al. (2004); 11, De Schutter et al. 2007; 12, Brand et al. (2006); 13, Brown et al. (2006); 14, Rohila et al. (2006); 15, Van Leene et al. (2007); 16, Liu et al. (2002); 17, http://www.psb.ugent.be/gateway/; 18, Karimi et al. (2005); 19, Marjanac et al. (2007). Other useful resources include: http://www.biology.wustl.edu/pikaard/Vectors%20homepage.html; http://www.cambia.org/daisy/cambia/home.html; and http://www.unizh.ch/botinst/Devo_Website/curtisvector/. RLUC, Renilla LUC; hRLUC, humanized LUC. Lastly, small high-copy E. coli plasmids are routinely introduced into plant cells or protoplasts via methods that do not require Agrobacterium-mediated delivery, such as particle bombardment, polyethylene glycol/Ca2+ transfection, or electroporation. These plasmids have also been adapted for plant transgene construction via Gateway recombinational cloning (Table II Gateway high-copy plant destination vectors All vectors carry the ampicillin bacterial selectable marker. RfA or RfB, Gateway reading frames. References are: 1, Karimi et al. (2002); 2, Tzfira et al. (2005); 3, http://www.bio.utk.edu/vonarnim/BRET/BRET-vectors.html; 4, Walter et al. (2004); 5, Ehlert et al. (2006); 6, M. Karimi, S. Bernacki, P. Hilson, and D. Robertson, unpublished data; 7, Karimi et al. (2005). Gateway high-copy plant destination vectors All vectors carry the ampicillin bacterial selectable marker. RfA or RfB, Gateway reading frames. References are: 1, Karimi et al. (2002); 2, Tzfira et al. (2005); 3, http://www.bio.utk.edu/vonarnim/BRET/BRET-vectors.html; 4, Walter et al. (2004); 5, Ehlert et al. (2006); 6, M. Karimi, S. Bernacki, P. Hilson, and D. Robertson, unpublished data; 7, Karimi et al. (2005). Because the Gateway system has proven efficient, flexible, and reliable, several laboratories have constructed a wide range of Gateway destination vectors for the analysis of plant genetic elements. An overview of the structure of these vectors and their applications according to the type of functional assays for which they were designed is given below. In destination vectors designed for gene overexpression, a standard Gateway cassette (attR1-ccdB-attR2) is placed downstream of the CaMV 35S promoter and upstream of a standard terminator (Tables I, nos. 1–6 and II, no. 1). A similar configuration can be obtained with MultiSite Gateway binary vectors for two or three fragment recombination (Table I, nos. 84–88) with the added advantage that any strong, inducible, or tissue-specific promoter captured in an entry vector (attL4-promoter-attR1) can be fused with any gene of interest present in another entry clone (attL1-gene-attL2). Already, a collection of sequence-validated promoters (including CaMV 35S, Cassava vein mosaic virus [CsVMV], A. tumefaciens nopaline synthase [nos], maize [Zea mays] ubiquitin [ubi], and A. rhizogenes rolD) is available for MultiSite LR reaction that is complemented with terminator entry clones (attR2-terminator-attL3) including CaMV 35S, nos, octopine synthase, and gene7 (Karimi et al., 2007). For certain experiments gene expression has to be induced at a chosen time, for example to characterize loss- or gain-of-function mutations that cause embryo lethality. Vectors that have been constructed for the inducible transcriptional activation of a gene of interest are described below. An alternative system has been devised for misexpression restricted to clonal sectors. In this configuration, the gene of interest is cloned in a Gateway cassette placed downstream of the 35S CaMV promoter, but separated from it by a spacer containing a gene coding for the ENHANCED GFP (EGFP) flanked by loxP sites. Another transgene carried within the same T-DNA expresses the CRE recombinase under the control of the promoter of the heat shock protein gene HSP18.2 of Arabidopsis (Arabidopsis thaliana). To avoid any unwanted CRE activity, the CRE enzyme is fused to the mammalian glucocorticoid receptor (GR) domain that localizes the hybrid protein into the nucleus only in the presence of dexamethasone (DEX). Thereby, the gene of interest is constitutively activated upon combined heat and DEX treatments, resulting in the excision of the EGFP spacer by CRE recombination of the two loxP sites. The activation sectors are marked by the absence of the GFP fluorescence (Joubès et al., 2004; Table I, no. 66). CRE-GR-mediated activation was used successfully in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, but not in Arabidopsis (L. De Veylder, personal communication). A similar system, proven to be reliable in Arabidopsis root tissues, was constructed in which CRE activity does not require DEX treatment (De Schutter et al., 2007; Table I, no. 67). Independently, binary destination vectors have also been created for cloning any promoter of choice upstream of the CRE ORF (Marjanac et al., 2007; Table I, no. 89). The transcriptional activity of a promoter can be characterized by the temporal and spatial expression patterns of a reporter protein. In such studies, the promoter sequence must first be cloned upstream of the corresponding reporter gene. Alternatively, the presence of a protein of interest can be monitored if it carries a tag at its C or N terminus, after transcription under the control of its own promoter (Tables I, nos. 10–13,17–20 and II, no. 2) or of a heterologous strong promoter, such as CaMV 35S (Tables I, nos. 29–63 and II, nos. 3–18). When the added domain in a translational fusion codes for a fluorescent protein or an epitope tag, the protein of interest can be localized subcellularly by microscopic analysis of living or fixed cells (Citovsky et al., 2006). Binary destination vectors have been designed in which a standard Gateway cassette (attR1-ccdB-attR2) precedes the ORF coding for an enzyme (luciferase [LUC] or GUS), a fluorescent protein (GFP, yellow fluorescent protein [YFP], cyan fluorescent protein [CFP], or red fluorescent protein [RFP]), with or without a nuclear localization signal, a purification tag (polyhistidine [6xHis]), an epitope tag (hemagglutinin [HA], FLAG, c-Myc, or AcV5), or a combination of these (Table I, nos. 9–20). The same final configuration can be obtained via MultiSite Gateway recombination (Table I, no. 21). In some of these constructs, the spacer at the 3′ end of the inserted fragment (with regard to transcription) is structured to enable translational fusion so that a protein of interest encoded in a DNA sequence with its native promoter can be expressed with an additional domain at its C terminus (Table I, nos. 11–20). This type of arrangement is necessary to study cis-regulatory sequences located in introns or exons. The MultiSite Gateway system can also be used to assemble a promoter of choice (attL4-promoter-attR1), the selected reporter tag or and the gene coding for the protein of interest or in a compatible destination vector (Table I, no. sequence-validated reporter entry clones are available for such LR cloning or epitope or or enzyme or of or (Karimi et al., 2007). In Gateway constructs translational the or captured in the entry clones are assembled in the same frame register and are separated by attB sites coding for for and for that the encoded in attB sites are than encoded in multiple restriction cloning sites, or In the or of a functional assay involving translational fusion does not on a particular cloning but on a The ORF structure in the entry clones to be constructs are designed for expression of For it be to a tag at the N terminus of the characterized protein at or its C terminus that are for at of the Arabidopsis protein coding sequences a et al., that not be functional fused to an additional tag or cause the tag to be the protein. the other the of a tag of the original from the on downstream ORF entry clones have to be generated in open or (with native et al., 2006). systems have been developed for gene activation or They an that codes for an transcription expressed in restricted tissues, at or upon or and a in which an promoter the transcription of a gene under activated by the transcription these is in research projects requiring the combination of multiple and by transformation or via The plant system has been developed for the of gene expression by the and adapted to the Gateway When to and the transcription that of three DNA transcriptional activation and receptor et al., 2000). In the vector (Table I, no. any promoter or sequence can be cloned upstream of the ORF et al., 2006). In another the transgene is located in the same T-DNA as a gene that the domain of activation (Table I, no. vectors (Table I, nos. an promoter upstream of a Gateway cassette (attR1-ccdB-attR2) for transcriptional of any gene of to a bacterial ampicillin resistance gene and a origin of that can be used for (Table I, nos. of another system have been for MultiSite Gateway cloning (Karimi et al., 2007). is based on of two transcription of the activation domain fused to the bacterial domain et al., that is constitutively et al., and that the domain and is inducible by DEX treatment et al., 2005). and to and as as a promoter have been captured in entry Thereby, the and at the core of this system can be assembled via MultiSite cloning (Karimi et al., 2007). the introduction of plant transformation technology, plant gene was by at from transgenic via a et al., Gateway vectors be used to this In have been to gene via 2007). In particular, that the of small are in plant cells et al., 2000). it is not to a for the transcription of corresponding to the two of the same DNA an of the Gateway LR recombination this et al., The sequence for captured in an entry clone is in a single LR clonase reaction in a destination vector two independent Gateway separated by an spacer The Gateway are that their attR1 and recombination sites are with to so that the two of the sequence are to in the resulting expression clone. binary destination vectors have been constructed according to this They in structure of the and promoter the transcription of the (Table I, nos. a vector has been created for the of advantage of the system et al., 2005; Table I, no. Alternatively, the of vectors for the expression of under the control of any promoter of choice can also be facilitated by a only involving MultiSite Gateway cloning, as described M. Karimi, M. A. A. D. S. and P. Hilson, unpublished The LR clonase reaction is most with destination vectors in which the sequence the attR sites (including the ccdB is inserted as a (Table I, nos. of an and A of based on the constructs described is that the attB1 and sites that are to expression of an independent transgene including the same Gateway sites and P. Hilson, unpublished have also been used to plant genes et al., et al., 2006). These are engineered from (such as in which the two short sequences that the which are and by sequences chosen to be as specific as to the et al., 2005). via specific and can be induced under the control of specific the original described by et al. to an in a single PCR that three DNA can be adapted to the Gateway cloning by of the attB1 and recombination sites restriction at the of the The resulting can be captured in an entry clone into a destination vector as any other via a standard or MultiSite LR clonase reaction, for transcription in different tissues, at various or upon The of the attB sites at the of an sequence does not its M. Karimi, M. A. A. D. S. and P. Hilson, unpublished data; and personal communication). gene has also been used to gene expression in et al., 2004; Robertson, 2004). In are with a vector a fragment from a gene. have the advantage to the to for but are on its is to certain tissues, but this restriction be by the of the vectors have been to be compatible with Gateway cloning (Tables I, no. and II, no. functional such as the of require the into selected of an DNA binary destination vectors have been designed for such fragment (Table I, nos. In some of the T-DNA also a reporter (Table I, no. enabling the transformed and cells in tissues, such as upon A. transformation et al., et al., 2006). The in living cells can be with methods that require the of via translational For purification the tag codes for a domain that and with to or multiple fixed on and with cell et al., of the of the the in the can be by For fluorescence two hybrid are of a fluorescent or When the they a functional fluorescent protein that is of two via fluorescence relies on the a different fluorescent In such the donor and (such as and into a in the fluorescence of the donor fluorescence and fluorescence have already been in plant cells through Gateway destination vectors for the of with various et al., Van Leene et al., fluorescent et al., or and et al., et al., I, nos. and II, nos. and systems in which the protein upon is a transcriptional and have also been adapted for plant cell assays et al., 2006). In this the translational are expressed for in polyethylene from two independent Gateway expression clones and under the control of the 35S CaMV The for the domain or the activation domain of and a nuclear localization The reporter is encoded in a in which a promoter containing sites the transcription of the enzyme (Table II, nos. can be to multiple in a single MultiSite Gateway recombinational cloning can this Binary destination vectors have been created for the expression of two or three genes under the control of different strong plant promoters (Table I, nos. and the final expression gene must be first captured in a entry clone or and a Gateway destination The two or three destination are located in the T-DNA region of a plant binary destination vector and are flanked by different promoter and terminator sequences (Karimi et al., 2007). Another vector system has been designed for the of multiple into the same binary destination vector via of LR involving two of entry clones et al., 2006). advantage of different of attL and attR sites, and of two bacterial selection markers and In this system, added in a transgene also carries the Gateway cassette the is recombined in the in a of that Gateway cloning in (for 2007). only and MultiSite Gateway the of PCR hybrid (including attB and for the creation of an entry clone in a BP reaction an attB in an appropriate vector the and the creation of an expression clone resulting from a or a MultiSite LR clonase reaction, based on the recombined entry and destination All generated are as sequence and with the entry clones genetic are because the same multiple laboratories can be used in any assay for which destination vectors The Gateway system is for systematic cloning projects because the specific BP and LR on sites of to to by and can be regardless of the sequence of the it is robust for and large-scale clone have already been containing Arabidopsis genetic captured in Gateway clones (for Hilson, Arabidopsis 2007). They and in which the original is or to enable the of a sequence coding for an tag, and sequence used for et al., 2004; et al., 2007). of Arabidopsis promoters et al., and are or will be available in a as as similar resources on the sequence of other plant the of of constructs is not an researchers can novel functional assays based on cloned sequence laboratories have already developed genetic advantage of the and of the Gateway cloning Another example of applications is a Gateway cassette including sequences for the of in plant et al., 2007; M. Karimi and A. unpublished to no has on the of recombination att site sequences in for the creation of The Gateway system is commercialized for research only and is by of the in transgenic as a research not as a product an important the transformation of many constructs into plants. For certain studies, such as transcriptional or plant assays in or transformed cells have proven useful to genetic et al., 2004; De et al., 2005; et al., 2005; Ehlert et al., et al., 2007). assays in many are also in alternative heterologous systems for which Gateway destination vectors are such as and to protein et al., et al., and transcriptional et al., in vitro protein for the of protein et al., more in for the of in large necessary for or the same genetic for plant assays also in other and to the functional the many a plant the recent development of plant transformation vectors and large-scale clone plant researchers have adopted the Gateway cloning In the this platform will remain an important asset in projects requiring systematic cloning, modular assembly, and expression in various contexts. To advantage of the of the system, experiments the DNA fragments they are not already available as Gateway entry In this context, and are to the of resources in a and for and De for in the