2 Gateway Vectors for Plant Genetic Engineering : Overview of Plant Vectors , Application for Bimolecular Fluorescence Complementation ( BiFC ) and Multigene Construction

Yuji Tanaka1, Tetsuya Kimura2, Kazumi Hikino3, Shino Goto3,4, Mikio Nishimura3,4, Shoji Mano3,4 and Tsuyoshi Nakagawa1 1Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 2Department of Sustainable Resource Science, Graduate School of Bioresources, Mie University, 3Department of Cell Biology, National Institute for Basic Biology, 4Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies, Japan


Introduction
Transgenic technologies for the genetic engineering of plants are very important for basic plant research and biotechnology.For example, promoter analysis with a reporter such as green fluorescent protein (GFP) is typically used to determine the expression pattern of genes of interest in basic plant research.Moreover, downregulation or controlled expression studies of target genes are used to determine the function of these genes.In plant biotechnology, overexpression of heterologous genes by transgenic methods is widely used to improve industrially important crop plants.Recently, genome projects focusing on various higher plants have provided abundant sequence information, and genome-wide studies of gene function and gene regulation are being carried out.In these areas of research, transgenic analyses using genetically modified plants will become more essential.For example, high-throughput promoter analysis to examine the temporal and spatial regulation of gene expression, the subcellular localization of the gene products based on reporter genes, and ectopic expression of cDNA clones and RNAi will reveal the functions of a variety of genes.For gene manipulation in plants, the binary system of Agrobacteriummediated transformation is most widely used.This system consists of two plasmids derived from Ti plasmids, namely disarmed Ti plasmids and binary vectors (Bevan, 1984).The former contains most genes for T-DNA transfer from Agrobacterium tumefaciens to plants, whereas the latter is composed of a functional T-DNA and minimal elements for replication both in Escherichia coli and in A. tumefaciens.M os t of t h e wi de l y u s e d b i n a r y ve c t or s established in the 1990s were constructed by a traditional restriction endonuclease based method.Therefore, it was time consuming and laborious to construct modified genes on

Basic Ti-binary vector for Agrobacterium-mediated transformation and Gateway cloning
Transformation mediated by the soil bacterium A. tumefaciens is widely used for gene manipulation of plants.This bacterium has huge Ti-plasmids (larger than 200 kb) and the ability to transfer the T-DNA region of the Ti-plasmid to infect plant chromosomes.The natural Ti-mediated transformation system can be applied to transfer novel genes into a plant genome.To be useful for gene manipulation, binary vectors possessing the T-DNA region were developed.The vectors must possess a plant selection marker gene, a bacterial antibiotic resistance gene, a site for cloning foreign genes, T-DNA border sequences for gene transfer to the plant genome, an origin of replication (ori) for a broad host range of the plasmid and an ori for E. coli.Although binary vectors are much smaller than native Tiplasmids, they are still large and cause difficulties in gene cloning by traditional methods.Gateway Technology (available from Invitrogen) is based on the site-specific recombination system between phage lambda and E. coli DNA.This system was modified to improve its specificity and efficiency to utilize it as a universal cloning system.The advantages of Gateway cloning are as follows: it is free from the need for restriction endonucleases and DNA ligase, has a simple and uniform protocol, and offers highly efficient and reliable cloning and easy manipulation of fusion constructs.Therefore, the development of a variety of Gateway cloning compatible vectors for many purposes will expand the usefulness of this system in plant research.

Ti-binary vector for Agrobacterium-mediated plant transformation
A. tumefaciens harboring a Ti-plasmid can transfer a specific segment of the plasmid, the T-DNA region, which is bounded by a right border (RB) and a left border (LB) sequence, to the genome of an infected plant (Figure 1).Expression of the T-DNA genes causes the overproduction of phytohormones in the infected cells, which causes crown gall tumors.Although T-DNA genes are required for crown gall tumor formation, other genes called the vir genes outside of the T-DNA region are essential for transfer of T-DNA into the host plant genome.These vir genes work even when they reside on another plasmid in A. tumefaciens.Based on these findings, a Ti-binary vector system was developed to overcome the difficulty of manipulating the original Ti plasmids in vitro by recombinant DNA methods due to their huge size (Bevan, 1984).A wide range of shuttle vectors for E. coli and A. tumefaciens was constructed that contain T-DNA border sequences flanking multiple restriction sites for foreign DNA cloning and marker genes for selection in plant cells.Using this vector system, DNA manipulation and vector construction can be done in E. coli; the vector is then transferred to A. tumefaciens harboring an artificial Ti-plasmid in which the T-DNA has been deleted.The vector is maintained stably in A. tumefaciens, and the cloned foreign DNA and marker gene between RB and LB can be transferred to the host plant genome by the transformation system encoded by vir genes on the T-DNA deletion Ti-plasmid.In early studies, several dicot plants were transformed by an Agrobacterium method.However, various dicot and monocot plants can now be transformed by co-cultivation of leaf slices or cultured calli with chemicals inducing expression of vir genes.Transformed cells are selected by marker gene phenotype such as antibiotic resistance and regenerated to transgenic plants.The most important model plant, Arabidopsis thaliana, can be easily transformed by A. tumefaciens using a floral dip procedure.

Outline of Gateway cloning
Gateway cloning technology is based on the lambda phage infection system, in which sitespecific reversible recombination reactions occur during phage integration into and excision from E. coli genome (Figure 2).In this process, the attP site (242 bp) of lambda phage and the attB site (25 bp) of E. coli recombine (in a BP reaction) and the lambda phage genome is integrated into the E. coli genome.After the recombination reaction, the lambda phage genome is flanked by the attL (100 bp) and attR (168 bp) sites.In the reverse reaction, the www.intechopen.comGenetic Engineering -Basics, New Applications and Responsibilities 38 phage DNA is excised from the E. coli genome by recombination between the attL and attR sites (in an LR reaction).The BP reaction needs two proteins, the phage integrase (Int) and the E. coli integration host factor (IHF).The mixture of these two proteins is called BP clonase in the Gateway system.In the LR reaction, Int, IHF and one more phage protein, excisionase (Xis), are required, and this mixture is called LR clonase.The Gateway cloning method uses these att sites and clonases for construction of recombinant DNA in vitro.Fig. 2. BP and LR reactions in lambda phage infection of E. coli.The site-specific reversible BP and LR recombination reactions occur during lambda phage integration into and excision from the E. coli genome Basic strategies for application of Gateway technology to plasmid construction are shown in Figure 3.For the basic Gateway system, four pairs of modified att sites were generated for directional cloning.They are attB1 and attB2, attP1 and attP2, attL1 and attL2, and attR1 and attR2; a recombination reaction can occur only in the combinations of attB1 and attP1, attB2 and attP2, attL1 and attR1, or attL2 and attR2, since recombination strictly depends on att sequences (Hartley et al., 2000;Walhout et al., 2000).In addition to these att sites, the negative selection marker ccdB, the protein product of which inhibits DNA gyrase, and a chloramphenicol-resistance (Cm r ) marker are used for selection and maintenance of Gateway vectors.Usually, att1 is located at the 5' end of the open reading frame (ORF) and att2 is located at the 3' end.This orientation is maintained in all cloning steps.First, the gene of interest should be cloned in an entry vector by TOPO cloning (pENTR/ D-TOPO), a BP reaction (pDONR221), or restriction endonuclease and ligase (pENTR1A).Each vector is available from Invitrogen.To make an entry clone by a BP reaction, the attB1 and attB2 sequences are added to the 5' and 3' ends, respectively, of the ORF by adapter PCR.The product (attB1-ORF-attB2) is subjected to a BP reaction with a donor vector, pDONR221, which possesses an attP1-ccdB-Cm r -attP2 cassette.Because of the negative selection marker ccdB between attP1 and attP2, only transformants harboring the recombined vectors carrying attL1-ORF-attL2 (the entry clone) can grow on the selection plate.Once the entry clone is in hand, the ORF is transferred to a destination vector that possesses an attR1-Cm r -ccdB-attR2 cassette.Since destination vectors also contain ccdB between attR1 and attR2, and have a selection marker gene that is different from the entry clone, only the recombined destination vectors carrying attB1-ORF-attB2 will be selected.Gateway cloning is designed so that the smallest att sequence, attB (25 bp), appears in the final product to minimize the length of cloning junctions after the clonase reaction.In N-or C-terminal fusion constructs, the ORF is linked to a tag with eight or more amino acids encoded by the attB1 or attB2 sites.Because the reading frame of attB1 and attB2 is unified in the Gateway system, any entry clone incorporated into a destination vector is correctly fused to the tag sequence.Fig. 3. Schematic illustration of Gateway cloning.An entry clone is constructed by TOPO directional cloning, a BP reaction or restriction digestion and ligation.For construction using the BP reaction, the ORF region is amplified by adapter PCR and the resulting attB1-ORF-attB2 fragment is cloned into pDONR221 by a BP reaction to generate an entry clone containing attL1-ORF-attL2.Subsequently, the ORF is cloned into destination vectors by an LR reaction to generate expression clones including tagged fusion constructs.For D-TOPO cloning, CACC is added to the ORF by adapter PCR, and the resulting CACC-ORF fragment is cloned into pENTR/ D-TOPO.B1, attB1; B2, attB2; P1, attP1; P2, attP2; L1, attL1; L2, attL2; R1, attR1; R2, attR2; Pro, promoter; Ter, terminator; Cm r , chloramphenicol resistance marker; ccdB, negative selection marker in E. coli.; Km r , kanamycin-resistance marker

Binary vectors compatible with Gateway cloning
A large number of binary vectors compatible with Gateway cloning, known as destination vectors, have been developed and are summarized in a review (Karimi et al., 2007b).Gateway compatible binary vectors for promoter analysis have the general structure attR1-Cm r -ccdB-attR2-tag-terminator, and after an LR reaction with an attL1-promoter-attL2 entry clone, they yield an attB1-promoter-attB2-tag-terminator binary construct.Gateway compatible binary vectors for expression of tagged fusion proteins have the general structure promoter-tag-attR1-Cm r -ccdB-attR2-terminator (for N-terminal fusions) or promoter-attR1-Cm r -ccdB-attR2-tag-terminator (for C-terminal fusions).After an LR reaction with an attL1-ORF-attL2 entry clone, they respectively yield promoter-tag-attB1-ORF-attB2terminator or promoter-attB1-ORF-attB2-tag-terminator.The tag added to the N-terminus of the ORF is linked by the peptide encoded by the attB1 sequence (XSLYKKAGX), and the tag added to the C-terminus is linked by the peptide encoded by the attB2 sequence (XPAFLYKVX).Gateway compatible binary vectors for RNAi analysis (Helliwell & Waterhouse, 2003;Hilson et al., 2004;Karimi et al., 2002;Miki & Shimamoto, 2004) generally have the inverted structure of cassettes: promoter-attR1-ccdB-attR2-linker-attR2-ccdB-attR1terminator.By an LR reaction with an attL1-trigger-attL2 entry clone, the trigger sequence is incorporated into both sites in opposite orientations, yielding a promoter-attB1-trigger-attB2-linker-attB2-(complementary trigger)-attB1-terminator construct.When the construct is introduced into plants, hairpin RNA is expressed and processed into small interfering RNA that functions in gene silencing.
Among many Gateway compatible binary vector series, the pW (Karimi et al., 2002), pMDC (Brand et al., 2006;Curtis & Grossniklaus, 2003) and pEarleyGate (Earley et al., 2006) series contain vectors available for many kinds of experiments in plants.The pW series consists of vectors for overexpression or antisense repression by the cauliflower mosaic virus 35S promoter (P 35S ), for promoter analysis using luciferase (LUC), β-glucuronidase (GUS), or GFP-GUS as reporters, and for construction of gene fusions with GFP, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) or red fluorescent protein (RFP).The pMDC series consists of vectors for cloning, for overexpression by P 35S , for inducible expression by heat shock or estrogen treatment, for promoter analysis using GFP-6xHis or GUS as reporter, and for gene fusions with GFP, GFP-6xHis, or GUS.The pEarleyGate is a BASTA ®resistance binary vector series consisting of vectors for overexpression by P 35S , for promoter analysis using HA, FLAG, Myc, or AcV5, and for gene fusions with YFP, HA, FLAG, Myc, AcV5, tandem affinity purification (TAP) tags, YFP-HA, or GFP-HA.
The vectors described above are useful tools; however, sometimes it is necessary to use a different series if an existing one does not have a vector of the required type.In order to carry out most experiments within the same series (having a unified backbone and a unified junction sequence), we constructed a comprehensive Gateway compatible binary vector system carrying many reporters and tags based on the same backbone, as mentioned in next section.

Development of Gateway binary vector (pGWB) series
To make Gateway compatible binary vectors efficiently, we first tried to establish a systematic method for construction of a vector series.For this purpose, we designed a construction method for introducing a tag sequence by blunt end ligation to save time and labor caused by restriction sites in the tag sequence.Based on this notion, platform vectors pUGW0 and pUGW2 (Nakagawa et al., 2007a) were made using pUC119 as the backbone.As described below, many Gateway binary vector (pGWB) series were constructed from intermediate plasmid pUGWs, which were made with pUGW0 or pUGW2.The characteristics and accession nos. of each pGWB are summarized in Information of Gaeway Binary Vectors (pGWBs) (http:/ / shimane-u.org/nakagawa/ gbv.htm).

Platform vectors pUGW0 and pUGW2 for construction of pGWB series
The platform vectors pUGW0 and pUGW2 include P 35S and the nopaline synthase terminator (Tnos), as shown in Figure 4.A pUGW0 was the starting vector for N-terminal fusions, with the structure HindIII-P 35S -XbaI-ATG-Aor51HI-attR1-Cm r -ccdB-attR2-SacI-Tnos.A tag (reporter or epitope tag) sequence amplified by blunt-end PCR was introduced into the Aor51HI site (blunt end) to yield HindIII-P 35S -XbaI-ATG-tag-attR1-Cm r -ccdB-attR2-SacI-Tnos.In the case of a small epitope tag, an oligonucleotide could be introduced directly into the Aor51HI site.Translation is initiated at the ATG just upstream of the Aor51HI site.pUGW2 was the starting vector for C-terminal fusions, with the structure HindIII-XbaI-HindIII-P 35S -XbaI-attR1-Cm r -ccdB-attR2-Aor51HI-SacI-Tnos.Tag sequences were introduced by the same method used for pUGW0.The P 35S region could be easily removed by digestion with XbaI followed by self-ligation for construction of promoter-less pUGWs.Because there is no need to digest the tag fragment with restriction enzymes to introduce it into the Aor51HI site of pUGW0 and pUGW2, any tag fragment can be cloned by the same method.With these simple procedures, a pUGW series containing a variety of tags was efficiently generated.They were sources of Gateway cassettes including tag sequences, and were used for construction of a Gateway binary vector (pGWB).Moreover, the pUGWs are Gateway compatible plant vectors useful for transient expression analysis after particle bombardment or protoplast transformation.Because of their small size and high copy number in E. coli, preparation and handling of pUGW plasmids are very easy.

The pGWB series (pGWBxx and pGWB2xx) based on the pBI plasmid
Initially, pGWB was constructed on the backbone of modified pBI carrying a nopaline synthase promoter (Pnos) driven neomycin phosphotransferase II (NPTII) and P 35S -driven hygromycin phosphotransferase (HPT), which confer kanamycin-resistance (Km r ) and hygromycin-resistance (Hyg r ), respectively, to plants (Mita et al., 1995).The initial pGWB series (pGWBxx) consists of 36 vectors designed for simple cloning of genes (pGWB1), for overexpression of ORF clones (pGWB2), and for fusion with a variety of tags (pGWB3 through pGWB45) as shown in the Complete List of pGWB (http:/ / shimaneu.org/nakagawa/ gbv.htm).GUS, TAP and LUC are available for C-fusion, and 10 other tags, sGFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP), are available for both N-and C-fusion.The promoter-less C-fusion vectors can be used for promoter analysis.By an LR reaction with a promoter entry clone, a binary construct of promoter:tag is created.The remaining N-and C-fusion vectors contain P 35S for constitutive expression.By an LR reaction with an ORF entry clone, binary constructs expressing tag-ORF or ORF-tag are easily obtained (Figure 5).With the pGWBs, promoter activity, detection of tagged proteins, and subcellular localization of proteins can be analyzed effectively (Nakagawa et al., 2007a).represents a variety of acceptor sites (R1-R2) described in the box.The pGWB series includes plasmids with no promoter and no tag, or with no promoter and a C-tag.These are used for expression controlled by a gene's own promoter.The pGWB plasmids also include the following types: a 35S promoter and no tag, a 35S promoter and a C-tag, and a 35S promoter and an N-tag.These are used for constitutive expression using the 35S promoter.After an LR reaction with the entry clone, the expression clones indicated in the right panel are obtained.The tag is fused via the attB sequence.B1, attB1; B2, attB2; L1, attL1; L2, attL2; R1, attR1; R2, attR2; Tnos, nopaline synthase terminator; M, selection marker for plant; Cm r , chloramphenicol-resistance marker; ccdB, negative selection marker in E. coli.; P 35S , 35S promoter We also constructed pGWBs carrying the Pnos:HPT:Tnos marker instead of P 35S :HPT:Tnos (pGWB1-45) to avoid a possible effect of the P 35S sequence on the expression pattern and strength of the cloned gene (Zheng et al., 2007).These vectors are named pGWB203, 204, 228 and 235, and their characters are shown at the bottom of the Complete List of pGWB (http:/ / shimane-u.org/nakagawa/ gbv.htm).In early experiments, when the phosphate transporter PHT1 promoter was used for promoter analysis in A. thaliana, GUS activity in plant extracts was 5-fold higher with pGWB3 than with pGWB203 (Nakagawa et al., 2007a).

R4L1 Gateway binary vector (R4L1pGWB) series (R4L1pGWB4xx and R4L1pGWB5xx) for promoter analysis
Due to establishment of the R4pGWB system, many kinds of attL4-promoter-attR1 entry clones were constructed and have been used as a resource for expression of ORFs in plants.We plan to also utilize these resources of attL4-promoter-attR1 entry clones for efficient promoter:tag experiments, and developed an R4L1 Gateway binary vector (R4L1pGWB) (Nakamura et al., 2009) containing attR4-Cm r -ccdB-attL1-tag-Tnos.By the simple bipartite LR reaction with attL4-promoter-attR1 and R4L1pGWB, an attB4-promoter-attB1-tag-Tnos construct used for promoter assays can be easily obtained in this system (Figure 8, right).The tags in R4L1pGWBs are G3GFP-GUS, GUS, LUC, EYFP, ECFP, G3GFP and TagRFP as shown in the Complete List of R4L1pGWB (http:/ / shimane-u.org/nakagawa/ gbv.htm).

Application of the pGWB system
Because Gateway cloning is efficient, precise, flexible and simple to use, its application will continue to grow in plant research.In this section, we briefly describe two recent advances in our pGWB system, a split reporter for interaction analysis and recycling cloning for multigene constructs.

Gateway vectors for bimolecular fluorescence complementation (BiFC) assay
BiFC is based on the reconstitution of a fluorescent signal when two interacting proteins or peptides, which are fused to either an N-or C-fragment of a split fluorescent protein, interact.Due to its relative technical simplicity and the ability to use fluorescence microscopes for observation, a growing number of publications describe the use of BiFC to analyze protein-protein interactions.In addition to monitoring protein-protein interactions, this method has expanded to wider application, such as multicolor B iF C to investigate protein complexes (Hu & Kerppola, 2003;Kodama & Wada, 2009;Lee et al., 2008;Waadt et al., 2008), detection in vivo (Bracha-Drori et al., 2004;Walter et al., 2004) and combined with bioluminescence resonance energy transfer (BRET; Chen et al., 2008;Gandia et al., 2008;Xu et al., 2007).To date, several BiFC vectors dedicated to plant research have been constructed.Among our efforts in development of Gateway technology, we have generated various destination vectors for BiFC assays.In this section, we introduce our Gateway technologybased BiFC vectors, and describe their application.

Detection of protein-protein interactions in plant cells by BiFC assay
The investigation of protein-protein interactions provides valuable information in cell biology.In addition to BiFC, several other techniques detect protein-protein interactions, such as co-immunoprecipitation assays (Co-IP), in vitro binding assays, the yeast two-hybrid system (Y2H; James et al., 1996), the mating-based split-ubiquitin system (mbSUS; Ludewig et al., 2003;Obrdlik et al., 2004), BRET (Chen et al., 2008;Xu et al., 2007), fluorescence resonance energy transfer (FRET; Day et al., 2001), fluorescence lifetime imaging microscopy (FLIM; Bastiaens & Squire, 1999) and fluorescence correlation spectroscopy (FCS; Hink et al., 2002).The imaging-based approaches such as BiFC and FRET have been utilized in plant research because they enable detection in plant cells, in contrast to Y2H and mbSUS, which are functional only in yeast cells, and because they do not require specific antibodies or purification of proteins, unlike Co-IP and in vitro binding assays.
The BiFC assay is one of the most convenient techniques among the image-based approaches.Although FRET and FLIM are useful and powerful techniques for detection of protein-protein interactions, FRET requires complicated analysis such as of acceptor bleaching and an exclusive device is necessary for FLIM.Although several considerations are required even for BiFC assays, special devices are not required for detection, and complicated analysis is not necessary after obtaining image data.In addition, the BiFC assay provides information on subcellular location of the interacting proteins.We used our Gateway vector construction system (Hino et al., 2011;Nakagawa et al., 2008;Nakagawa et al., 2007b) to make destination vectors for BiFC assays.Using these vectors, it is easy to make constructs for detection of protein-protein interactions.These Gateway vectors have worked well in plant cells (Goto et al., 2011;Hino et al., 2011;Singh et al., 2009).

Principles of the BiFC assay
In BiFC assays, a fluorescent reporter, such as CFP, GFP, YFP and RFP, is split into two nonfluorescent fragments, N-and C-fragments (Figure 9A,B).Two proteins or peptides, which are to be tested for interaction, are fused at the N-or C-terminus of each fragment.After expression of both fusion genes simultaneously, if an interaction occurs between the two proteins, the non-fluorescent fragments are reconstituted and behave as an unsplit fluorescent protein.Therefore, the detection of fluorescence means the target proteins interact (Figure 9A).
Once the interaction occurs, the reconstituted molecule does not dissociate into nonfluorescent fragments, leading to enhancement of fluorescence due to accumulation of reconstituted fluorescent proteins.
There are eight potential combinations to be tested for protein-protein interactions in a BiFC assay, taking into account which protein of the two partners tested is fused to the N-or Cterminal end of which N-or C-fragment (Figure 9C).However, improper fusion of a split fragment sometimes abolishes protein function and masks information on subcellular targeting.For example, the peroxisome targeting signal 2 (PTS2) must be fused to the Nterminus of the split fluorescent protein (Singh et al., 2009;Figure 10B).In contrast, PTS1 must be fused to the C-terminus of a split fluorescent protein, because its location at the Cterminus is necessary for its function.In these cases, the number of combinations tested is fewer.However, if there is no information on protein function, all combinations should be tested.Viewed in this light, our destination vectors are useful for construction of several fusion genes at the same time.

Destination vectors for the multicolor and in vivo BiFC assays
Various BiFC vectors have been developed and used in plant research (Bracha-Drori et al., 2004;Diaz et al., 2005;Ding et al., 2006;Goto et al., 2011;Hino et al., 2011;Loyter et al., 2005;Maple et al., 2005;Marrocco et al., 2006;Ohad et al., 2007;Singh et al., 2009;Waadt et al., 2008;Walter et al., 2004;Zamyatnin et al., 2006).All the vectors, including ours, use P 35S to  (Hu & Kerppola, 2003;Waadt et al., 2008), the CFP, GFP and YFP in our system were split between residues 174 and 175, and mRFP1, which contains an amino acid substitution of the 66th glutamine to threonine, was split between residues 154 and 155.Amino acids in CFP and YFP that were converted from GFP are depicted in white.In the case of RFP, amino acids that are different from GFP are not represented, since there are many substitutions.(C) Potential combination of two fragments.There are eight possible configurations in the BiFC assay.Each target protein (gray and black) can be fused at its N-or C-terminus to the N-or C-terminal fragment of the fluorescent protein (light green) express a fusion gene.There are two ways to insert a target gene into the 5' or 3' end of a split fragment of fluorescent protein gene: (1) cloning into a multicloning site using digestion and ligation, and (2) Gateway technology (Hino et al., 2011;Walter et al., 2004).Our BiFC vectors were developed to be compatible with Gateway technology.We generated four kinds of destination vectors for BiFC assays (Figure 10A), enabling the transfer of a gene of interest from the entry clone to the 5' or 3' end of each split fragment.Therefore, researchers are able to easily fuse a gene of interest to the 5' or 3' end of the split fragment, leading to various convenient constructs.
The BiFC vectors were initially generated using YFP (Hu et al., 2002).However, other fluorescent proteins, BFP (Hu & Kerppola, 2003), CFP (Kodama & Wada, 2009;Lee et al., 2008), GFP (Hu et al., 2002;Kodama & Wada, 2009), Venus, (Lee et al., 2008), Cerulean (Lee et al., 2008), DsRed-monomer (Kodama & Wada, 2009), mRFP1 (Jach et al., 2006), mCherry (Fan et al., 2008), and a far-red fluorescent protein, mLumin (Chu et al., 2009), have reportedly been useful for BiFC assay.We adopted CFP, GFP, YFP and mRFP1 to generate vectors (Figure 9B), and verified their usefulness for detection of protein-protein interactions www.intechopen.comGateway Vectors for Plant Genetic Engineering: Overview of Plant Vectors, Application for Bimolecular Fluorescence Complementation (BiFC) and Multigene Construction 49 (Figure 10B-E).PTS2-containing proteins are directed to peroxisomes after binding to a receptor, PEX7, in the cytosol (Hayashi & Nishimura, 2006;Mano & Nishimura, 2005).We were able to observe reconstituted CFP fluorescence as punctate structures, when allowing interaction of nCFP-PEX7 and PTS2-cCFP (Figure 10B), which agrees with a previous report (Singh et al., 2009).Lesion simulating disease 1 (LSD1), a negative regulator of programmed cell death, is a zinc finger protein that forms homodimers.We also tried to detect LSD1 homooligomerization using the combination of LSD1-nYFP and LSD1-cYFP.Reconstituted YFP signals were observed in the cytosol and nucleus (Figure 10C), a result that coincided with previous data (Walter et al., 2004).The localization and interaction of one of the plasma membrane intrinsic proteins, PIP2, which belongs to the aquaporin family, with other PIP members were demonstrated by FRET and FLIM assays in maize cells (Zelazny et al., 2007).An Arabidopsis PIP2 gene, PIP2;1, was also fused to split fragments of mRFP1 and used for investigation of homooligomerization (Figure 10D).We were able to detect reconstituted RFP signals at the plasma membrane.(Hu & Kerppola, 2003;Kodama & Wada, 2009;Lee et al., 2008;Waadt et al., 2008).We also investigated which combinations among different fragments in our BiFC vectors are practical for reconstitution of signals using nXFP-PEX7 and PTS2-cXFP (XFP means CFP, GFP, YFP or RFP).Combinations among split fragments of CFP, GFP and YFP enabled the reconstitution of fluorescence (Table 1, Figure 10E), although some combinations did not give reproducible results.In contrast, a reconstituted RFP signal was observed only between split fragments from RFP (Table 1 10A) to binary vectors for in vivo BiFC assays (Figure 11A).Using these binary vectors, researchers are able to easily generate transgenic plants expressing a fusion gene of the N-or C-fragment with a gene of interest.We prepared two kinds of binary vectors, containing either Km r or Hyg r markers.Therefore, after crossing transgenic plants expressing either the N-or C-fragment, it will be easier to obtain transgenic plants expressing both N-or C-fragments from screening on medium with both kanamycin and hygromycin.
Agroinfiltration is a powerful technique to express an alien gene in planta, and it has been reported that this technique is functional in BiFC assays (Bracha-Drori et al., 2004;Waadt et al., 2008;Walter et al., 2004).We examined whether our binary vectors could also work well in agroinfiltration using nYFP-Peroxin 6 (PEX6) and cYFP-ABERRANT PEROXISOME MORPHOLOGY 9 (APEM9) (Figure 11B).We already reported the interaction of PEX6 and APEM9 using transient expression of these fusion genes in onion epidermal cells (Goto et al., 2011).We mixed three cultures of A. tumefaciens (strain C58C1Rif R ) haboring nYFP-PEX6, cYFP-APEM9 or CFP-PTS1 as peroxisomal markers, and then co-infiltarted into the leaf cells of Nicotiana tobaccum.Reconstituted YFP signal was observed as punctate structures (Figure 11C), and these signals surrounded the CFP-labeled peroxisome matrix (Figure 11C-E), showing that BiFC occurs at the peroxisomal membrane, as reported previously (Goto et al., 2011).These results demonstrated that our binary vectors for BiFC assays work well.

Special considerations for BiFC assays using our vectors
In BiFC assays, fluorescence is derived from reconstituted fluorescence or artificial noise.The same is true for our B iF C vectors.F luorescence is sometimes observed even in combination with an untagged vector as a negative control.Therefore, it is necessary to test expression using a negative control vector.Conversely, when fluorescence is not observed after expression of two fusion genes, there are two views about the result.One is that the interaction does not occur, although the two fusion genes are expressed properly.The other is that gene expression is inefficient or that the genes were inefficiently introduced into the We always express an additional gene, such as CFP-PTS1 in Figure 11, to investigate the efficiency of gene expression in transient assays.At the same time, this helps visualize cells and organelles so that it is easier to observe introduced cells that are bombarded or agro-infiltrated.The alternative method is the detection of fusion protein by immunoblotting.Some vectors are developed to add the epitope tag to split fragments so that the detection of accumulation of fusion proteins is carried out by immunoblotting (Bracha-Drori et al., 2004;Waadt et al., 2008;Walter et al., 2004).Of course, if specific antibodies against target protein are possible to obtain, they are useful for verification of protein accumulation.

Perspectives
Our BiFC vectors have wide application to analysis of protein-protein interactions.Future introduction of the R4pGWB system (Nakagawa et al., 2008) to these BiFC vectors will allow regulation of each fusion gene under a specific promoter, leading to examination of the interaction with tissue or developmental stage specificity.Additionally, inducible promoters will be used for transient expression in transgenic plants harboring R4pGWB-based BiFC fragments.Since a great variety of fluorescent proteins with different properties, such as large Stokes' shift, are available, more various combinations for the multicolour BiFC assay will be generated by adopting our Gateway technology system to new fluorescent proteins, revealing the relationship among several factors in a complex.

Recycling cloning system for multigene constructs
Multigene transformation of plants is a powerful technology for molecular breeding because it can simultaneously improve multiple enzymes and factors constituting biological pathways (Ha et al., 2010;Nakayama et al., 2000;Naqvi et al., 2009;Ye et al., 2000).For multigene transformation, methods such as re-transformation, co-transformation, and crossfertilization are available (Dafny-Yelin & Tzfira, 2007), but the most practical method is the utilization of a multigene construct, a vector carrying multiple expression units (Chen et al., 2006).In this section, we introduce a recycling cloning system for cloning multiple expression units by simple repetitive LR reactions.The gene of interest is cloned into the MCS of pRED (gene/ pRED) and subjected to an LR reaction with the destination vector containing R1-R2 acceptor sites.In this step, a binary construct carrying gene-attR4-RCS-attR3 is obtained.Next, conversion vector pCON, containing attL4-attR1-RCS-attR2-attL3, is subjected to an LR reaction to introduce the attR1-RCS-attR2 acceptor site into the resulting binary construct, and the binary construct obtained, which carries attR1-RCS-attR2, is recycled for the next round of the cloning cycle, together with another gene/ pRED clone (Figure 12).Before the LR reactions, binary constructs are digested by a rare cutter to suppress colonies derived from non-recombinants.With these simple repetitive reactions, genes are introduced sequentially into one vector.Using this recycling cloning system, we made a multigene construct containing four expression units of reporter genes and confirmed expression of all four reporters in transformed tobacco BY-2 cells (Kimura, unpublished results).

Conclusions
Gateway cloning is an efficient, reliable, easy and flexible technology, so many types of vectors have been developed and used worldwide.Our pGWBs series consists of many vectors with a variety of tags and four resistance markers.They are constructed on the same vector backbone and provide unified experimental conditions in transgenic research.
Because the introduction of a tag sequence into pUGW is very easy (Figure 4), the number of vectors for fusion with new tags is growing in our Gateway vector system.Among them, vectors for fusion with split fluorescent proteins are very important tools for BiFC assays.Our Gateway technology-based BiFC vectors are useful when several fusion genes must be generated for detection of protein-protein interactions among several factors in transient or in vivo assays.Introduction of the R4pGWB system (Nakagawa et al., 2008) to these BiFC vectors will lead to wider applications.Recycling cloning has the potential to introduce many expression units in high efficiency and will open a new way for genetic engineering of plants.

Distribution and information updates
All vectors described in this chapter are available for non-commercial research purposes, although the permission of original developers is required for some tags.The e-mail addresses for requesting the vectors are mano@nibb.ac.jp (for distribution of BiFC vectors) and tnakagaw@life.shimane-u.ac.jp (for distribution of other pGWBs).

Fig. 1 .
Fig. 1.Ti-binary vector system for Agrobacterium-mediated plant transformation.A binary vector, in which a target gene and plant selection marker gene are cloned between the two border sequences (RB and LB), is transformed into A. tumefaciens harboring a disarmed Ti-plasmid without the T-DNA region.Plant cells are infected by the transformed A. tumefaciens and then the target gene and marker gene are transferred into a plant chromosome by the vir genes on Ti-plasmid

Fig. 4 .
Fig. 4. Procedure for construction of pUGWs.pUGW0 and pUGW2 are the starting vectors for construction of new pUGW derivatives.The tag sequence amplified by blunt-end PCR is introduced into the Aor51HI site of pUGW0 or pUGW2, which yields pUGWs for N-fusion or C-fusion.The region between P 35S and Tnos is indicated.The nucleotide sequence corresponding to the region from attR1 to attR2 is underlined.Cm r , chloramphenicol resistance marker; ccdB, negative selection marker in E. coli.; P 35S , 35S promoter

Fig. 5 .
Fig. 5. Cloning into pGWB by LR reaction.The Gateway region in pGWB (top of the figure)represents a variety of acceptor sites (R1-R2) described in the box.The pGWB series includes plasmids with no promoter and no tag, or with no promoter and a C-tag.These are used for expression controlled by a gene's own promoter.The pGWB plasmids also include the following types: a 35S promoter and no tag, a 35S promoter and a C-tag, and a 35S promoter and an N-tag.These are used for constitutive expression using the 35S promoter.After an LR reaction with the entry clone, the expression clones indicated in the right panel are obtained.The tag is fused via the attB sequence.B1, attB1; B2, attB2; L1, attL1; L2, attL2; R1, attR1; R2, attR2; Tnos, nopaline synthase terminator; M, selection marker for plant; Cm r , chloramphenicol-resistance marker; ccdB, negative selection marker in E. coli.; P 35S , 35S promoter

Fig. 9 .
Fig. 9. Principles of the BiFC assay.(A) Nonfluorescent fragments (YN and YC) of a fluorescent protein are brought together through interaction of the tested proteins or peptides (a, b and c) to which they are fused.The interaction of the two proteins causes reconstitution of a fluorescent signal.(B) Diagram of amino acid substitutions among CFP, GFP, YFP and mRFP1, and the positions where they were fragmented.Although there are alternative positions to split a fluorescent protein into two fragments(Hu & Kerppola, 2003;Waadt et al., 2008), the CFP, GFP and YFP in our system were split between residues 174 and 175, and mRFP1, which contains an amino acid substitution of the 66th glutamine to threonine, was split between residues 154 and 155.Amino acids in CFP and YFP that were converted from GFP are depicted in white.In the case of RFP, amino acids that are different from GFP are not represented, since there are many substitutions.(C) Potential combination of two fragments.There are eight possible configurations in the BiFC assay.Each target protein (gray and black) can be fused at its N-or C-terminus to the N-or C-terminal fragment of the fluorescent protein (light green)

Fig. 10 .
Fig. 10.Schematic representation of the multicolor BiFC vectors and examples of transient expression.(A) Four kinds of destination vectors for transient expression were generated to be compatible with Gateway technology.nXFP and cXFP, the N-or C-fragment, respectively, of CFP, GFP, YFP or RFP; ColE1 ori, ColE1 replication origin; Amp r , ampicillinresistance marker used for selection in bacteria; Cm r , chloramphenicol-resistance marker; ccdB, negative selection marker used in bacteria; P 35S , 35S promoter; Tnos, nopaline synthase terminator; R1, attR1; R2, attR2.(B-E) Fluorescence images of onion epidermal cells expressing the fusion genes indicated above each panel were acquired 18-24 hr after particle bombardment.Bars = 50 µm Multicolor BiFC assays have been developed to examine protein-protein interactions among various factors, since some combinations of N-and C-fragments of different fluorescent proteins allow reconstitution of signals(Hu & Kerppola, 2003;Kodama & Wada, 2009;Lee et al., 2008;Waadt et al., 2008).We also investigated which combinations among different fragments in our BiFC vectors are practical for reconstitution of signals using nXFP-PEX7

Fig. 11 .
Fig. 11.Schematic representation of the binary vectors for the BiFC assay and examples of an in vivo BiFC experiment using an Agrobacterium-infiltration technique.(A) Four kinds of destination vectors for an in vivo BiFC assay were generated to be compatible with Gateway technology.nXFP and cXFP, the N-or C-fragment, respectively, of CFP, GFP, YFP or RFP; cXFP; sta, region conferring stability in Agrobacterium; rep, broad host-range replication origin; bom, cis-acting element for conjugational transfer; ori, ColE1 replication origin; Cm r , chloramphenicol-resistance marker; ccdB, negative selection marker used in bacteria; P 35S , 35S promoter; Tnos, nopaline synthase terminator; R1, attR1; R2, attR2; Black arrowheads indicate right border and left border.(B-E) An example of an in vivo BiFC experiment.(B) Three fusion genes, nYFP-PEX6, cYFP-APEM9 and CFP-PTS1 as peroxisome markers were expressed in Nicotiana tobaccum.(C-E) Fluorescence images of leaf epidermal cells were acquired 3 days after infiltration.(C) Reconstituted YFP signals.(D) Peroxisomes visualized with CFP.(E) A merged image of (C) with (D).Insets represent magnified images of a peroxisome.Bars = 20 µm and 1 µm for each inset cells.We always express an additional gene, such as CFP-PTS1 in Figure11, to investigate the efficiency of gene expression in transient assays.At the same time, this helps visualize cells and organelles so that it is easier to observe introduced cells that are bombarded or agro-infiltrated.The alternative method is the detection of fusion protein by immunoblotting.Some vectors are developed to add the epitope tag to split fragments so that the detection of accumulation of fusion proteins is carried out by immunoblotting(Bracha-Drori et al., 2004;Waadt et al., 2008;Walter et al., 2004).Of course, if specific antibodies against target protein are possible to obtain, they are useful for verification of protein accumulation.

Fig. 12 .
Fig. 12. Schematic illustration of recycling cloning.The pRED vector has the structure L1-MCS-R4-RCS-R3-L2.The gene of interest is cloned into the MCS of pRED419 and subsequently subjected to an LR reaction with a destination vector.In this step, the DNA fragment of gene-R4-RCS-R3 is incorporated into the destination vector and a binary clone carrying B1-gene-R4-RCS-R3-B2 is obtained.Next, the resulting binary clone is subjected to an LR reaction with pCON to introduce the R1-RCS-R2 sequence into the binary clone.The binary clone carrying B1-gene-B4-R1-RCS-R2-B3-B2 is recycled for introduction of another gene by LR reaction with another gene/ pRED in a second cycle.The marker gene (M) is transcribed in the opposite orientation to the cloned gene.B1, attB1;B2, attB2; B3, attB3; B4,  attB4; L1, attL1; L2, attL2; L3, attL3; L4, attL4; R1, attR1; R2, attR2; R3, attR3; R4, attR4; M, selection marker for plant; Cm r , chloramphenicol-resistance marker; ccdB, negative selection marker in E. coli.; MCS, multiple cloning site; RCS, rare cutter site As shown in the right panel of Figure12, two vectors are used for each cloning cycle in this system.The recycle donor vector pRED has four att sites, a multiple cloning site (MCS) and a rare cutter site (RCS) in the following order: attL1-MCS-attR4-RCS-attR3-attL2.The RCS

Table 1 .
). Summary of the detection of reconstituted signals using various combinations of split fragments from different fluorescent proteins.cC, cG, cY and cR represent the C-fragment of CFP, GFP, YFP and RFP, respectively.nC, nG, nY and nR indicate the N-fragment of CFP, GFP, YFP and RFP, respectively.'+' and '-' denote detection of interaction and inability for interaction, respectively.''±'' indicates that reproducible results could not be obtained We adapted our BiFC vectors for transient expression (Figure