- Email: [email protected]
Modular cloning in plant cells
Update
TRENDS in Plant Science
Vol.10 No.3 March 2005
Research Focus
Modular cloning in plant cells Mansour Karimi, Bjo¨rn De Meyer and Pierre Hilson Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent, Belgium
New plant genes are being discovered at a rapid pace. Yet, in most cases, their precise function remains elusive. The recent advent of recombinational cloning techniques has significantly improved our ability to investigate gene functions systematically. For example, proteins fused with diverse fluorescent tags can be expressed at will using versatile cloning cassettes. In addition, novel binary T-DNA vectors are now available to assemble multiple DNA fragments simultaneously, which greatly facilitate plant cell and protein engineering. Introduction Understanding how genes, together with the other elements that make living cells, act collectively as plants develop and propagate in their environment is a major challenge. Gene functional analyses require the isolation and assembly of various DNA fragments extracted from the genome (e.g. open reading frames and promoters). Ideally, these should be available for all known genes and formatted in a way that permits their modular assembly [1]. Simultaneous cloning of multiple fragments With recombinational cloning (RC) protocols, DNA fragments are transferred between plasmids regardless of their sequence [2,3] and recombinant DNA constructs can be generated reliably on a large scale [4,5]. This innovation has also facilitated the use of large backbone plasmids such as Agrobacterium tumefaciens binary T-DNA vectors designed for the transformation of plant cells [6–9]. Recently, the Gatewaye technology (Invitrogen; http:// www.invitrogen.com/) has been extended with the addition of novel recombination sites with unique specificities to assemble multiple DNA segments in a single highly efficient and specific in vitro LR clonasee reaction. The segments are cloned in an expression vector in a predefined order, orientation and translational reading frame [10,11]. Taking advantage of these improvements, plant binary destination vectors have been created with RC Multisite Gatewaye cassettes (Figure 1a), in which two or three segments can be transferred from source entry clones and pasted contiguously in a single step (Figure 2). Furthermore, collections of functional elements built as Gatewaye entry clones, such as promoters [12,13], open reading frames [14] and/or diverse tags, can be combined at will in the Multisite Gatewaye cassettes, simplifying the design and construction of complex recombinant DNA molecules [10,11]. Corresponding author: Hilson, P. ([email protected]).
Diverse fluorescent protein tags The localization of a protein is key to its function and the expression of translational fusions with the green fluorescent protein (GFP) is routinely used to determine protein distribution in subcellular compartments. Plant binary Gatewaye destination vectors are available for that purpose [6,7]. Yet, for specific applications, such as co-localization or fluorescence resonance energy transfer, different proteins need to be distinguished and must be tagged with fluorescent markers that emit light at distinct wavelengths. Therefore, a series of plant destination vectors has been created for the expression of cyan, green, yellow and red fluorescent proteins (CFP, GFP, YFP and RFP [15], respectively). They were designed so that any of the four tags could be expressed with a protein of interest fused at its amino (N) or carboxyl (C) terminus (Figure 1b). All constructs depicted schematically in Figure 1 carry one of three plant selectable markers coding for resistance to kanamycin (nptII), hygromycin (hpt) or glufosinate ammonium (bar). All RC expression cassettes are also (a)
LB
R4
R2 RB
p*m42WG LB
R4
R3 RB
p*m43WG R2
LB
R4 RB
p*7m24WG R3
LB
R4 RB
p*7m34WG (b)
R2
LB
R1
RB
p*7XWG2 R2
LB
R1
RB
p*7WGX2 T-DNA borders
attR sites
Plant selectable marker
ccdB
nos terminator
CmR
nos promoter
35S terminator
35S promoter
Egfp, Ecfp, Eyfp, rfp TRENDS in Plant Science
Figure 1. Plant binary vectors designed for recombinational cloning. (a) T-DNA containing a plant selectable marker gene and a Multisite Gatewaye cassette compatible with the simultaneous cloning of two (R2–R4) or three (R3–R4) ordered contiguous DNA fragments. Both cassette types are available with or without an adjacent 35S terminator. The order of the R sites determines the orientation of the fragments transferred in the final LR clonase product. (b) T-DNA containing a plant selectable marker gene and a Gatewaye cassette for the expression of translational fusion with one of four fluorescent tags. The vector series enables expression of each tag either at the N- or C-terminus of a protein of interest.
www.sciencedirect.com 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.01.008
104
Update
(a)
TRENDS in Plant Science
Vol.10 No.3 March 2005
Two-fragment recombination BP reactions
B4
B1
B1 Fragment 1
ccdB
ccdB
P1 pDONR221 Km
pDONR-P4P1R P1R Km
P4
Fragment 1 L4
B2 Fragment 2
pENTR1
P2
Fragment 2 R1
L1
L2
pENTR2 Km
Km
LR reaction
attB ccdB
R4
R2
p*m42WG, p*7m24WG Sp
attP attL
B2
B1
B4 Fragment 1
attR
Fragment 2
BP recombination LR recombination
Expression clone Sp
(b)
Three-fragment recombination BP reactions
B4
B1
B1
Fragment 1
B2
B2 Fragment 2
B3 Fragment 3
ccdB
ccdB
ccdB
P4 pDONR-P4P1R P1R Km
P1
pDONR221 Km
Km pENTR2
L1
P2R pDONR-P2RP3 Km
P2
P3
L2
Fragment 2
Fragment Fragment 33
Fragment 1
LR reaction
L4
R2
R1
pENTR1 Km
pENTR3 Km
L3
ccdB
R4
R3
p*m43WG, p*7m34WG Sp
Fragment 1
B3
B2
B1
B4
Fragment 2
Fragment 3
Expression clone Sp
TRENDS in Plant Science
Figure 2. Multisite Gatewaye recombinational cloning strategy. In both the two- (a) and three-fragment (b) schemes, each of the entry clones is produced in vitro in a BP clonasee reaction that transfers a PCR amplicon or plasmid segment flanked by the appropriate attB sites in one of the three compatible donor vectors (pDONR P4-P1R, pDONR 221 or pDONR P2R-P3; http://www.invitrogen.com/). Subsequently, the two or three fragments are assembled in vitro in a single Multisite LR clonasee reaction, by transfer from the two or three entry plasmids in the destination vector to form the expression clone. The products of the BP or LR reactions are introduced into Escherichia coli cells and the entry or expression vectors are selected in bacteria grown on kanamycin (Km) or spectinomycin (Sp) medium, respectively.
available in high-copy number plasmids. This collection of vectors is a useful resource for the plant research community and can be obtained on line (http://www.psb. ugent.be/gateway). The website also includes other accessions and provides RC instructions, as well as experimentally verified sequences, maps and Vector NTI files (Invitrogen) for each vector. Acknowledgements We thank Kris Van Poucke, Rudy Vanderhaeghen and Danny Geelen for their assistance in testing the described resources, and Martine De Cock for help in preparing the manuscript. www.sciencedirect.com
References 1 Brasch, M.A. et al. (2004) ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list. Genome Res. 14, 2001–2009 2 Hartley, J.L. et al. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 3 Liu, Q. et al. (2000) Rapid construction of recombinant DNA by the univector plasmid-fusion system. Methods Enzymol. 328, 530–549 4 Walhout, A.J.M. et al. (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287, 116–122 5 Hilson, P. et al. (2004) Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. Genome Res. 14, 2176–2189
Update
TRENDS in Plant Science
6 Karimi, M. et al. (2002) GATEWAYe vectors for Agrobacteriummediated plant transformation. Trends Plant Sci. 7, 193–195 7 Curtis, M.D. and Grossniklaus, U. (2003) A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 8 Guo, H. and Ecker, J.R. (2003) Plant responses to ethylene gas are mediated by SCFEBF1/EBF2-dependent proteolysis of EIN3 transcription factor. Cell 115, 667–677 9 Helliwell, C. and Waterhouse, P. (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30, 289–295 10 Sasaki, Y. et al. (2004) Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system. J. Biotechnol. 107, 233–243
Vol.10 No.3 March 2005
11 Cheo, D.L. et al. (2004) Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome Res. 14, 2111–2120 12 Hilson, P. et al. (2003) European consortia building integrated resources for Arabidopsis functional genomics. Curr. Opin. Plant Biol. 6, 426–429 13 Dupuy, D. et al. (2004) A first version of the Caenorhabditis elegans promoterome. Genome Res. 14, 2169–2175 14 Gong, W. et al. (2004) Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol. 135, 773–782 15 Van Damme, D. et al. (2004) In vivo dynamics and differential microtubule-binding activities of MAP65 proteins. Plant Physiol. 136, 3956–3967
Gordon Conference on Photosynthesis 3–8 July 2005 Bryant College, Smithfield, RI, USA For more information see http://www.grc.org/programs/2005/photosyn.htm
Gordon Conference on Plant Metabolic Engineering 10–15 July 2005 Tilton School, Tilton, NH, USA For more information see http://www.grc.uri.edu/programs/2005/plant.htm
Annual Meeting ASPB 16 July–20 July 2005 Seattle, WA, USA For more information see http://www.aspb.org/meetings/pb-2005/index.cfm
12th International Congress on Molecular Plant–Microbe Interactions 17–22 July 2005 Cancu´n, Me´xico For more information see http://www.sodio.net/XIICONGRESS_IS-MPMI/
XVII International Botanical Congress 17–23 July 2005 Vienna, Austria For more information see http://www.ibc2005.ac.at/ www.sciencedirect.com
105
TRENDS in Plant Science
Vol.10 No.3 March 2005
Research Focus
Modular cloning in plant cells Mansour Karimi, Bjo¨rn De Meyer and Pierre Hilson Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent, Belgium
New plant genes are being discovered at a rapid pace. Yet, in most cases, their precise function remains elusive. The recent advent of recombinational cloning techniques has significantly improved our ability to investigate gene functions systematically. For example, proteins fused with diverse fluorescent tags can be expressed at will using versatile cloning cassettes. In addition, novel binary T-DNA vectors are now available to assemble multiple DNA fragments simultaneously, which greatly facilitate plant cell and protein engineering. Introduction Understanding how genes, together with the other elements that make living cells, act collectively as plants develop and propagate in their environment is a major challenge. Gene functional analyses require the isolation and assembly of various DNA fragments extracted from the genome (e.g. open reading frames and promoters). Ideally, these should be available for all known genes and formatted in a way that permits their modular assembly [1]. Simultaneous cloning of multiple fragments With recombinational cloning (RC) protocols, DNA fragments are transferred between plasmids regardless of their sequence [2,3] and recombinant DNA constructs can be generated reliably on a large scale [4,5]. This innovation has also facilitated the use of large backbone plasmids such as Agrobacterium tumefaciens binary T-DNA vectors designed for the transformation of plant cells [6–9]. Recently, the Gatewaye technology (Invitrogen; http:// www.invitrogen.com/) has been extended with the addition of novel recombination sites with unique specificities to assemble multiple DNA segments in a single highly efficient and specific in vitro LR clonasee reaction. The segments are cloned in an expression vector in a predefined order, orientation and translational reading frame [10,11]. Taking advantage of these improvements, plant binary destination vectors have been created with RC Multisite Gatewaye cassettes (Figure 1a), in which two or three segments can be transferred from source entry clones and pasted contiguously in a single step (Figure 2). Furthermore, collections of functional elements built as Gatewaye entry clones, such as promoters [12,13], open reading frames [14] and/or diverse tags, can be combined at will in the Multisite Gatewaye cassettes, simplifying the design and construction of complex recombinant DNA molecules [10,11]. Corresponding author: Hilson, P. ([email protected]).
Diverse fluorescent protein tags The localization of a protein is key to its function and the expression of translational fusions with the green fluorescent protein (GFP) is routinely used to determine protein distribution in subcellular compartments. Plant binary Gatewaye destination vectors are available for that purpose [6,7]. Yet, for specific applications, such as co-localization or fluorescence resonance energy transfer, different proteins need to be distinguished and must be tagged with fluorescent markers that emit light at distinct wavelengths. Therefore, a series of plant destination vectors has been created for the expression of cyan, green, yellow and red fluorescent proteins (CFP, GFP, YFP and RFP [15], respectively). They were designed so that any of the four tags could be expressed with a protein of interest fused at its amino (N) or carboxyl (C) terminus (Figure 1b). All constructs depicted schematically in Figure 1 carry one of three plant selectable markers coding for resistance to kanamycin (nptII), hygromycin (hpt) or glufosinate ammonium (bar). All RC expression cassettes are also (a)
LB
R4
R2 RB
p*m42WG LB
R4
R3 RB
p*m43WG R2
LB
R4 RB
p*7m24WG R3
LB
R4 RB
p*7m34WG (b)
R2
LB
R1
RB
p*7XWG2 R2
LB
R1
RB
p*7WGX2 T-DNA borders
attR sites
Plant selectable marker
ccdB
nos terminator
CmR
nos promoter
35S terminator
35S promoter
Egfp, Ecfp, Eyfp, rfp TRENDS in Plant Science
Figure 1. Plant binary vectors designed for recombinational cloning. (a) T-DNA containing a plant selectable marker gene and a Multisite Gatewaye cassette compatible with the simultaneous cloning of two (R2–R4) or three (R3–R4) ordered contiguous DNA fragments. Both cassette types are available with or without an adjacent 35S terminator. The order of the R sites determines the orientation of the fragments transferred in the final LR clonase product. (b) T-DNA containing a plant selectable marker gene and a Gatewaye cassette for the expression of translational fusion with one of four fluorescent tags. The vector series enables expression of each tag either at the N- or C-terminus of a protein of interest.
www.sciencedirect.com 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.01.008
104
Update
(a)
TRENDS in Plant Science
Vol.10 No.3 March 2005
Two-fragment recombination BP reactions
B4
B1
B1 Fragment 1
ccdB
ccdB
P1 pDONR221 Km
pDONR-P4P1R P1R Km
P4
Fragment 1 L4
B2 Fragment 2
pENTR1
P2
Fragment 2 R1
L1
L2
pENTR2 Km
Km
LR reaction
attB ccdB
R4
R2
p*m42WG, p*7m24WG Sp
attP attL
B2
B1
B4 Fragment 1
attR
Fragment 2
BP recombination LR recombination
Expression clone Sp
(b)
Three-fragment recombination BP reactions
B4
B1
B1
Fragment 1
B2
B2 Fragment 2
B3 Fragment 3
ccdB
ccdB
ccdB
P4 pDONR-P4P1R P1R Km
P1
pDONR221 Km
Km pENTR2
L1
P2R pDONR-P2RP3 Km
P2
P3
L2
Fragment 2
Fragment Fragment 33
Fragment 1
LR reaction
L4
R2
R1
pENTR1 Km
pENTR3 Km
L3
ccdB
R4
R3
p*m43WG, p*7m34WG Sp
Fragment 1
B3
B2
B1
B4
Fragment 2
Fragment 3
Expression clone Sp
TRENDS in Plant Science
Figure 2. Multisite Gatewaye recombinational cloning strategy. In both the two- (a) and three-fragment (b) schemes, each of the entry clones is produced in vitro in a BP clonasee reaction that transfers a PCR amplicon or plasmid segment flanked by the appropriate attB sites in one of the three compatible donor vectors (pDONR P4-P1R, pDONR 221 or pDONR P2R-P3; http://www.invitrogen.com/). Subsequently, the two or three fragments are assembled in vitro in a single Multisite LR clonasee reaction, by transfer from the two or three entry plasmids in the destination vector to form the expression clone. The products of the BP or LR reactions are introduced into Escherichia coli cells and the entry or expression vectors are selected in bacteria grown on kanamycin (Km) or spectinomycin (Sp) medium, respectively.
available in high-copy number plasmids. This collection of vectors is a useful resource for the plant research community and can be obtained on line (http://www.psb. ugent.be/gateway). The website also includes other accessions and provides RC instructions, as well as experimentally verified sequences, maps and Vector NTI files (Invitrogen) for each vector. Acknowledgements We thank Kris Van Poucke, Rudy Vanderhaeghen and Danny Geelen for their assistance in testing the described resources, and Martine De Cock for help in preparing the manuscript. www.sciencedirect.com
References 1 Brasch, M.A. et al. (2004) ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list. Genome Res. 14, 2001–2009 2 Hartley, J.L. et al. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 3 Liu, Q. et al. (2000) Rapid construction of recombinant DNA by the univector plasmid-fusion system. Methods Enzymol. 328, 530–549 4 Walhout, A.J.M. et al. (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287, 116–122 5 Hilson, P. et al. (2004) Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. Genome Res. 14, 2176–2189
Update
TRENDS in Plant Science
6 Karimi, M. et al. (2002) GATEWAYe vectors for Agrobacteriummediated plant transformation. Trends Plant Sci. 7, 193–195 7 Curtis, M.D. and Grossniklaus, U. (2003) A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 8 Guo, H. and Ecker, J.R. (2003) Plant responses to ethylene gas are mediated by SCFEBF1/EBF2-dependent proteolysis of EIN3 transcription factor. Cell 115, 667–677 9 Helliwell, C. and Waterhouse, P. (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30, 289–295 10 Sasaki, Y. et al. (2004) Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system. J. Biotechnol. 107, 233–243
Vol.10 No.3 March 2005
11 Cheo, D.L. et al. (2004) Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome Res. 14, 2111–2120 12 Hilson, P. et al. (2003) European consortia building integrated resources for Arabidopsis functional genomics. Curr. Opin. Plant Biol. 6, 426–429 13 Dupuy, D. et al. (2004) A first version of the Caenorhabditis elegans promoterome. Genome Res. 14, 2169–2175 14 Gong, W. et al. (2004) Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol. 135, 773–782 15 Van Damme, D. et al. (2004) In vivo dynamics and differential microtubule-binding activities of MAP65 proteins. Plant Physiol. 136, 3956–3967
Gordon Conference on Photosynthesis 3–8 July 2005 Bryant College, Smithfield, RI, USA For more information see http://www.grc.org/programs/2005/photosyn.htm
Gordon Conference on Plant Metabolic Engineering 10–15 July 2005 Tilton School, Tilton, NH, USA For more information see http://www.grc.uri.edu/programs/2005/plant.htm
Annual Meeting ASPB 16 July–20 July 2005 Seattle, WA, USA For more information see http://www.aspb.org/meetings/pb-2005/index.cfm
12th International Congress on Molecular Plant–Microbe Interactions 17–22 July 2005 Cancu´n, Me´xico For more information see http://www.sodio.net/XIICONGRESS_IS-MPMI/
XVII International Botanical Congress 17–23 July 2005 Vienna, Austria For more information see http://www.ibc2005.ac.at/ www.sciencedirect.com
105