GATEWAY vectors for Agrobacterium-mediated plant transformation

Research Update
TRENDS in Plant Science Vol.7 No.5 May 2002
193
What’s next?
Although, the functional question raised
by Levings and Siedow [1] remains
unanswered, knowledge of thioredoxin o
and the associated NTR enriches the
field and opens the door to the
identification of new regulatory events
in plant mitochondria.
Acknowledgement
Y.B. gratefully acknowledges the support
of a fellowship from the Swiss National
Science Foundation.
References
1 Levings, C.S. and Siedow, J.N. (1995)
Regulation by redox poise in chloroplast. Science
268, 695–696
2 Laloi, C. et al. (2001) Identification and
characterization of a mitochondrial thioredoxin
system in plant. Proc. Natl. Acad. Sci. U. S. A.
98, 14144–14149
3 Buchanan, B.B. (1980) Role of light in the
regulation of chloroplast enzymes. Annu. Rev.
Plant Physiol. 31, 341–374
4 Schürmann, P. and Jacquot, J-P. (2000) Plant
thioredoxin system revisited. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 51, 371–400
5 Buchanan, B.B. et al. The ferredoxin/thioredoxin
system: from discovery to molecular structures
and beyond. Photosynth. Res. (in press)
6 Williams, C.H., Jr et al. (2000) Thioredoxin
reductase: two modes of catalysis have evolved.
Eur. J. Biochem. 267, 6110–6117
7 Arner, E.S.J. and Holmgren, A. (2000)
Physiological functions of thioredoxin and
thioredoxin reductase. Eur. J. Biochem.
267, 6102–6109
8 Fabianeck, R.A. et al. (1998) The active-site
cysteines of the periplasmic thioredoxin-like
protein CcmG of Escherichia coli are important
but not essential for cytochrome c maturation
in vivo. J. Bacteriol. 180, 1947–1950
9 Carmel-Harel, O. and Storz, G. (2000) Roles of
the glutathione- and thioredoxin-dependent
reduction systems in the Escherichia coli and
Saccharomyces cerevisiae response to oxidative
stress. Annu. Rev. Microbiol. 54, 439–461
10 Pedrajas, J.R. et al. (1999) Identification and
functional characterization of a novel mitochondrial
thioredoxin system in Saccharomyces cerevisiae.
J. Biol. Chem. 274, 6366–6373
11 Miranda-Vizuete, A. et al. (2000) The
mitochondrial thioredoxin system.
Antioxid. Redox Signal. 2, 801–810
12 Rabilloud, T. et al. (2001) The mitochondrial
antioxidant defense system and its response to
oxidative stress. Proteomics 1, 1105–1110
13 Rouhier, N. et al. (2001) Isolation and
characterization of a new peroxiredoxin from
poplar sieve tubes that uses either glutaredoxin
or thioredoxin as proton donor. Plant Physiol.
127, 1299–1309
14 Cabrillac, D. et al. (2001) The S-locus receptor
kinase is inhibited by thioredoxins and activated
by pollen coat proteins. Nature 410, 220–223
15 Besse, I. and Buchanan, B.B. (1996) Thiocalsin:
a thioredoxin-linked substrate-specific protease
dependent on calcium. Proc. Natl. Acad. Sci.
U. S. A. 93, 3169–3175
16 Mestres-Ortega, D. and Meyer, Y. (1999)
The Arabidopsis thaliana genome encodes at
least four thioredoxins m and a new
prokaryotic-like thioredoxin. Gene 240,
307–331
17 Scheibe, R. (1991) Redox modulation of
chloroplast enzymes. A common principle for
individual control. Plant Physiol. 96, 1–2
18 Ruelland, E. and Miginiac-Maslow, M. (1999)
Regulation of chloroplast enzyme activities by
thioredoxins: activation or relief of inhibition?
Trends Plant Sci. 4, 136–141
19 Trebitsh, T. and Danon, A. (2001) Translation of
psbA mRNA is regulated by signals initiated by
both photosystems II and I. Proc. Natl. Acad. Sci.
U. S. A. 98, 12289–12294
20 Bodenstein-Lang, J. et al. (1989) Animal and
plant mitochondria contain specific thioredoxins.
FEBS Lett. 258, 22–26
21 Marcus, F. et al. (1991) Plant thioredoxin h: an
animal-like thioredoxin occurring in multiple
cell compartments. Arch. Biochem. Biophys. 287,
195–198
22 Hammel, K.E. et al. (1983)
Ferredoxin/flavoprotein-linked pathway for the
reduction of thioredoxin. Proc. Natl. Acad. Sci.
U. S. A. 80, 3681–3689
Yves Balmer
Bob B. Buchanan*
Dept of Plant and Microbial Biology,
University of California, Berkeley, CA 94720,
USA.
*e-mail: view@nature.berkeley.edu
Techniques & Applications
GATEWAY vectors for Agrobacterium-mediated plant
transformation
Mansour Karimi, Dirk Inzé and Ann Depicker
Agrobacterium tumefaciens is the preferred
method for transformation of a wide range
of plant species. Commonly, the genes to be
transferred are cloned between the left and
right T-DNA borders of so-called binary
T-DNA vectors that can replicate both in
E. coli and Agrobacterium. Because these
vectors are generally large, cloning can be
time-consuming and laborious. Recently,
the GATEWAY conversion technology
has provided a fast and reliable alternative
to the cloning of sequences into large
acceptor plasmids.
Published online: 11 April 2002
The GATEWAY conversion technology
(Invitrogen, Gaithersburg, MD, USA) is
based on the site-specific recombination
reaction mediated by phage λ. DNA
fragments flanked by recombination sites
(att) can be transferred into vectors that
contain compatible recombination sites
(attB × attP or attL × attR) in a reaction
mediated by the GATEWAY BP
Clonase or LR Clonase Enzyme Mix
(Invitrogen). The entry clones, which can
be considered general donor plasmids, are
made by recombining the DNA fragment
of interest with the flanking attB sites
into the attP site pDONR201 mediated
by the GATEWAY BP Clonase
Enzyme Mix. Subsequently, the fragment
in the entry clone can be transferred to
any destination vector that contains the
attR sites by mixing both plasmids and by
using the GATEWAY LR Clonase
Enzyme Mix.
Here we describe a set of GATEWAYcompatible
binary T-DNA destination
vectors for a wide range of different
applications (Fig. 1). Details can be found
on the web site (http://www.plantgenetics.
rug.ac.be/gateway/), which provides the
complete DNA sequence, a map, and a
Vector NTI view of all constructs. The web
site will be updated regularly by adding
new constructs and relevant information.
Backbone of GATEWAY-compatible vectors
The backbone of all described
GATEWAY-compatible vectors is the
plasmid pPZP200 [1]. This plasmid is
relatively small (6.7 kb), contains an
origin for replication in E. coli (ColE1) and
in Agrobacterium (pVS1), has a pBR322
bom site for mobilization from E. coli to
http://plants.trends.com
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(02)02251-3

194 Research Update
TRENDS in Plant Science Vol.7 No.5 May 2002
Overexpression or
antisense of a gene
Overexpression of
a gene together with
a visible marker
Double-stranded
RNA (post-transcriptional
gene silencing)
C-terminal gfp fusion
N-terminal gfp fusion
N, C-terminal gfp fusion
Promoter analysis
(gfp–gus fusion)
Transformation or
complementation with
genomic sequences
T-DNA borders
plant selectable markers
nos terminator
Agrobacterium, and possesses a
streptomycin and/or spectinomycin
resistance gene for plasmid selection.
The T-DNA border regions are derived
from the nopaline plasmid pTiT37 and
contain the 25 bp repeat and outer border
regions but no internal T-DNA sequences;
therefore, natural T-DNA sequences are
not transferred into transformed plants.
LB
LB
LB
LB
LB
LB
LB
LB
LB
nos promoter
35S promoter
attR1
Fig. 1. Maps of GATEWAY destination vectors.
The vector names were assembled with the following
abbreviations and symbols: *, plant selectable marker
(nptII, hpt or bar); 2, promoter of 35S; 7, terminator of
35S; GW, GATEWAY cassette (attR1 with or without
CmR, ccdB, R2 orientation); WG, GATEWAY cassette
(R2, ccdB, with or without CmR, R1 orientation); D, greenfluorescent
protein (GFP) cassette (pRolD–EgfpER–t35S);
F, enhanced GFP (Egfp; Clontech Laboratories, Palo Alto,
CA, USA); S, β-glucuronidase (GUS) sequence.
LB
R1
R2
R2
R2
R1
R2
R1
p*2GW7
R2
p*2WG7
p*7WG2
p*7WG2D
LB R1 R2 R2 R1
RB
p*7WGF2
R2
p*7FWG2
R2
p*7FWGF2
p*GWFS7
p*WG
R2
R1
R1
p*7GWIWG2(II)
attR2
ccdB
CmR
R2
p*7GWIWG2(I)
R1
R1 RB
R2
R1
R1
R1
R2
RB
RB
RB
TRENDS in Plant Science
Destination vectors
All types of T-DNA destination vectors are
available with three plant selectable marker
genes: the neomycin phosphotransferase II
(nptII ), the hygromycin phosphotransferase
(hpt), and the bialaphos acetyltransferase
(bar) genes, which confer resistance against
kanamycin, hygromycin and glufosinate
ammonium, respectively. All three selectable
markers are under transcriptional
regulation of the nopaline synthase (nos)
promoter and nos terminator [2] and are
placed at the left side of the T-DNA.
The GATEWAY recombination site
for introduction of the fragment of interest
was placed at the right side of the T-DNA.
For construction of all vectors in this
collection we used the reading frameA (rfA)
RB
RB
35S terminator
Egfp
rolD promoter
R1
RB
RB
RB
intron
gus
RB
of the GATEWAY vector conversion
system (Invitrogen).
For overexpression of a DNA sequence,
the GATEWAY site was placed between
the promoter and the terminator of the
cauliflower mosaic virus (CaMV) 35S
transcript [3] because that promoter is
highly active in most plant cells of
transgenic plants. Downstream of the 35S
promoter part, the tobacco mosaic virus
(TMV) Ω leader ensures efficient translation
of the inserted coding sequences [4]. When
both overexpression and cosuppression of a
cloned sequence are desired, the p*2GW7
and p*2WG7 destination vectors should
be used. In these vectors, the inserted
sequences are transcribed to the outside of
the T-DNA. Single T-DNA inserts will then
usually result in overexpression of the
transgene whereas specially inverted
T-DNA repeats about the right border will
trigger post-transcriptional silencing of the
transgene and concomitant cosuppression of
homologous endogenes. By contrast, when
only overexpression is desired, p*7WG2 is
the vector of choice. All overexpression
vectors can also be used for antisense
expression of a particular sequence.
A T-DNA destination overexpression
or antisense vector (p*7WGD2) with an
additional screenable marker was made
essentially for the transformation of
tobacco Bright Yellow 2 (BY2), because
early visualization of transgenic calli is
important. The visible marker cassette
contained the rolD promoter fused to the
coding sequences of the enhanced
green-fluorescent protein (GFP) linked to
the endoplasmic reticulum-targeting
signal (EgfpER) and 35S terminator.
For cosuppression of plant endogenes, two
series of destination vectors p*7GWIWG2(I)
and p*7GWIWG2(II) were designed in a way
similar to that of the GATEWAY cassette
described by S. Varsha Wesley et al. [5].
The intron cloned between the inverted
GATEWAY fragments was selected from
an Arabidopsis database (ac007123.em_pl)
on the basis of ideal features for efficient
splicing (A + T-rich with a branch site close
to the consensus). In the p*7GWIWG2 (II)
series, the chloramphenicol resistance
(CmR) marker was removed from the
GATEWAY cassette and placed inside
the intron. In plants, these T-DNA
destination expression vector constructs
will produce double-stranded RNA
(hairpin RNA) from the inserted sequence
of interest, triggering post-transcriptional
gene silencing in an efficient way.
http://plants.trends.com

Research Update
TRENDS in Plant Science Vol.7 No.5 May 2002
195
The p*GWFS7 vectors were
constructed for promoter analysis.
In these vectors, a frame fusion between
the regions coding for EgfpER and
β-glucuronidase (gus) were cloned
downstream of the GATEWAY cassette.
For the localization of particular proteins,
another series of T-DNA destination
vectors was designed to allow the N-, C-,
or N- and C-termini of the protein of
interest to be fused to the GFP protein.
Finally, the T-DNA destination vector
p*WG allows the rapid cloning of any gene
or sequence to be transformed into plants.
As a test, the gus-coding sequence was
successfully recombined in all sets of T-DNA
destination vectors. Where applicable, the
expected expression was observed in
transformed hairy roots or BY2 cells
(for an update see http://www.plantgenetics.
rug.ac.be/gateway/).
References
1 Hajdukiewicz, P. et al. (1994) The small, versatile
pPZP family of Agrobacterium binary vectors for
plant transformation. Plant Mol. Biol. 25, 989–994
2 Hellens, R.P. et al. (2000) pGreen: a versatile and
flexible binary Ti vector for Agrobacterium-mediated
plant transformation. Plant Mol. Biol. 42, 819–832
3 Odell, J.T. et al. (1985) Identification of DNA
sequences required for activity of the cauliflower
mosaic virus 35S promoter. Nature 313, 810–812
4 Gallie, D.R. et al. (1989) Visualizing mRNA
expression in plant protoplasts: factors
influencing efficient mRNA uptake and
translation. Plant Cell 1, 301–311
5 Wesley, S.V. et al. (2001) Construct design for
efficient, effective and high-throughput gene
silencing in plants. Plant J. 27, 581–590
Mansour Karimi
Dirk Inzé*
Ann Depicker
Dept of Plant Systems Biology, Flanders
Interuniversity Institute for Biotechnology (VIB),
Ghent University, K.L. Ledeganckstraat 35,
B-9000 Gent, Belgium.
*e-mail: diinz@gengenp.rug.ac.be
Signal transduction
The special series of Trends in Plant Science articles focussing on
multiple aspects of signal transduction concludes with the May
issue. The complete series is listed below:
• Hirschi, K. (2001) Vacuolar H + /Ca 2+ transport: who’s directing the
traffic? Trends Plant Sci. 6, 100–104
• Pickett, J.A. and Poppy, G.M. (2001) Switching on plant genes
by external chemical signals. Trends Plant Sci. 6, 137–139
• Corpas, F.J., Barroso, J.B. and del Río, L.A. (2001) Peroxisomes
as a source of reactive oxygen species and nitric oxide signal
molecules in plant cells. Trends Plant Sci. 6, 145–150
• Wendehenne, D., Pugin, A., Klessig, D.F. and Durner, J. (2001)
Nitric oxide: comparative synthesis and signaling in animal
and plant cells. Trends Plant Sci. 6, 177–183
• Paul, M., Pellny, T. and Goddijn, O. (2001) Enhancing
photosynthesis with sugar signals. Trends Plant Sci. 6, 197–200
• Memelink, J., Verpoorte, R. and Kijne, J.W. (2001) ORCAnization
of jasmonate-responsive gene expression in alkaloid
metabolism. Trends Plant Sci. 6, 212–219
• Munnik, T. (2001) Phosphatidic acid: an emerging plant lipid
second messenger. Trends Plant Sci. 6, 227–233
• Knight, H. and Knight, M.R. (2001) Abiotic stress signalling
pathways: specificity and cross-talk. Trends Plant Sci. 6, 262–267
• Schmülling, T. (2001) CREam of cytokinin signalling: receptor
identified. Trends Plant Sci. 6, 281–284
• Bartels, D. (2001) Targeting detoxification pathways: an efficient
approach to obtain plants with multiple stress tolerance? Trends
Plant Sci. 6, 284–286
• Stals, H. and Inzé, D. (2001) When plant cells decide to divide.
Trends Plant Sci. 6, 359–364
• Nürnberger, T. and Scheel, D. (2001) Signal transmission in the
plant immune response. Trends Plant Sci. 6, 372–379
• Kim, T-H. , Hoffman, K., von Arnim, A.G. and Chamovitz, D.A.
(2001) PCI complexes: pretty complex interactions in diverse
signaling pathways. Trends Plant Sci. 6, 379–386
• Innes, R.W. (2001) Mapping out roles of MAP kinases in plant
defense. Trends Plant Sci. 6, 392–394
• Reed, J.W. (2001) Roles and activities of Aux/IAA proteins in
Arabidopsis. Trends Plant Sci. 6, 420–425
• Harrar, Y., Bellini, C. and Faure, J-D. (2001) FKBPs: at the crossroads
of folding and transduction. Trends Plant Sci. 6, 426–431
• Brownlee, C. (2001) The long and the short of stomatal density
signals. Trends Plant Sci. 6, 441–442
• Clouse, S.D. (2001) Integration of light and brassinosteroid
signals in etiolated seedling growth. Trends Plant Sci. 6, 443–445
• Bachmair, A., Novatchkova, M., Potuschak , T. and Eisenhaber, F.
(2001) Ubiquitylation in plants: a post-genomic look at a
post-translational modification. Trends Plant Sci. 6, 463–470
• Rodermel, S. (2001) Pathways of plastid-to-nucleus signaling.
Trends Plant Sci. 6, 471–478
• Lahaye, T. and Bonas, U. (2001) Molecular secrets of bacterial
type III effector proteins. Trends Plant Sci. 6, 479–485
• Hedden, P. (2001) Hormones at Mendel’s birthplace.
Trends Plant Sci. 6, 498–500
• Zhang, S. and Klessig, D.F. (2001) MAPK cascades in plant
defense signaling. Trends Plant Sci. 6, 520–527
• Muday, G.K. and Delong, A. (2001) Polar auxin transport:
controlling where and how much. Trends Plant Sci. 6, 535–542
• Carré, I.A. (2002) ELF3: a circadian safeguard to buffer effects of
light. Trends Plant Sci. 7, 4–6
• Seo, M. and Koshiba, T. (2002) Complex regulation of ABA
biosynthesis in plants. Trends Plant Sci. 7, 41–48
• Lindsey, K., Casson , S. and Chilley, P. (2002) Peptides: new
signalling molecules in plants. Trends Plant Sci. 7, 78–83
• Lee, J. and Rudd, J.J. (2002) Calcium-dependent protein
kinases: versatile plant signalling components necessary for
pathogen defence. Trends Plant Sci. 7, 97–98
• Drøbak , B.K. and Heras, B. (2002) Nuclear phosphoinositides
could bring FYVE alive. Trends Plant Sci. 7, 132–138
• Fankhauser, C. (2002) Light perception in plants: cytokinins
and red light join forces to keep phytochrome B active.
Trends Plant Sci. 7, 143–145
• Balmer, Y. and Buchanan, B. (2002) Yet another plant
thioredoxin. Trends Plant Sci. 7, 191–193
• Briggs, W.R. and Christie , J.M. (2002) Phototropins 1 and 2:
versatile plant blue-light receptors. Trends Plant Sci. 7, 204–210
• Weber, H. (2002) Fatty acid-derived signals in plants. Trends
Plant Sci. 7, 217–224
• Xing, T., Ouellet, T. and Miki, B.L. (2002) Towards genomics and
proteomics studies of protein phosphorylation in
plant–pathogen interactions. Trends Plant Sci. 7, 224–230
http://plants.trends.com