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Breakthrough Technologies
A Set of Modular Binary Vectors for Transformation
of Cereals1[W][OA]
Axel Himmelbach
2
,UweZierold
2
,Go
¨tz Hensel, Jan Riechen, Dimitar Douchkov,
Patrick Schweizer, and Jochen Kumlehn*
Leibniz Institute of Plant Genetics and Crop Plant Research, D–06466 Gatersleben, Germany
Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well
as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been
compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After
having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum
vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as
for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated
promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination.
Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of
further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated
by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in
barley.
Cereals represent crops of foremost economic im-
portance worldwide (http://faostat.fao.org). Conse-
quently, they are major targets in plant research,
biotechnology, and commercial crop plant improve-
ment, especially in the context of global climate
changes and the rapidly growing demand for human
nutrition.
A vast amount of different genetic resources has
been generated and collected in databases worldwide
(Alonso and Ecker, 2006; Stein, 2007). Assemblies of
large EST datasets (approximately 855,000 ESTs for
wheat [Triticum aestivum] and approximately 437,000
ESTs for barley [Hordeum vulgare]) provided important
insight into the genome organization and led to in
silico prediction of about 50,000 unique genes for
wheat and barley, respectively (Zhang et al., 2004;
Stein, 2007). Although bioinformatics, transcriptome
analysis (Close et al., 2004; Druka et al., 2006; Zierold
et al., 2005), transient overexpression, virus-induced
gene silencing (VIGS; Lacomme et al., 2003), and
transient-induced gene silencing (TIGS; Douchkov
et al., 2005) have greatly extended the information on
putative roles of genes, we are left with the major
challenge of elucidating gene functions by modulation
of their expression in planta. The recent development
of reliable and efficient Agrobacterium-mediated trans-
formation technologies for cereals (for review, see
Shrawat and Loerz, 2006; Goedeke et al., 2007) has
stimulated a variety of strategies toward functional
gene characterization, thereby paving the way for
deeper understanding of crop plant biology in cereals.
Comprehensive analyses of gene function include
stable transformation with sequences for overexpres-
sion or knock-down of plant genes. Binary vectors
used for generation of transgenic cereal species are
typically cumbersome due to their large size and the
rather limited number of useful restriction sites. To by-
pass laborious preparation of constructs, GATEWAY
technology (Invitrogen) is used especially for binary
vectors generating knock-down lines. GATEWAY-
derived cloning systems are based on the site-specific
recombination system from bacteriophage l(Landy,
1989) and circumvent traditional cloning methods in-
volving restriction and ligation of DNA sequences. A
number of GATEWAY-based binary vector sets for
plant functional genomics have been developed,
thereby allowing overexpression or knock-down of
effector genes, expression of fusion proteins (Karimi
et al., 2002; Curtis and Grossniklaus, 2003; Chung
et al., 2005; for review, see Earley et al., 2006), and
transformation of multiple genes (Chen et al., 2006).
Most overexpression studies employ a strong, con-
stitutive promoter, such as the cauliflower mosaic
virus (CaMV) 35S promoter (Odell et al., 1985), fol-
lowed by phenotypic analysis of the transgenic plant.
In many cases, ectopic expression experiments gave
1
This work was supported by the German Federal Ministry
of Research and Education (project PRO-GABI and Deutsche
Forschungsgemeinschaft Forschergruppe 666).
2
These authors contributed equally to the article.
* Corresponding author; e-mail kumlehn@ipk-gatersleben.de.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jochen Kumlehn (kumlehn@ipk-gatersleben.de).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a sub-
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.111575
1192 Plant Physiology, December 2007, Vol. 145, pp. 1192–1200, www.plantphysiol.org Ó2007 American Society of Plant Biologists
important insight into gene function (Jack et al., 1994).
However, as possible consequence of ubiquitous over-
expression and misdirection of gene products, unde-
sirable pleiotropic effects on the plant may be caused.
In addition, strong accumulation of unnecessary pro-
teins leads to wasteful energy consumption, which
could, in turn, generate phenotypes that are not di-
rectly correlated with the recombinant protein itself.
To avoid such unwanted pleiotropic effects that oc-
clude phenotypic analysis, transgene expression can
be controlled temporally and spatially by the use of
cell- and tissue-specific (Luo et al., 2006; Vickers et al.,
2006) or chemically inducible (Holtorf et al., 1995; Zuo
and Chua, 2000; Deveaux et al., 2003; Maizel and
Weigel, 2004) promoters. Most promoters available to
date are derived from dicotyledonous plants. Unfor-
tunately, such promoters are typically dysfunctional in
cereal species. Thus, expression of transgenes in ce-
reals has been largely driven by ubiquitous promoters,
such as those from the maize (Zea mays) ubiquitin
1(Ubi1; Christensen et al., 1992; Oldach et al., 2001) or
the rice (Oryza sativa)actin1gene(Act1; McElroy et al.,
1990; Vickers et al., 2006). However, a few specific
promoters derived from cereal species have been
characterized and used to drive transgene expression.
To confine transgene expression to the cereal seeds, sev-
eral grain-specific promoters, such as the oat (Avena
sativa)AsGlo1 (Vickers et al., 2006), the barley hordein
(Hor2-4,Hor3-1; Patel et al., 2000; Cho et al., 2002), and
the rice glutelin B1 (GluB1; Patel et al., 2000) promoters,
have been employed recently. Even though drought-
inducible promoters were described for barley and rice
(Xiao and Xue, 2001), stress-induced expression sys-
tems that are functionally verified in cereals or other
monocotyledonous species are not yet available. As a
consequence, there is growing demand for transforma-
tion technology that permits controllable expression of
transgenes in cereals.
Knock-down approaches aim at perturbation of
gene function due to the elimination of transcripts
using antisense RNA, RNA interference (RNAi), or the
generation of dominant-negative effects by interfering
with protein complexes (Olive et al., 1996; Ramirez-
Parra et al., 2003). Again, temporal and spatial control
of effector-gene expression largely supports the inter-
pretation of transgene-induced phenotypes.
At present, two major transformation strategies for
monocotyledonous plants are established. Compared
to biolistic techniques (Stoeger et al., 1999; Bhalla et al.,
2006) Agrobacterium-mediated transformation offers
several advantages (Tzfira and Citovsky, 2006), such
as simpler integration patterns resulting in lower
mutational consequences for the transgenic plant
(Latham et al., 2006) and limited transgene silencing
via cosuppression. In addition, the option for fine
tuning the Agrobacterium-based transformation proto-
cols renders more and more cereal species amenable
for efficient genetic engineering (Shrawat and Loerz,
2006; Conner et al., 2007). These advantages prompted
us to implement the GATEWAY cloning system and
expression cassettes into a vector set for Agrobacterium-
mediated transformation, thereby providing the ver-
satility to use the vector set for the transformation of a
large panel of cereal species and genotypes.
Although GATEWAY-based binary vectors have
been developed for dicotyledonous plants (e.g. Wesley
et al., 2001; Curtis and Grossniklaus, 2003; Tzfira et al.,
2005), these are typically not useful for monocotyle-
dons, mainly because of the limited functionality of
promoters that are used to drive either the gene of
interest or the plant selection marker. But also, other
specific vector elements, such as the plant-selectable
marker and the origin of replication, may impede the
amenability of a binary vector. The pVS1 origin of
replication derived from Pseudomonas aeruginosa con-
ferred high plasmid stability in Agrobacterium even
under nonselective conditions (Itoh et al., 1984), thus
ensuring the persistence of an effective population of
transformation-competent bacteria during the entire
period of cocultivation with target plant cells. This
may be especially crucial in transformation systems
for atypical Agrobacterium hosts, such as monocotyle-
donous plants. However, some binary GATEWAY
destination vectors have been generated especially for
use in monocotyledons (Miki and Shimamoto, 2004;
www.bract.org). Unfortunately, these do not allow
convenient and comprehensive modification with re-
gard to the promoters and the plant selection marker to
tailor derivatives for further specific approaches.
Here, we provide a set of generic binary vectors that
is made available for phenotypic studies in stably
transformed cereal species. Its modular configuration
permits convenient insertion of promoter and effector
sequences, as well as of plant selection marker cas-
settes of choice. The insertion of effector sequences
into the binary overexpression and knock-down vector
series is facilitated by the highly efficient GATEWAY
recombination system. The spectrum of applications
is further extended by the options to test constructs in
transient expression assays (e.g. in barley) prior to
starting the laborious stable transformation procedure
and by the option to transform monocotyledonous
and dicotyledonous plants using the same binary vec-
tor. Vector derivatives with strong, constitutive pro-
moters, such as the maize ubiquitin promoter (ZmUbi1;
Furtado and Henry, 2005), the double-enhanced CaMV
35S promoter (d35S; Furtado and Henry, 2005), or the
rice actin promoter (OsAct1; McElroy et al., 1990;
Vickers et al., 2006), are provided. In addition, the
wheat glutathione S-transferase promoter (TaGstA1;
Altpeter et al., 2005) permits the expression of trans-
genes confined to leaf epidermis in a constitutive
manner. With the availability of a combination of the
highly efficient GATEWAY cloning system, a selection
of cereal promoters controlling the expression of genes
of interest, different plant selection markers, together
with the option of further convenient vector modifi-
cations, the functional characterization of DNA se-
quences will be greatly facilitated in cereal species.
Binary Vector Set for Cereal Transformation
Plant Physiol. Vol. 145, 2007 1193
RESULTS AND DISCUSSION
GATEWAY Compatibility of Binary Destination Vectors
Traditional cloning of DNA sequences for overex-
pression or RNAi knock-down experiments into bi-
nary plant transformation vectors is laborious and
time consuming. To facilitate generation of binary
vectors for cereal species, we used the GATEWAY
system for recombinational cloning (Fig. 1A). GATE-
WAY technology takes advantage of a modified bac-
teriophage lrecombination system, thereby allowing
a highly efficient, site-specific, and reliable exchange of
DNA fragments between plasmids. The recombina-
tion reaction requires an entry vector containing a
gene of interest flanked by appropriate recombination
sites (e.g. attL1 and attL2), a recombination enzyme
(Clonase), and a binary destination vector. The binary
destination vector contains compatible recombination
sites (e.g. attR1 and attR2) integrated downstream of
the plant promoter of choice. For generation of RNAi
constructs, an inverted repeat of such GATEWAY in-
sertion cassettes is required. To enable the formation
of an RNA hairpin structure, the inverted repeat of in-
sertion cassettes is recommended to be separated by a
spacer or intron sequence. To this end, we used a wheat
RGA2 intron in this study (Douchkov et al., 2005).
To allow for efficient introduction of gene sequences
of interest into the entry vector, plasmid pIPKTA38
was used (Douchkov et al., 2005), which lacks the
negative bacterial selection marker ccdB but contains
the multiple cloning site (MCS) instead. The SwaI
restriction site present in the MCS permits the highly
Figure 1. Schematic representation of the modular binary destination vectors generated. A, There are two basic vector types
designed for overexpression (OE) and for RNAi-mediated knock-down (RNAi). The GATEWAY destination cassettes of the OE
vectors consist of R1 (attR1 recombination attachment site), Cmr(chloramphenicol acetyltransferase gene), ccdB (negative
selection marker), and R2 (attR2 recombination attachment site) sequences. The RNAi vectors further contain the wheat RGA2
intron (I) separating the inverted repeat of the GATEWAY destination cassettes. Transcription is terminated either by the A.
tumefaciens nos (t) or the CaMV 35S termination signal (T). All vectors can be digested using endonuclease Sfi I to obtain two
fragments, one containing the GATEWAYexpression cassette and the other comprising all other components of the vector; i.e. RB
(right border), ColE1 (origin of replication for E. coli ), pVS1 (origin of replication for A. tumefaciens), Spec r(streptomycin/
spectinomycin bacterial resistance), LB (left border), and the plant selection marker Hptr(hygromycin phosphotransferase)
controlled by the maize ZmUbi1 promoter. The latter SfiI fragment can be readily exchanged by respective fragments of
compatible binary vectors (e.g. available from DNA Cloning Service) that carry other plant selectable marker expression
cassettes. B, Expression of the GATEWAY cassettes of the binary vectors pIPKb001 to pIPKb010 is driven by the promoters
specified below (i.e. the constitutive ZmUbi1,OsAct1, and d35S promoters, and the epidermis-specific TaGstA1 promoter). To
enable convenient integration of further promoters, vectors pIPKb001 and pIPKb006 contain a MCS1 upstream of the GATEWAY
destination cassettes. Sequences of all binary destination vectors presented are available under the GenBank accession numbers
given.
Himmelbach et al.
1194 Plant Physiol. Vol. 145, 2007
efficient introduction of sequences by using a cyclic-
cut-ligation reaction involving the concurrent use of
the SwaI restriction endonuclease and T4 DNA ligase
(Douchkov et al., 2005). To obtain the successfully
recombined destination vector with high efficiency,
two bacterial selection schemes were imposed. Entry
vector (kanamycin resistance) and destination vector
(spectinomycin and chloramphenicol resistance) con-
tain different antibiotic selection markers. In addition,
upon recombination, the gene of interest replaces the
ccdB negative selection marker that poisons most
Escherichia coli strains (Bernard and Couturier, 1992).
In addition to pIPK38, any appropriate GATEWAY-
compatible entry vector containing a DNA sequence of
interest can be used to generate a respective binary
vector for overexpression or knock-down approaches.
Interchangeability of Promoter Sequences
In the vectors presented, transgene expression is
driven either by several strong, constitutive promoters
(ZmUbi1,d35S, and OsAct1) or the epidermis-specific
wheat glutathione S-transferase promoter (TaGstA1).
To permit future extensions of the range of promoters
controlling the gene of interest, MCS1 was introduced
to create the generic destination vectors pIPKb001 and
pIPKb006 (Fig. 1B). Additional promoter sequences
can thus be incorporated directly into these plasmids
prior to or following a GATEWAY recombination
reaction. Thus, versatility is provided that is required
to employ the vectors to functionally test new pro-
moters or other regulatory elements or to integrate
known promoter sequences that possess particularly
useful properties.
Interchangeability of Plant Selection Markers
Although the hygromycin phosphotransferase (hpt)
selection marker of the binary plasmid 6U (DNA Clon-
ing Service) is widely employed for barley and wheat
(Goedeke et al., 2007; Hensel et al., 2008), different
plant selection markers, such as phosphinothricin-
N-acetyl transferase (pat), may be preferred for some
target species or required for iterative gene-stacking
approaches (Halpin, 2005). Compatible binary vectors,
such as 7U (DNA Cloning Service), containing fur-
ther expression cassettes of plant selection markers
have been conveniently swapped to some of the bi-
nary vectors by using the rare-cutting enzyme SfiI (see
Fig. 1A). Likewise, binary plasmids available from the
DNA Cloning Service with further plant selection gene
cassettes, such as neomycin phosphotransferase II
(nptII; plasmid 9U) or dihydrofolate reductase (dhfr;
plasmid 5U), can be readily combined with any of
the binary vectors presented here. In addition, any of
the binary vectors available can be used to create a
respective vector devoid of a plant selectable marker
expression cassette. Those vectors might be useful in
exceedingly efficient transformation systems that do
not necessarily require the application of selective
conditions. The benefit of such an approach would
be to obtain transgenic plants instantly free of a se-
lectable marker gene. To this end, the SfiI fragment
containing the plant selection marker has to be ex-
changed with the compatible markerless fragment of
the binary vector B-BA (DNA Cloning Service). Fur-
thermore, the vector series provides the potential to
introduce alternative plant selection markers, such as
the phospho-mannose-isomerase gene (Reed et al., 2001;
Goldstein et al., 2005) or sequences for site-specific
recombination-mediated marker deletion strategies (Cre/
loxP; Darbani et al., 2007) through the generation of
respective SfiI-compatible plasmids.
The vector set also allows the expression units
for the plant resistance marker as well as the over-
expression/knock-down cassettes to be juxtaposed in
two orientations. By using the binary plasmid 6U, both
transcription units are oriented convergently, whereas
the plasmid 65U (DNA Cloning Service) permits the
cassettes to be positioned in tandem.
Vector Elements Facilitating Transgenic Plant Analysis
Phenotypic characterization of transgenic plants
often includes analysis of integration patterns of
T-DNA within the plant genome. This involves deter-
mination of the integration events of the T-DNA se-
quences as well as verification of complete T-DNA
transfer to the plant, especially when sequences are
used that cause negative selection pressure. To sim-
plify screening for plants harboring the complete
T-DNA sequence, primer pairs spanning the overex-
pression cassette were generated. To verify the inte-
gration of entire hairpin constructs (derivatives of
pIPKb007–pIPKb010), the sense and antisense repeats
of the hairpin cassette can be detected independently
by specific primer pairs, the first spanning the region
between the promoter and the RGA2 intron, and the
second the region between the RGA2 intron and the
terminator, respectively. This feature proved to be
highly beneficial because, in our experience, not all
generated transgenic plants surely contain both in-
verted sequence repeats of a given hairpin construct.
All of the available primers can be employed regardless
of the sequence of interest introduced to the destination
vector because they are designed to anneal with se-
quences flanking the GATEWAY cassettes. Primer
sequences and information on their target templates
are available (see Supplemental Table S1; Fig. 1). By
using these primers for PCR followed by DNA se-
quence analysis, the integrity of the T-DNA was con-
veniently verified in a large number of transgenic lines
(data not shown).
Functional Analysis of Binary Overexpression Vectors
Functionality of plasmids with respect to integrity of
the destination cassette, promoter strength, and gen-
eral transformation efficiency was tested by introduc-
tion of the gus reporter in the overexpression vector
Binary Vector Set for Cereal Transformation
Plant Physiol. Vol. 145, 2007 1195
series (pIPKb002–pIPKb005) followed by transforma-
tion of barley and subsequent expression analysis.
Transgenic barley lines carrying overexpression se-
quences were generated using methods based on the
cocultivation of immature barley embryos with Agro-
bacterium followed by regeneration under antibiotic
(hygromycin) selection. This procedure yielded trans-
formants with an efficiency ranging between about
30% and 60% (related to the number of barley embryos
used), as was previously observed for plasmid 6U
derivatives without GATEWAY cassettes (Hensel et al.,
2008). Following transformation using the overexpres-
sion vectors presented here, the gus reporter was ver-
ified by PCR in .90% of the T0plants tested and was
inherited to the T1generation according to Mendelian
rules (data not shown). Expression of the gus reporter
under the control of the ZmUbi1 promoter in barley
leaf segments of a segregating T1population obtained
by gene transfer using pIPKb002_GUS is shown in
Figure 2A.
For quantification of promoter strength in trans-
genic barley lines, the specific GUS activity generated
under the control of the ZmUbi1 promoter (pIPKb002_
GUS), the OsAct1 promoter (pIPKb003_GUS), the CaMV
d35S promoter (pIPKb004_GUS), and the TaG s tA 1
promoter (pIPKb005_GUSI) was measured (Fig. 2B).
Analysis of T1seedling pools from independently
derived primary transgenic lines revealed the stron-
gest average specific GUS activity (97 676 fluores-
cence units [FU] h21mg21) obtained by the ZmUbi1
promoter, followed by the OsAct1 promoter (40 630
FU h21mg21), the TaGstA1 promoter (26 619 FU h21
mg21), and the CaMV d35S promoter (15 69FUh
21
mg21). In contrast to the overexpression lines, wild-
type plants only showed background GUS activity
(5 62FUh
21mg21). The moderate average expression
obtained by the TaGstA1 promoter has to be assigned
to its specificity for the epidermis that represents only
a minor proportion of the leaf. In an additional exper-
iment using the same transgenic plants carrying the
Figure 2. Overexpression of GUS in transgenic bar-
ley. The amount of GUS protein is dependent on the
promoter controlling the overexpression cassette. A,
Barley transformants (T1generation; 10 d old) ex-
pressing the gus reporter under the control of the
ZmUbi1 promoter (pIPKb002_GUS) were analyzed
for GUS activity. The photograph represents leaf
segments as typical examples of constitutive gus
reporter activity in a segregating population. B, Ten-
day-old seedlings from independently generated
transgenic barley lines expressing the gus reporter
under the control of the ZmUbi1 (pIPKb002_GUS),
OsAct1 (pIPKb003_GUS), CaMV d35S (pIPKb004_
GUS), and TaGstA1 (pIPKb005_GUSI) promoters,
respectively, were pooled (15 T0plants each) for
quantitative fluorimetric GUS measurements. Wild-
type barley plants (white bars) served as controls.
Specific GUS enzyme activity is shown. Quantifica-
tion was reproduced twice with very similar results.
Himmelbach et al.
1196 Plant Physiol. Vol. 145, 2007
gus reporter under the control of the TaGstA1 pro-
moter, reporter expression was compared in epidermis
pealed off from the abaxial side of leaves to that of
the corresponding leaf remnants with the upper epi-
dermis still attached to their adaxial surface (because
upper epidermis cannot be removed appropriately).
Fluorescence spectroscopy revealed that GUS activity
in isolated epidermis was, on average, 10 times as
strong as in the corresponding leaf remnants. This
result not only confirmed our earlier finding that the
TaGstA1 promoter drives specific expression in barley
leaf epidermis (Altpeter et al., 2005), but also that this
particular promoter specificity is retained in the con-
text of pIPKb005. Furthermore, the epidermis repre-
sents an important tissue for plant defense against
pathogens that enter the plant via a direct mode of
penetration through the epidermis (e.g. powdery mil-
dew fungus [Blumeria graminis] and Fusarium head
blight [Fusarium culmorum]). The epidermis-specific
wheat TaGstA1 promoter could therefore be used to
control the expression of antifungal effector genes,
thereby interfering with pathogen infection.
Derivatives of pIPKb002 to pIPKb005 with various
genes of interest integrated in the GATEWAY destina-
tion site were successfully used to produce stable
transgenic barley and wheat plants. The molecular
and phenotypic characterization of these plants will
be published elsewhere. Moreover, the vectors
pIPKb002_GUS and pIPKb004_GUS carrying the gus
reporter under the control of the ZmUbi1 and the d35S
promoter, respectively, were used to stably transform
tobacco (Nicotiana tabacum). Expectedly, these plants
showed ubiquitous expression of the gus reporter as
revealed by fluorescence spectroscopy (data not shown).
This result indicates that the vector set presented here
provides the opportunity to transform both mono- and
dicotyledonous plant species with the same binary
vector.
Functional Analysis of Binary RNAi Vectors
The discovery of RNAi triggered by double-
stranded RNA paved the way for the high-throughput
production of loss-of-function mutants for functional
genomics in plants, including cereals (Waterhouse
et al., 1998). The set of binary destination vectors for
cereals described here allows the constitutive expres-
sion of RNAi sequences under the control of the d35S
promoter, as well as of the ZmUbi1 and the OsAct1
promoter. Furthermore, we provide a destination vec-
tor containing the wheat TaGstA1 promoter, which
permits the epidermis-specific knock-down of gene
expression.
To test binary RNAi vectors for their performance in
the TIGS system (Nielsen et al., 1999; Schweizer et al.,
1999, 2000), we transiently knocked down the barley
mildew resistance locus o(Mlo gene), which encodes a
negative regulator of resistance to the powdery mil-
dew fungus. Barley leaf segments were challenge
inoculated with powdery mildew fungus (B. graminis
f. sp. hordei) and scored for their resistance phenotype
at the single-cell level in successfully transformed
epidermis cells, marked by expression of the gus
reporter. Introduction of the empty binary vectors
revealed an expected susceptibility to powdery mil-
dew ranging from 18% to 38%. However, delivery of
the binary RNAi constructs directed against the Mlo
gene greatly reduced susceptibility (,5%) in all pro-
moters tested (Fig. 3). Similar results were obtained
with control vector (pIPKTA36; Douchkov et al., 2005)
particularly designed for TIGS experiments targeting
the Mlo gene. The results suggest that the function of
the negative regulator of resistance (Mlo) has been
eliminated or at least largely reduced by the RNAi
constructs, thereby leading to a phenocopy of the loss-
of-function mlo resistance allele.
Derivatives of pIPKb007 to pIPKb010 with frag-
ments from various genes of interest integrated in the
GATEWAY destination sites were successfully used to
produce stable transgenic barley and wheat plants.
Molecular and phenotypic characterization of these
plants will be published elsewhere.
CONCLUSION
A series of modular binary plasmids for stable
Agrobacterium-mediated transformation of cereals
Figure 3. TIGS of the Mlo gene caused increased resistance to powdery
mildew (B. graminis f. sp. hordei) infection. Binary RNAi constructs
targeting the Mlo gene were cobombarded together with a gus expres-
sion plasmid (ZmUbi1-promoter-gus fusion) followed by challenge
inoculation with powdery mildew (B. graminis f. sp. hordei) 3 d post-
bombardment. Expression of the RNAi sequence was controlled by the
ZmUbi1 (pIPKb007_Mlo), OsAct1 (pIPKb008_Mlo), CaMV d35S
(pIPKb009_Mlo), and TaGstA1 (pIPKb010_Mlo) promoters, respec-
tively. Cells transformed with the corresponding empty vectors
(pIPKb007, pIPKb008, pIPKb009, and pIPKb010) served as controls.
Plasmid pIPKTA36 was utilized as a positive control for RNAi-mediated
gene silencing of the Mlo gene. Haustorium formation of powdery
mildew was scored 48 h postinoculation. The susceptibility index
represents the number of GUS-positive cells harboring at least one
haustorium divided by the total number of GUS-stained cells. The
mean SD of three independent experiments is shown.
Binary Vector Set for Cereal Transformation
Plant Physiol. Vol. 145, 2007 1197
such as barley and wheat is made freely available for
noncommercial use. Vector derivatives are provided
for overexpression studies or knock-down analyses.
Modular configuration of the presented vectors allows
for convenient introduction of coding sequences to be
overexpressed or knocked down, any promoter se-
quence to drive the gene of interest, as well as any
preferred plant selectable marker cassette. This pro-
vides the opportunity to generate vector derivatives
tailored for the particular requirements of various
plant transformation systems and for the ultimate
elucidation of the function of any particular candidate
DNA sequence. The introduction of genes of interest in
these generic vectors is greatly facilitated by the im-
plementation of the GATEWAY recombinational cloning
system in both the overexpression and the knock-
down vectors presented. Beside the highly beneficial
simplification of cloning RNAi constructs, a major
advance derives from the opportunity to easily gener-
ate overexpression and knock-down binary vectors
using entire GATEWAY-compatible cDNA libraries.
High versatility of the vector set is further provided
through construction of derivatives with promoters
functional in cereal species, which drive ubiquitous or
epidermis-specific expression of transgenes.
Although data providing direct functional proof of
the newly developed binary vectors are presented here
only for barley, it can be anticipated that the vector set
will also be useful for any further cereal or monocot-
yledonous species. Moreover, some of the vectors
generated have been shown to be amenable to the
genetic transformation of both mono- and dicotyle-
donous plants.
Eventually, the presented vector set provides a
potential basis for the implementation of further use-
ful features, such as the integration of affinity or
screenable tags that can be N- or C-translationally
attached to the coding sequence, or for the develop-
ment of systems that permit conditional gene expres-
sion or directed T-DNA insertion mutagenesis in
cereal species.
MATERIALS AND METHODS
Plasmid Construction
All molecular biological manipulations were performed according to
standard protocols (Sambrook and Russel, 2001). The constructs that involved
PCR and synthetic oligonucleotides were verified by sequencing. Details of
plasmid constructs are available in Supplemental Materials and Methods S1.
Plants and Powdery Mildew Fungus
Barley (Hordeum vulgare ‘Ingrid’ and ‘Golden Promise’) and powdery
mildew (Blumeria graminis DC Speer f. sp. hordei) were cultivated as described
elsewhere (Zimmermann et al., 2006). Leaf segments of plants were challenge
inoculated with powdery mildew at a density of 150 to 200 conidia mm22.
Generation of Transgenic Plants
Immature barley ‘Golden Promise’ embryos were transformed with the
GUS overexpression vector series using the Agrobacterium tumefaciens strain
AGL1 as described elsewhere (Hensel et al., 2008). The resulting plantlets
were selected on medium containing hygromycin (50 mg L21).
Transient Expression and TIGS
Binary plasmids were transiently expressed in bombarded barley leaf
epidermal cells of ‘Ingrid’ by using a PDS-1000/He System (Bio-Rad) essen-
tially as described previously (Douchkov et al., 2005). To monitor the transfor-
mation of epidermal cells, a plasmid expressing the gus reporter under the
control of the maize (Zea mays)Ubi1 promoter (pUbiGUS) was cotransformed
together with the binary vector DNA. For TIGS, bombarded leaf segments were
challenge inoculated with powdery mildew (B. graminis DC Speer f. sp. hordei)
48 h postbombardment. The interaction phenotype, represented by the fraction
of GUS-specifically stained epidermis cells harboring at least one haustorium
(susceptibility indexin percent), was examined by light microscopy (Douchkov
et al., 2005).
Protein and GUS Measurements
Leaf or peeled epidermis was ground in liquid nitrogen and 10 mg of
material was resuspended in incubation buffer (50 mMsodium phosphate, pH
7.2, 1 mMEDTA, 0.1% [w/v] Triton X-100, 10 mMb-mercaptoethanol). GUS
enzyme activity was measured in the soluble protein fraction by using
4-methylumbelliferyl-b-D-glucoside (2 mM) as a substrate. Fluorescence was
recorded at 365-nm excitation and 456-nm emission wavelength using a
luminescence spectrometer (GIBCO TEC Synergy HT). Protein concentration
was determined employing standard methods (Bradford, 1976). Leaf seg-
ments were stained histochemically for GUS activity as described elsewhere
(Jefferson et al., 1987; Schweizer et al., 1999).
Generation of Entry Vectors (Cyclic-Cut-Ligation) and
Clonase Reaction
DNA fragments were ligated into the SwaI site of plasmid pIPKTA38 as
previously described (Douchkov et al., 2005). Briefly, the ligation reaction
containing T4 DNA ligase and the restriction endonuclease SwaI was incu-
bated at 25°C for 1 h. Enzymes were then inactivated by heating to 65°C for
15 min. Additional SwaI enzyme was added for 1 h at 25°C to quantitatively
eliminate religated plasmid. The resulting recombinant pIPKTA38 clones were
transformed into Escherichia coli (DH10B) cells and verified by restriction
analysis. Positive pIPKTA38 clones were used as entry vector in the LR
reaction of the GATEWAY system with the binary destination plasmids
pIPKb001 to pIPKb010. The Clonase reaction was essentially performed as
described elsewhere (Douchkov et al., 2005).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers EU161567 to EU161576 (pIPKb001 to
pIPKb010) and EU161577 (pSB156; supplemental data).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. PCR-based detection of overexpression or
knock-down GATEWAY cassettes in transgenic plants.
Supplemental Table S1. Primer sequences for the PCR-based detection of
overexpression or knock-down cassettes integrated in the plant ge-
nome.
Supplemental Materials and Methods S1. Further information on mate-
rials and methods.
ACKNOWLEDGMENTS
The expert technical assistance of Heike Bu
¨chner and Cornelia Marthe is
gratefully acknowledged. We further thank Dr. Sylvia Broeders for providing
plasmid pSB156.
Received October 23, 2007; accepted October 25, 2007; published November 2,
2007.
Himmelbach et al.
1198 Plant Physiol. Vol. 145, 2007
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