Plant Biotechnology Journal (2003) 1, pp. 51–57
A hybrid Bacillus thuringiensis delta-endotoxin gives
resistance against a coleopteran and a lepidopteran pest
in transgenic potato
Blackwell Science, Ltd
Samir Naimov1, Stefan Dukiandjiev1 and Ruud A. de Maagd2*
1
Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24 Tsar Assen Street, Plovdiv, Bulgaria
2
Business Unit Cell Cybernetics, Plant Research International BV, PO Box 16, 6700 AA Wageningen, the Netherlands
Received 13 June 2002; revised 25
September 2002; accepted 1 October 2002.
*Correspondence (fax: +31 (0)317 418094;
e-mail [email protected])
Summary
Expression of Bacillus thuringiensis delta-endotoxins has proven to be a successful
strategy for obtaining insect resistance in transgenic plants. Drawbacks of expression of
a single resistance gene are the limited target spectrum and the potential for rapid
adaptation of the pest. Hybrid toxins with a wider target spectrum in combination with
existing toxins may be used as tool to mitigate these problems.
In this study, Desiree potato plants were genetically modified to resist attack by insect
species belonging to the orders Coleoptera and Lepidoptera, through the insertion of such
a hybrid gene, SN19. Transgenic plants were shown to be resistant against Colorado potato
beetle larvae and adults, potato tuber moth larvae, and European corn borer larvae. These
Keywords: Bacillus thuringiensis,
Colorado potato beetle, European corn
borer, hybrid Cry1 protein, potato
tuber moth, transgenic potato plants.
are the first transgenic plants resistant to pests belonging to two different insect orders. In
addition, the target receptor recognition of this hybrid protein is expected to be different
from Cry proteins currently in use for these pests. This makes it a useful tool for resistance
management strategies.
Introduction
The coleopteran Colorado potato beetle (Leptinotarsa
decemlineata Say; CPB) and the lepidopteran potato tuber
moth (Phthorimaea operculella Ziller; PTM) are some of
the most destructive pests of cultivated potato. Their life cycle,
feeding habits and demonstrated ability to develop resistance
to chemical insecticides have made the control of CPB and
PTM an increasing agricultural problem (Forgash, 1985; Trivedi
and Rajagopal, 1992). Another important agricultural pest on
potato is the European corn borer (Ostrinia nubilalis Hübner,
ECB) (Hanzlik et al., 1997). At the present time the control of
these insect pests is accomplished primarily by the use of
chemical insecticides, through the use of different insecticidal
Cry proteins originating from Bacillus thuringiensis in sprays,
or by the expression of Cry proteins in transgenic plants.
The cry gene family is a large, still growing family of homologous genes, with each gene encoding a protein active on
insect larvae of a subset of species usually belonging to the
same order (Schnepf et al., 1998). Cry1 proteins are generally
© 2003 Blackwell Publishing Ltd
active against lepidopterans (larvae of moths and butterflies).
Cry1Ba also has some activity against coleopterans (beetles),
although its toxicity for CPB is much lower than that of Cry3Aa,
the most active natural toxin (Bradley et al., 1995). Somewhat
higher activity against CPB was reported for Cry1Ia (Tailor
et al., 1992). A cry1Ba/cry1Ia hybrid gene (SN19) encoding a
protein consisting of domains I and III of Cry1Ba and domain
II of Cry1Ia, with high activity against CPB was constructed
and described earlier by us (Naimov et al., 2001). Both parental
proteins, Cry1Ba and Cry1Ia, are highly toxic for European
corn borer and potato tuber moth (Van Frankenhuyzen and
Nystrom, 2002), suggesting that the hybrid protein may also
have these properties.
Cry toxins have been expressed in a number of plant
species. Expression of one member of this family usually results
in resistance against a single pest insect or against a few
relatively closely related insect species within one order. Cry3Aaexpressing potatoes with resistance to CPB (Perlak et al.,
1993), and Cry1Ia-expressing potatoes with resistance to PTM
(Douches et al., 2001) are examples of this.
51
52 Samir Naimov et al.
In this manuscript we describe production of the first
transgenic potato plants with leaves protected against
attack by a number of insect pests, which are members of two
different orders – Coleoptera (CPB) and Lepidoptera (PTM and
ECB) – by expression of the previously described SN19 hybrid
gene under the control of a chrysanthemum ribulose-1,5bisphosphate carboxylase /oxygenase small subunit (Rubisco
SSU) promoter and terminator.
For the reconstruction of the gene encoding the toxic
as high as 0.25% of total soluble plant protein. None of
the 16 plants transformed with SN32 (unmodified domain II)
had detectable Cry protein expression (lower detection limit
= 0.025% of total soluble leaf protein), although presence of
the SN19 gene could be demonstrated by PCR (results not
shown). Only the plants transformed with SN48 (modified
domain II) expressing detectable levels of SN19 showed
some level of resistance to neonate CPB larvae, with 10 of
the 20 transformed lines leading to more then 80% CPB
mortality.
Plants transformed with the SN48 construct were analysed
in more detail with regard to the correlation between protein
expression and CPB resistance. As can be seen in Figure 2,
expression of the modified SN19 (construct SN48) gene to
0.2% or more of total soluble protein in potato leaves is
fragment of SN19, a synthetic and truncated (2090 bp)
cry1Ba gene (Desai, 1999), optimized for expression in
plants, was used as the scaffold for insertion of the domain
II-encoding part of cry1Ia. A sequence analysis of this domain
II-encoding part identified a number of potential RNA
instability elements and polyadenylation sites (Figure 1A).
By a combination of PCR with mutagenic primers and
recombination of overlapping fragments via PCR (overlap
extension) (Figure 1B) 28 single nucleotides were changed,
eliminating putative polyadenylation sites, mRNA instability
sequences, and consecutive C + G and A + T stretches. The
mosaic SN19 with unmodified domain II-encoding DNA,
and SN19 with modified domain II-encoding DNA, were
cloned between a promoter and terminator fragment
derived from the chrysanthemum Rubisco SSU gene (obtained
from N.S. Outchkourov, manuscript in preparation) in the
binary transformation vector pBINPLUS. This resulted in
transformation vectors pSN32 and pSN48, respectively.
sufficient to give complete resistance against CPB larvae with
100% mortality and no visible damage in a leaf feeding assay
(Figure 3A,E). Two of the SN48 lines (lines 8 and 11, having
on average 0.25% and 0.23% Cry protein expression,
respectively) were tested for resistance against adult CPB.
In contrast to control leaves, the transgenic leaves were
completely undamaged, even after 10 days (Figure 3B,F). After
this period, most of the Colorado potato beetles which had
been placed on transgenic potato leaves were still alive, but
not feeding and smaller in size than control beetles.
The SN48 lines 11 and 16 (having on average 0.23% and
0.25% Cry protein expression, respectively) were tested
for resistance against potato tuber moth and European
corn borer – both Lepidopteran pests. Leaf infestation with
10 neonate PTM larvae, in three separate experiments
resulted in extensive tunnelling by four or five live larvae in
control leaves after 4 days (Figure 3C). In contrast, no live
PTM larvae could be recovered from the SN19-expressing
These expression cassettes were introduced in potato cultivar
‘Desiree’ by Agrobacterium tumefaciens-mediated transformation. Sixteen and 20 transgenic potato lines per construct, respectively, were obtained and successfully adapted
to greenhouse conditions.
plants in all three experiments. No signs of tunnelling or other
visible damage were recorded in these leaves (Figure 3G).
Control leaves infested with 2-day-old ECB larvae in three
separate experiments resulted in tunnelling in the leaf stem
with subsequent wilting of the leaves. ECB mortality was
zero after 3 days in control leaves (Figure 3D). In contrast,
SN19-expressing leaves remained healthy, and caused a
100% mortality of the ECB larvae in all three experiments.
Only small wounds on the leaf surface, presumably from
feeding attempts, could be observed here (Figure 3H).
Results
Domain II modification and plant transformation
vectors
Insect resistance of transgenic plants
Leaves of transgenic plants were analysed by immunochemically estimating the expression of the protein, and by testing
for resistance to CPB larvae in a single bioassay. As expected,
the highest accumulation of the protein of interest was
obtained with the fully modified hybrid gene. The use of the
strong green tissue-specific promoter of the chrysanthemum
Rubisco SSU in combination with a modification of the open
reading frame resulted in levels of SN19 protein which were
Discussion
Although current transgenic plants expressing a Cry protein
are effectively protected against one or a few relatively related
pests, their activity spectrum is limited.
© Blackwell Publishing Ltd, Plant Biotechnology Journal (2003), 1, 51– 57
Coleopteran and lepidopteran resistant transgenic potato 53
Figure 1 Modification of domain II of the SN19 hybrid gene. (A) Sequence of the cry1Ia domain II-encoding fragment and (below) the primers used for
mutagenesis. Only the nucleotides to be altered are shown in the primer sequences. (B) Mutagenesis and recombination via PCR. In separate
amplifications, four fragments of the SN19 gene were amplified using mutagenic primers. Denatured fragments annealing in the overlapping primer
regions were extended. This resulted in a full-length product which was amplified using the two flanking primers (1B1Id1for, 1IRsRIIRev), and
subsequently used as a template for the second round of mutagenic PCR reactions.
Expression of the SN19 gene in potato was an approach
designed to prove our hypothesis that the effective expression of a single hybrid delta-endotoxin gene could provide
effective resistance against a coleopteran and a lepidopteran
pest simultaneously. Expression of SN19 resulted in complete
protection of transgenic potato leaves against CPB larvae
© Blackwell Publishing Ltd, Plant Biotechnology Journal (2003), 1, 51–57
and adults, PTM larvae, and ECB larvae. A strong correlation
between insect resistance and SN19 expression was found
for CPB larvae. In our hands, up to 0.25% expression of SN19
was reached, which was higher than the protein level
required for complete plant protection. In agreement with
results reported in previous studies (Perlak et al., 1991) we
54 Samir Naimov et al.
Figure 2 Correlation between delta-endotoxin SN19 expression and CPB
larval mortality of transgenic potato plants in independent SN48
transgenic lines expressing the SN19 hybrid gene with modified domain
II-encoding DNA. Only two of the lines with undetectable protein levels
are shown. Error bars depict standard deviation from the means of three
experiments.
found that a significant modification of the Bacillus thuringiensis delta-endotoxin encoding hybrid gene SN19 was
necessary for successful expression in plants. Although the
optimization of codon usage in the domain II encoding part
could possibly enhance expression of the transgene even
further (Perlak et al., 1991), in our case the partial modification
of domain II seems to be sufficient for expression of the SN19
hybrid gene in potatoes. We conclude that the expression of
SN19 in transgenic potato plants could provide excellent
protection against several major potato pests in the field.
Whereas an expanded host range has economic advantages,
it may also have disadvantages in the form of increased
effects on nontarget and/or beneficial insects. Pre-release
testing for these effects would have to be included in the
safety assessment of any such crop, as indeed it was for
already commercialized insect-resistant transgenic crops.
The relatively low homology of SN19 with Cry3Aa and
Cry1Ab suggests that SN19 may bind to midgut receptors
that are different from those for Cry3Aa in CPB and for
Cry1Ab in PTM or ECB, respectively. Indeed, it has been
shown that Cry1Ba and Cry1Ab do not compete for the same
binding site in ECB (Denolf et al., 1993) and PTM (Escriche
et al., 1994). In contrast, the more homologous Cry1Aa,
Cry1Ab and Cry1Ac show a high degree of overlap of binding
specificities in many insects, including PTM (Escriche
et al., 1997) and ECB (Denolf et al., 1993; Hua et al., 2001).
Changes in toxin binding sites is the most commonly occurring resistance mechanism against Cry proteins in insects
(Ferré and Van Rie, 2002), and occur in Cry3Aa-resistant CPB
(Loseva et al., 2002). For this reason ‘pyramiding’ or ‘stacking’ of two genes encoding proteins with different receptor
a
b
c
d
e
f
g
h
Figure 3 Leaf feeding assays comparing control lines (A–D) and SN19-expressing (E–H) line 11 (expressing 0.23% of total soluble protein). A, E: CPB
larvae; B, F: CPB adults; C, G: PTM larvae; D, H: ECB larvae.
© Blackwell Publishing Ltd, Plant Biotechnology Journal (2003), 1, 51– 57
Coleopteran and lepidopteran resistant transgenic potato 55
recognition properties (Roush, 1998), or deploying mixtures
of seeds with two different toxins (Caprio, 1998) have been
considered as resistance management strategies. Cry7 and
Cry8, which have relative a low homology with Cry3’s, and
which have been shown to be active against CPB (Van
Frankenhuyzen and Nystrom, 2002) may be alternative ‘second
genes’ for resistance management, but in contrast to SN19
their use in transgenic plants has not so far been demonstrated. Although the use of a single hybrid toxin would not
have an advantage over the use of a single wild type toxin,
SN19 may well play the role of the second toxin for resistance
management, simultaneously with Cry1Ab against ECB and
PTM and with Cry3Aa against CPB in transgenic potatoes.
This would reduce the number of transgenes necessary to be
transferred from four to three.
Experimental procedures
Modification of domain II of cry1Ia
For the modification of DNA motifs with a possible negative
effect on gene expression in plants, site directed mutagenesis
and recombination via PCR were performed. Eight different primer sets (Eurogentec) containing the desired mutations were used (Figure 1A). In order to introduce multiple
nucleotide changes, the following modifications of a previously described protocol (Ho et al., 1989) were made: (i) four
different DNA fragments were assembled during the first
combinatorial PCR, and (ii) the amplified product from the
first combinatorial PCR was used as a template for a second
round of primary PCRs, followed by a second combinatorial
PCR (see Figure 1B). The PCR products were separated on
0.8% agarose gel and purified using a QIAEX II agarose gel
replacement of the domain II-encoding part with the
corresponding part of cry1Ia as a MunI–Rsr II fragment (Naimov
et al., 2001) with or without modifications, giving pSN45
and pSN24, respectively. The open reading frames were cut
from pSN24 and pSN45 by BspHI and Bgl II digestion and
ligated into the NcoI–Bgl II sites of pUCRBC1, between the
Chrysanthemum ribulose-1,5-bisphosphate carboxylase /
oxygenase small subunit promoter and terminator (N.S.
Outchkourov, manuscript in preparation). Resulting expression cassettes RBC-unmodified SN19 (pSN28) and RBCmodified SN19 (pSN46), were cloned in pBINPLUS (van
Engelen et al., 1995) using the HindIII and EcoRI sites
flanking the cassettes. Resulting binary vectors pSN32 and
pSN48, respectively, were subsequently introduced in
Agrobacterium tumefaciens strain Agl0 (Lazo et al., 1991)
by electroporation (Mersereau et al., 1990). A. tumefaciens
mediated potato transformation was performed following
the previously described protocol (Lauterslager et al., 2001).
The obtained transgenic lines were subsequently multiplied
and adapted to greenhouse conditions: 25 °C and a 16 h
light/8 h dark cycle.
Insect bioassays
All bioassays were performed on detached fully grown
potato leaves stuck in water agar. For the CPB bioassays, 10
neonate larvae were placed on the upper leaf surface. After
2 days, the leaves were replaced by fresh ones, and mortality
was scored after 4 days. For adult CPB, four newly emerged
insects were placed on leaves for up to 10 days. Potato
tuber moth bioassays were performed as described earlier
(Mohammed et al., 2000) by placing 10 neonate larvae on
the back surface of potato leaves. For testing of European
extraction kit (Qiagen). For all PCR steps, Pfu-Turbo DNA
polymerase (Stratagene) was used. After the final amplification step the product was digested with MunI and RsrII and
used for replacement of the corresponding part of SN23 (see
below), resulting in SN45, and sequenced in both directions.
corn borer resistance, 2-day-old larvae were used, grown on
an artificial diet at 28 °C. Larvae were allowed to feed on
potato leaves for 2 days. Plants transformed with the
empty pBINPLUS vector were used as a negative control. All
bioassays were performed threefold on separate dates.
Construction of binary vectors
Protein quantification
A BamHI–Bgl II fragment containing the truncated, synthetic
cry1Ba gene (2080 bp) (Desai, 1999) was cloned into a
Bgl II-site inserted in the SacI-site of pBluescript SK+ vector.
Using the Quick-Change™ Site Directed Mutagenesis Kit
(Stratagene), three restriction sites were subsequently
introduced: BspHI at base 1, MunI at bp 890, and Rsr II at bp
1480. The resulting pSN23 plasmid was subsequently used
for reconstruction of the SN19 hybrid encoding gene by
Leaf tissue (0.2 g) was ground with 400 µL of extraction
buffer (50 mM NaOH, 20 mM NaS2O5, 5 mM EDTA and 10%
Polyvinylpoly pyrrolidone), subsequently neutralized with
80 µL 1 M Tris-HCl, pH 5.5, and centrifuged at 16 000 g
for 10 min. The supernatant was transferred into a new
Eppendorf tube and additionally centrifuged at 16 000 g for
10 min. Protein concentrations in the supernatant were
determined by the Bradford method (Bio-Rad Laboratories).
© Blackwell Publishing Ltd, Plant Biotechnology Journal (2003), 1, 51–57
56 Samir Naimov et al.
The amount of Cry protein of interest was estimated by dotblot analysis as follows. Equal amounts of soluble leaf protein
(20 µg) were transferred to a nitrocellulose membrane using
an S&S Minifold Dot blotter (Schleicher & Schuell). Immunological detection was performed by treating the membrane
with blocking solution containing Tris buffered-saline (TBS:
10 mM Tris-HCl, pH 7.6, 150 mM NaCl), 5% (w/v) non-fat dry
milk, and 3% (w/v) Bovine serum albumin for 1 h, washed
three times with TBST buffer (TBS buffer, with 0.2% Tween20). 1 : 1000 diluted anti-Cry1Ba serum was applied and the
membrane was incubated for 1 h at room temperature. After
three washing steps with TBST, alkaline phosphatase conjugated anti-rabbit IgG (Sigma-Aldrich) was added (1 : 1000)
and incubated for 1 h. The membranes were washed three
times with TBST buffer, and once with carbonate buffer
(0.1 M NaHCO3, 1.0 mM MgCl2, pH 9.8). After 15 min incubation with 50 mL carbonate buffer, the membranes were
developed with 0.015% (w/v) 5-bromo-4-chloro-3-indolyl
phosphate (Sigma-Aldrich) and 0.03% (w/v) Nitro Blue
Tetrazolium (Sigma-Aldrich) in carbonate buffer. Serial
dilutions of trypsin-activated SN19 in phosphate buffed
saline (10 mM Na2HPO4 /KH2PO4, 0.8% (w/v) NaCl) added to
negative control plant extracts were used for comparison and
estimation of SN19 content in leaf tissues.
Acknowledgements
We would like to thank Syngenta Agribusiness Biotechnology Research, Inc. (Research Triangle Park, NC, USA) for
making available the synthetic cry1Ba gene. We also thank
Dr Silvia Arnone (ENEA C.R. Casaccia, Rome, Italy) for
supplying us with PTM eggs, and Dr Jeroen van Rie (Aventis
CropScience, Gent, Belgium) and Prof. Dr David Ellar (University of Cambridge, UK) for antisera. Furthermore, technical
assistance by Jos Molthoff, Petra Bakker and Mieke WeemenHendriks is acknowledged.
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