Toxicon 53 (2009) 122128

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Expression of a spider venom peptide in transgenic

tobacco confers insect resistance
Benjamn Hernandez-Campuzano a, Ramon Suarez a, Laura Lina a, Victor Hernandez a,
Elba Villegas a, Gerardo Corzo b, Gabriel Iturriaga a, *
a
b

n en Biotecnologa-UAEM, Av. Universidad 1001, Col. Chamilpa, Cuernavaca 62209, Mexico

Centro de Investigacio
Instituto de Biotecnologa-UNAM, Av. Universidad 2001, Col. Chamilpa, Cuernavaca 62210, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 August 2008
Received in revised form 8 October 2008
Accepted 16 October 2008
Available online 25 October 2008

Spider venom contains a mixture of peptide toxins, some able to kill insects specically to
those considered as important pest. In this study, a peptide toxin produced by the Macrothele gigas spider, Magi 6, was cloned and expressed in tobacco plants, as this toxin has
been shown to constitute an effective insecticide. For this purpose, a genetic construction
for the cDNA that codies for Magi 6 was subcloned in a plant expression vector using the
35S promoter and the 50 -end leader from tobacco mosaic virus, in order to transform
tobacco leaf disks. The resulting plants demonstrated the presence of Magi 6 gene in the
tobacco genome using PCR, and transcription of the cDNA was veried by means of RTPCR. The expression of the Magi 6 peptide in tobacco was demonstrated by Western blot,
which exhibited the expected size, thus suggesting a correct processing of the signal
peptide. No morphological alterations in the different transgenic lines were observed, nor
any change in plant growth. Subsequently, experiments were carried out challenging
detached leaves or whole plants with the herbivorous insect Spodoptera frugiperda. The
bioassays indicated that the transgenic lines were signicantly more resistant than the
wild type plants. This work demonstrated that the expression of Magi 6 peptide in
transgenic plants conferred resistance to insect attack and opens the possibility of
employing this peptide to improve the resistance of diverse plants.
2008 Elsevier Ltd. All rights reserved.

Keywords:
Macrothele gigas
Spider venom
Toxin
Transgenic tobacco

1. Introduction
Plagues are a serious threat to agriculture, causing huge
losses to farming. For a number of decades chemical
pesticides of diverse origin have been used to combat
insects, many of which are neurotoxic and carcinogenic, as
well as acting as persistent contaminants. Furthermore,
insect plagues have become resistant to many chemical
pesticides after many years of use.
A possible alternative solution to the problem posed by
agrochemicals is biological control or genetically modied
plants, in order to decimate insect plagues. To date, the

* Corresponding author. Tel.: 52 777 3297057; fax: 52 777 3297030.

E-mail address: iturri@buzon.uaem.mx (G. Iturriaga).
0041-0101/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2008.10.020

most common organism used as a bioinsecticide is Bacillus

thuringiensis (Bt), which codies for the insecticidal Cry
proteins (Bates et al., 2005). The Cry d-endotoxins form
crystals, which are proteolitically processed and dissolved
in the insect intestine, binding to the cellular membranes of
the epithelium, in order to form ion channels that induce
cell osmotic lysis and cause death to the insect (Bravo et al.,
2007).
During the last decade, the employment of transgenic
plants expressing Cry proteins has represented one of the
most important advances in agricultural biotechnology
(Ferry et al., 2006). Even though cases of insects resistant to
transgenic plants expressing cry genes have not been
detected in the eld, it is recommendable that a variety of
strategies are developed over the long term, in order to
avoid that insects become resistant. One option is

B. Hernandez-Campuzano et al. / Toxicon 53 (2009) 122128

pyramiding the expression of various Cry genes in plants

(Maqbool et al., 2001; Kurtz et al., 2007) or combining the
use of a variety of molecules to express hybrid Cry proteins
that manifest greater toxic effect on insects (Naimov et al.,
2003; Mehlo et al., 2005). Other examples of genes
different from the Bt toxins, for the production of transgenic plants resistant to insects are esculentin from
amphibians, chicken avidin and protease inhibitors from
both animals and plants (Christeller et al., 2002; Ponti et al.,
2003; Yoza et al., 2005; Abdeen et al., 2005). The overexpression of a Myb transcription factor gene involved in
the synthesis of secondary metabolites provides resistance
to insect plagues in maize (Johnson et al., 2007). Recently,
insect resistance of tobacco expressing a poisonous toxin
from the Hadronyche versuta spider was reported (Khan
et al., 2006).
The venom from insectivorous spiders represents
a complex mixture of molecules and peptides with a wide
range of mechanisms for activity at a biochemical level,
thus having a great biotechnological potential for
improving resistance to insects. An important constituent
of the spider venom consists of 410 kDa strong ligand
peptides, that are tightly folded by means of various
intramolecular disulde bridges and which include a great
diversity of antagonists, acting on the ion channels of
excitable membranes (Olivera et al., 1994; Grishin, 1999).
Among the great number of known spider species, the
Macrothele genus has been little studied and it is distributed in Iriomote a southern Japanese island. In a previous
work, it was described the purication and amino acid
sequencing of 6 peptides (Magi 16) that are found in
M. gigas poison (Corzo et al., 2003). Also, ten putative
peptides (Magi 716) whose sequence was deduced from
the cDNA of the venom glands of such spider were
described (Satake et al., 2004). The Magi 16 peptides were
fractionated by cation-exchange chromatography and some
of them act as neurotoxins or have site-specic afnity for
sodium channels. The six peptides were injected into
lepidoptera larvae Spodoptera litura, where both Magi 4 and
6 represented those inicting greatest lethality to insects
(Corzo et al., 2003).
In order to extend these observations and explore the
possible use of Magi 6 as a bioinsecticide, in this work the
cDNA of Magi 6 was cloned and expressed in transgenic
tobacco plants; moreover, their resistance to insect attack
was analyzed.
2. Materials and methods
2.1. Gene constructs and plant transformation
A 275-bp fragment from Magi 6 cDNA without leader
sequence was amplied using Expand High Fidelity PCR
System (Roche) by PCR with forward (50 -CATGCCATGGCTT
GCTGAAGGAAATGCAGC-30 ) and reverse (50 -GGGGTACCA
TCAACATCTCATGTTGCAGAGTACG-30 ) primers. The PCR
program consisted of 20 cycles of amplication (94  C,
0.5 min; 55  C, 0.5 min; 72  C, 0.5 min). The added CCATGG
sequence to create the NcoI site resulted in two extra amino
acids, proline and tryptophan, after the initiation codon
(underlined). The cDNA was digested with NcoI and KpnI

123

and cloned in a pBluescriptKS-derived plasmid (pJLU27)

containing the tobacco mosaic virus 50 -end leader sequence
(Dowson Day et al., 1993). The XbaIKpnI insert was excised
and subcloned in pBin19 vector (Bevan, 1984), containing
the 0.8 kb 35S promoter and 0.3-kb NOS polyadenylation
site. The construct was introduced by electroporation in
Agrobacterium tumefaciens LBA4404 strain and used to
transform Nicotiana tabacum L. cv. Petite Havana SR1
(Horsch et al., 1985). Regenerated tobacco transgenic plants
were selected on Murashige and Skoog medium (Sigma)
(Murashige and Skoog, 1962) containing 100 mg ml1
kanamycin (Sigma) and solidied with 0.8% phytagar
(Sigma) before being transferred to pots with soil and
grown in the greenhouse.
2.2. Genomic PCR and expression analysis
Genomic DNA from tobacco was isolated with a DNA
isolation kit (Puregene Gentra Systems) and 100 ng were
used for a PCR reaction using same oligonucleotides as
described above corresponding to Magi 6 cDNA. The same
PCR program was used except that amplication was of 35
cycles and one nal cycle at 72  C for 5 min. RT-PCR
experiments were performed using 2 mg of total RNA
extracted from tobacco plants using TRIZOL reagent
according to manufacturers instructions (Invitrogen), and
an oligo dT for 1st strand cDNA synthesis with SuperScript
II reverse transcriptase (Invitrogen) was used. PCR was
conducted using oligonucleotides corresponding to the
275 bp Magi 6 cDNA (described above) and the same
program except that only 25 cycles were used which corresponded to the linearity phase of the exponential reaction after comparison of the PCR products at different
cycles. PCR products were resolved in 1X Tris-acetic acidEDTA, 1% agarose gels stained with ethidium bromide.
2.3. Western blot
Protein extracts were prepared by homogenization of
100 mg plant tissue in a buffer containing 50 mM Tris-HCl
pH 7.0. Protein concentration was assayed using a protein
determination kit (Bio-Rad) based on the Bradford method
(Bradford, 1976). Proteins were precipitated overnight with
acetone and small proteins fractionated in a Microcon YM10 column (Millipore). Equal amounts of protein were
loaded per lane and separated in a 15% SDS-PAGE, and
transferred onto nitrocellulose Hybond-C membrane
(Amersham). Membranes were blocked with TBST buffer
(10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween 20),
containing 5% fat-free milk powder, incubated overnight at
4  C with Magi 6 mouse anti-serum diluted 1:100, washed
2 with TBST at 25  C, and incubated with a secondary
antibody conjugated to horseradish peroxidase diluted
1:1000. Immune complexes were detected by the chemioluminescence detection assay (Amersham).
2.4. Insect toxicity bioassays
Experimental assays on tobacco leaves were conducted
using Spodoptera frugiperda (Lepidoptera: Noctuidae)
larvae from neonatal to 6 instar. Five groups of ten larvae

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B. Hernandez-Campuzano et al. / Toxicon 53 (2009) 122128

from a single stage of development (neonatal and 23, 34,

45, and 56 instar) were placed in humid Petri dishes with
detached leaves from 8-week-old transgenic tobacco plants
from lines 56, 57 and 104, respectively. Detached leaves
from wild type tobacco were used as control. All Petri
dishes were maintained at 25  C with a relative humidity of
70%. The mortality of the neonatal larvae and the damage to
the leaves caused for such neonatal larvae were recorded
on the third day. The mortality of the instar larvae and the
damage to the leaves caused by them were assessed on the
seventh day. Concerning the assays using the entire plants,
the transgenic lines (lines 56, 57 and 104) and the wild
plants were infested with 20 larvae each and caged
together. Mortality of larvae and the amount of damage
were assessed after 6 days. All larvae toxicity bioassays
were repeated on three occasions and plant damage and
mortality of the larvae were evaluated 6 days later (Strizhov
et al., 1996). Entire plants were also maintained at 25  C
with a relative humidity of 70%.
2.5. Statistical analysis
Data were processed by analysis of variance using t
student test, followed by the Duncan-Waller mean analysis
(SAS Institute, 2002).
3. Results and discussion
3.1. Molecular characterization of Magi 6 gene
The Magi 6 cDNA clone was obtained from a M. gigas
venom gland library (Satake et al., 2004). The 313-bp
sequence (deposited in GenBank accession number
AB121203) revealed a 22-bp 50 -end leader sequence, and an
encoded peptide of 88 amino acids with a N-terminal
putative signal peptide of 25 amino acids and a 99-bp 30 end untranslated sequence containing a consensus
(AATAAA) polyadenylation sequence. Comparison of Magi 6
cDNA to the previously reported amino acid mature
sequence (Corzo et al., 2003) showed 100% identity with

the encoded peptide, and a predicted mature peptide of 37

amino acids with 8 cysteine residues suggesting the
formation of four intramolecular disulde bridges. No
homologous sequences to Magi 6 were found after analysis
in the public databases.
3.2. Expression of Magi 6 toxin in tobacco
To investigate the in vivo function of Magi 6 as bioinsecticide, its cDNA was overexpressed in tobacco plants.
The Magi 6 cDNA was subcloned without its native 50 -end
untranslated sequence in an Escherichia coli vector containing the tobacco mosaic virus 50 -end leader sequence to
enhance translation efciency in plants, since Magi 6 has
non-plant codon usage. (Dowson Day et al., 1993). The Magi
6 gene cassette was further subcloned in a plant transformation vector in front of the constitutive and strong 35S
promoter.
Seventy-one fully regenerated and kanamycin-resistant
tobacco plants were obtained after transformation with the
Agrobacterium tumefaciens system. The transgene integration event from each line was determined by genomic PCR
using Magi 6 gene as a target. Only 26 independent lines
had the gene insertion of the expected 0.3-kb size and 12
representative lines are shown (Fig. 1A). To assay for gene
expression of Magi 6 in transgenic plants, we used RT-PCR.
The 12 selected 35S::Magi 6 lines expressed the transgene
within a 3-fold range among the different lines (Fig. 1B).
Nine lines (lines 2, 26, 31, 56, 57, 66, 104, 107 and 109)
expressing moderate to high levels of Magi 6 transcript
were further analyzed for protein expression.
A western blot of total soluble protein extracted from
these 9 selected transgenic lines was conducted (Fig. 2).
The presence of Magi 6 was observed using an anti-Magi
6 mouse serum. Only in lines 56, 57 and 104 were
possible to detect a positive signal, showing a similar
level of Magi 6 expression among them. Magi 6 peptide
co-migrated with the synthetic toxin with an apparent
molecular weight of 4 kDa. Therefore it is likely that toxin
signal peptide was correctly processed as expected

Fig. 1. Molecular analyses of transgenic plants. (A) Genomic PCR of tobacco lines transformed with the 35S::Magi 6 construct (lines 2-160). The DNA band
corresponds to a 300-bp PCR fragment of Magi 6 gene. () represents an amplication control from pBin19 containing Magi 6. WT represents untransformed wild
type plants. (B) Gene expression analysis by RT-PCR of tobacco lines transformed with the 35S::Magi 6 construct (lines 2-160). The DNA band corresponds to
a 300-bp PCR fragment of Magi 6 gene.

B. Hernandez-Campuzano et al. / Toxicon 53 (2009) 122128

Fig. 2. Western blot analysis of Magi 6 peptide accumulation. (A) Equal

amounts (20 mg) of plant protein extract were loaded in each gel lane from
transformed (lines 56, 57 and 104) and untransformed (WT) tobacco. ()
corresponds to 0.7 mg of Magi 6 synthetic peptide. (B) Quantication of the
shown bands in arbitrary units.

(Fig. 2A). This process takes place in the endoplasmic

reticule, where disulde isomerase is also present
(Swanton and Bulleid, 2003). Thus it is probable that the
Magi 6 disulde bridges had formed.
The expression level of Magi 6 in the transformed plants
was estimated to be between 4 and 6% of soluble protein that
is equivalent to 1722 mg/g of plant tissue after comparison
with the amount of the control line of Magi 6 and a band
densitometric analysis (Fig. 2B). The three selected
35S::Magi 6 lines were grown in soil and T2 generation
selected. Comparison of 35S::Magi 6 lines with wild type
plants showed no morphological alterations or growth
retardation, suggesting that toxin expression in transgenic
tobacco had no apparent effect on plants (data not shown),
and it could also potentially mean that the expression of
Magi 6 in crops would not affect their yield. Transgenic lines
56, 57 and 104 were chosen for further analysis.
3.3. Resistance to insect damage of transgenic tobacco plants
Previous results have shown that Magi 6 was toxic to
larvae, however its mechanism of action is still unknown
(Corzo et al., 2003). Thus, once the presence of the peptide
Magi 6 was detected in transgenic tobacco plants, they
were tested for resistance against S. frugiperda as a model
for tobacco plague. S. frugiperda larvae from all instars were
reproduced and fed with tobacco plants, in order to
accustom them to this diet. When neonatal larvae were
used, a 100% mortality rate was observed on larvae fed on
leaves from wild type plants, as well as on those fed on
leaves from transgenic plants expressing Magi 6 (Fig. 3A).
This is not surprising since it is well known that tobacco has
insecticide properties per se, it is rich in alkaloids such as
nicotine, and because neonatal larvae are not used to a diet
based on this plant they die (Fig. 3A). However, at later
stages of larvae development they become adapted to the
tobacco leaves diet and the effect of Magi 6 peptide starts to

125

be clearly evident. The symptoms of mortality, consisted in

cessation of feeding and uncontrolled movements, followed by paralysis. In the case of larvae from the 23 instar,
mortality diminished by about 20%, but there was no
signicant difference (p > 0.05) in terms of the type of diet
based on wild type or transgenic leaves (Fig. 3A). In
contrast, the larvae of the 34 and 45 instar fed on wild
type plants demonstrated a mortality of 25% and 36%,
respectively, whilst the same instars fed on transgenic
plants demonstrated a mortality of 75% and 86%, respectively, representing a signicant (p < 0.05) 2.5 fold difference when compared to the non-transformed control
(Fig. 3A). It must be remarked that the death of larvae fed
on transgenic plants was caused by accid paralysis, similarly to previous observations when the puried Magi 6
toxin was injected in Spodoptera litura larvae (Corzo et al.,
2003), suggesting that the toxin is acting in transgenic
plants by probably the same mechanism. Thus, this data
suggests that Magi 6 expressed in tobacco acts as an
insecticide, which can be visually appreciated in isolated
leaves of tobacco, incubated over a period of seven days
with S. frugiperda larvae from the 45 instar (Fig. 3B).
Therefore, the transgenic plants are capable of resisting
damage from S. frugiperda (Fig. 3B).
In order to reinforce the previous analysis, bioassays
were undertaken for S. frugiperda using whole plants. Sixweek-old tobacco plants from the 56, 57 and 104 lines or
the wild type were inoculated with 45 instar larvae and
caged together. The results indicated a greater mortality
among the S. frugiperda larvae which had fed on 57 and 104
transgenic lines, when compared to the wild type, whereas
in the case of line 56, mortality was similar to that of the
non-transformed plant (Fig. 4A). On the other hand, the
insects showed a clear preference for non-transformed
plants (Fig. 4B), demonstrating the resistance to larvae
attack in the case of plants expressing the Magi 6 toxin.
Recently the peptide toxin u-ACTX-Hv1a (Hvt), from the
H. versuta spider, which functions as an antagonist of
calcium ion channels, was expressed in tobacco plants and
shown to confer insect resistance (Khan et al., 2006). In the
present study, we demonstrated that transgenic plants are
resistant to insect attack. We also showed that Magi 6
peptide was accumulated in leaves at 46% of soluble
protein that is equivalent to 1722 mg/g of plant tissue
(Table 1), and this was sufcient to make plants resistant to
insects (Fig. 3 and 4). Comparison of Magi 6 accumulation
in transgenic tobacco to the expression level of other bioinsecticide peptides reveals that this is one of highest levels
so far achieved (Table 1). Expression of different Cry
proteins in tobacco, tomato, alfalfa and potato was
comparatively low (Perlak et al., 1991, 1993; Strizhov et al.,
1996) to the expression of Magi 6 in tobacco plants. Other
peptides with bioinsecticide activity expressed in tobacco,
tomato or rice such as esculentin (Ponti et al., 2003),
proteinase inhibitors (Christeller et al., 2002; Abdeen et al.,
2005), avidin (Yoza et al., 2005) or the spider venom Hvt
toxin (Khan et al., 2006) accumulated at higher levels than
Cry proteins but still less than Magi 6 (Table 1). In all these
reports including the present study, the 35S promoter was
used; and in many instances, a leader with high translation
efciency was also part of the construct.

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B. Hernandez-Campuzano et al. / Toxicon 53 (2009) 122128

Fig. 3. Detached leaf toxicity assays. (A) Determination of mortality in insect larvae released on detached leaves of tobacco expressing Magi 6 (line 104) and
untransformed (WT) plants caged separately. (B) S. frugiperda larvae (4-5 instar) were released on the leaves and photographed after 7 days. White bars represent
wild type (WT), and black bars represent plant 104. The error bars indicate standard deviation. Asterisks indicate that the means of the samples are different at
the 0.05 level (p < 0.05).

An important question that remains unanswered is how

Magi 6 expressed in tobacco plants affects insects. First of
all, the oral administration of active insect neuropeptides
such as Magi 6 is unlikely to be successful since the insect
gut enzymes will probably degrade them. However, the
delivery of small peptides could be through a protein
carrier such as lectins. Previous studies have shown that
the snowdrop lectin from Galanthus nivalis and Nictaba
lectin from Nicotiana tabacum are resistant to gut proteolysis and can be detected in the haemolymph of orally
exposed insects (Fitches et al., 2004; Vandenborre et al.,
2008). Therefore, the ability of lectins to cross the gut
epithelium gives them the potential to act as a carrier to
deliver peptides to the circulatory system of target insect
species. Up to now, it has been demonstrated that the
snowdrop lectin delivers a fused insect neuropeptide to the
haemolymph of lepidopteran larvae following oral administration (Fitches et al., 2002; Edwards et al., 2002). In this
way, the ingestion of a spider neuropeptide together with

plant lectins could allow the translocation of such molecules from the digestive track to the insect hemolymph. In
the present work we show that insects fed on tobacco
expressing Magi 6 die, suggesting that this peptide toxin is
stable in the insect midgut. Also, it is worth mentioning
that Magi 6 resembles the structure of plant defensins
(Whetstone and Hammock, 2007). Plant defensins protect
organisms from pathogens or pest attack, and have structural features that are characteristic of the cysteine-knot
motif (Grubera et al., 2007), which is remarkably similar to
those of arachnid toxins, including Magi 6, in structure but
not in sequence. The mechanism of action of plant defensins is still unclear but certainly they could cross through
the digestive system.
Concerning other insect species that might be affected
by Magi 6 toxin, the oral ingestion and the translocation
from the digestive track to the insect hemolymph, would
depend on the insect midgut specic conditions. For
example, the biological activity of Bt toxins seems to

B. Hernandez-Campuzano et al. / Toxicon 53 (2009) 122128

127

Fig. 4. Whole plant toxicity assays. (A) Determination of mortality in insect larvae (4-5 instar) on different tobacco lines (56, 57 and 104) expressing Magi 6 and
on untransformed (WT) plants. (B) S. frugiperda larvae (4-5 instar) were released on transgenic (line 104) and wild type (WT) tobacco plants caged together. The
photograph was taken 6 days after insect release. White bars represent wild type plants (WT), and black bars represent transgenic plant 104. The error bars
indicate the standard deviation. Asterisks indicate that the means of the samples are different at the 0.05 level (p < 0.05).

Table 1
Expression level of peptide toxins in transgenic plants.
Protein

Plant

% Soluble mg/g
tissue
protein

CryIA

tobacco
tomato
potato
alfalfa
tobacco
tobacco
tobacco

0.20.3

NRa

NRa
0.010.2
0.10.2
NRa
0.5

0.0020.3 Perlak et al., 1993

NR
Strizhov et al., 1996

Cry3A
CryIC
Esculentin
Animal proteinase
inhibitor
Plant proteinase
inhibitor
Avidin
u-ACTX-Hv1a toxin
Magi 6
a

Not reported.

Reference
Perlak et al., 1991

12
NRa

Ponti et al., 2003

Christeller et al., 2002

tomato 1

NRa

Abdeen et al., 2005

rice
NRa
tobacco 0.10.25
tobacco 46

1
NRa
1722

Yoza et al., 2005

Khan et al., 2006
This work

depend on several biochemical characteristics of the insect

target such as midgut receptors, pH and protease concentration (Groot and Dicke, 2002). Therefore, other insect
species including benecial insects, might be affected by
Magi 6 depending on the biochemical characteristics of
their digestive track. These studies should be carried out
together with toxicity tests on mammals fed on plants that
express Magi 6 before any eld trial of a crop plant
expressing this gene is conducted. Perhaps the rst application of Magi 6 as a bioinsecticide could be carried out in
inedible crops, such as cotton or ornamental plants.
Acknowledgments
We thank Dr. Jorge L. Folch and Dr. Honoo Satake for
their kind gift of pJLU27 plasmid and Magi 6 cDNA,

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B. Hernandez-Campuzano et al. / Toxicon 53 (2009) 122128

respectively. G.I. received funding from CONACYT-Mexico

(No. 2004-CO1-46078), and G.C. from DGAPA-UNAM (No.
IN226006). One of the authors, Benjamn HernandezCampuzano, was a recipient of a CONACYT studentship.
Conict of interest
The authors declare that there are no conicts of
interest.
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