Download Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus

Biochem. J. (2009) 419, 309–316 (Printed in Great Britain)
309
doi:10.1042/BJ20081152
Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus
thuringiensis is due to improper processing of the protoxin
Raman RAJAGOPAL*, Naresh ARORA*, Swaminathan SIVAKUMAR*, Nagarjun G. V. RAO†, Sharad A. NIMBALKAR†
and Raj K. BHATNAGAR*1
*Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi 110 067, India, and †Division of Entomology, Dr. Panajbrao Deshmukh
Agricultural University (Dr. PDKV), Akola 444 104, India
The bacterium Bacillus thuringiensis produces ICPs (insecticidal
crystal proteins) that are deposited in their spore mother cells.
When susceptible lepidopteran larvae ingest these spore mother
cells, the ICPs get solubilized in the alkaline gut environment. Of
approx. 140 insecticidal proteins described thus far, insecticidal
protein Cry1Ac has been applied extensively as the main
ingredient of spray formulation as well as the principal ICP
introduced into crops as transgene for agricultural crop protection.
The 135 kDa Cry1Ac protein, upon ingestion by the insect, is
processed successively at the N- and C-terminus by the insect
midgut proteases to generate a 65 kDa bioactive core protein. The
activated core protein interacts with specific receptors located at
the midgut epithilium resulting in the lysis of cells and eventual
death of the larvae. A laboratory-reared population of Helicoverpa
armigera displayed 72-fold resistance to the B. thuringiensis
insecticidal protein Cry1Ac. A careful zymogram analysis of
Cry1Ac-resistant insects revealed an altered proteolytic profile.
The altered protease profile resulted in improper processing
of the insecticidal protein and as a consequence increased the LC50
concentrations of Cry1Ac. The 135 kDa protoxin-susceptible
insect larval population processed the protein to the biologically
active 65 kDa core protein, while the resistant insect larval
population yielded a mixture of 95 kDa and 68 kDa Cry1Ac polypeptides. N-terminal sequencing of these 95 and 68 kDa
polypeptides produced by gut juices of resistant insects revealed
an intact N-terminus. Protease gene transcription profiling by
semi-quantitative RT (reverse transcription)–PCR led to the
identification of a down-regulated HaSP2 (H. armigera serine
protease 2) in the Cry1Ac-resistant population. Protease HaSP2
was cloned, expressed and demonstrated to be responsible for
proper processing of insecticidal protoxin. The larval population
displaying resistance to Cry1Ac do not show an altered sensitivity
against another insecticidal protein, Cry2Ab. The implications of
these observations in the context of the possibility of development
of resistance and its management in H. armigera to Cry1Ac
through transgenic crop cultivation are discussed.
INTRODUCTION
insect pest with an LC50 of 12–25 ng/cm2 [2]. Concerns regarding
the possibility of an emergence of resistance against Cry1Ac
due to failure of implementing the recommended cultivation
regimen have been raised, thus questioning the sustained use
of these transgenics [3,4]. Laboratory-reared populations of
H. armigera showing resistance to Cry1Ac have been reported and
they show cross resistance to other effective insecticidal proteins,
e.g. Cry1Aa and Cry1Ab [5]. Isolated reports describing the
emergence of resistance under laboratory conditions with different
insects have identified receptor mutations or altered protease
profiles in the resistant population as the basis of resistance [6–9].
Though there have been no reports of field populations
of insects resistant to Cry1Ac in H. armigera, nevertheless,
such a possibility is a definite threat [4] given that laboratory
populations resistant to Cry1Ac have been isolated [8]. Since
insects have developed resistance to all the different chemical
insecticides used today, diverse resistance management strategies
have been recommended the world over to contain the resistance
development risk, which includes use of B. thuringiensis and
non-B. thuringiensis crop mixtures either as pure populations in
adjacent plots or as mixtures within the same plot [10]. This
strategy of providing a refuge is not being strictly practiced in
Bacillus thuringiensis spore crystal formulations have been
used commercially in agriculture and public health insect pest
management for the last thirty years very successfully. Within
the last few years, the 3.6 kb gene encoding the insecticidal
protein Cry1Ac has been integrated in the genomes of crop plants
such as maize, soybean and cotton to offer protection against
Helicoverpa armigera and other pests. The Cry1Ac transgenic
cotton plant (event 531, Monsanto, U.S.A.) has been cultivated
for the last 6–7 years in vast agricultural areas of the U.S.A.,
Argentina, Australia, China and India [1]. The adoption of
transgenic cropping has resulted in a significant reduction in the
usage and application of chemical insecticides.
The cotton bollworm, H. armigera, is the principal cotton pest
in the old world and inflicts major losses in India, China, Pakistan,
South and East Asia and parts of West Africa and Australia. Over
a period of time the insect has developed resistance and cross
resistance to various groups of chemical insecticides, including
organochlorines, organophosphates, carbamates and synthetic
pyrethroids. The insecticidal protein Cry1Ac is the most effective
of all B. thuringiensis insecticidal proteins tested against this
Key words: Cry 1Ac, insect, insecticide, pest management, toxin,
trypsin.
Abbreviations used: BBMV, brush border membrane vesicle; HaSP, Helicoverpa armigera serine protease; IB, inclusion bodies; ICP, insecticidal crystal
protein; IPTG, isopropyl β-D-thiogalactoside; LC50 , lethal concentration for 50 % mortality; ORF, open reading frame; RT, reverse transcription; UPGMA,
unweighted pair group method with arithmetic mean.
1
To whom correspondence should be addressed (email [email protected]).
The nucleotide sequence data reported for HaSP1, HaSP2, HaSP3, HaSP4, HaSP5 and HaSP6 will appear in the DDBJ, EMBL, GenBank® and GSDB
Nucleotide Sequence Databases under the accession numbers EF104918, EF104913, EF104914, EF104915, EF104916 and EF104917 respectively.
c The Authors Journal compilation c 2009 Biochemical Society
310
R. Rajagopal and others
India and elsewhere. The general argument in not planting refugia
is the wide availability of different non-transgenic host plants and
other alternate host crops in the immediate vicinity of cotton fields
[11]. India also has the distinction of growing large areas of cotton
with “illegal” B. thuringiensis cotton hybrids and simulation
studies have reported that development of resistance to Cry1Ac is
a distinct possibility [12]. However, for all suggested resistance
delay management strategies to be implemented, understanding
the mode of action of Cry1Ac on target insects and also the
molecular mechanism of resistance development against this toxin
is important [13,14]. Here we report analysis of a laboratory
reared population of H. armigera resistant to Cry1Ac. This
resistant population displayed an altered protease profile resulting
in improper processing of the Cry1Ac protoxin. Further, a specific
protease, HaSP2 (H. armigera serine protease 2), which is capable
of proper processing and activation of Cry1Ac polypeptide,
among the six different groups of serine proteases from
H. armigera midgut, displayed significantly reduced expression in
the resistant insect larvae. Such resistant insect larvae do not show
cross resistance to another insecticidal protein Cry2Ab, active
against H. armigera.
EXPERIMENTAL
Insects
H. armigera was reared and maintained on a semi-synthetic
artificial diet consisting of 300 g of chickpea flour, 10 g of
wheat germ, 5.3 g of ascorbic acid, 1.7 g of sorbic acid, 3.3 g
of methyl-p-hydroxybenzoate, 20 g of Wesson’s salt mixture,
2.5 g of aureomycin, 10 ml of vitamin mixture, 10 ml of
formaldehyde, 48 g of yeast and 16 g of agar in 1 litre of distilled water. Larvae and pupae of cotton bollworms were
collected randomly from cotton fields in and around Akola
(Maharastra, India) and reared to provide a homozygous
population. All the rearing procedures were carried out at 27 +
−
2 ◦C under 14:10 light/dark regime. These insects were reared
in the laboratory for three generations to obtain a homozygous
population. The insects were selected for resistance to Cry1Ac
protoxin by releasing 400 naı̈ve neonate H. armigera larvae on a
diet coated with 40 ng/cm2 of diet at one larva per well. After five
days, the 18 survivors were transferred to a toxin-free diet and
allowed to complete the life cycle. At each generation, neonate
larvae were released on a diet containing 40 ng of Cry1Ac/cm2
and survivors after 5 days were transferred to a toxin-free diet.
This procedure was continued for 10 generations.
Insect bioassays
ICPs (insecticidal crystal proteins) of Cry1Ac was purified from
a Escherichia coli strain JM 103 expressing the protoxin of
Cry1Ac (cloned in pKK 223-3 expression vector). Similarly, a
synthetic gene of Cry2Ab was constructed (GeneArt, Regensburg,
Germany), cloned and expressed in pQE30 expression vector. The
expression of Cry1Ac and Cry2Ab into the inclusion bodies of
E. coli cells was induced by 5 mM IPTG (isopropyl β-D-thiogalactoside) and allowed to grow for 3 h following induction.
Insecticidal protein concentration was estimated by densitometric
scanning (Alpha System, Midlothian, VA, U.S.A.) and comparing
with band intensities of BSA at different concentrations.
Bioassays were conducted on neonate larvae by placing them on
Cry protoxin incorporated diet in plastic vials. Cry1Ac inclusion
bodies were mixed in 2 ml of distilled water and the stock was
assayed in artificial diet at various concentrations such as 0.01,
0.05, 0.1, 0.5, 1.0, 2.5, 5 and 10 μg/ml diet. All the assays were
c The Authors Journal compilation c 2009 Biochemical Society
replicated four times and 30 larvae per treatment were used.
Mortality was recorded every 24 h after treatment for 3 days.
Pooled data were subject to statistical analysis. Median lethal
concentration (LC50 , lethal concentration for 50 % mortality) was
calculated by Probit analysis by using the computer software
Indo-Stat (Indo-Stat Services, India).
Gut extracts
Fifth instar 2nd day H. armigera larvae from the F10 generation
(both resistant and susceptible populations) were chilled on ice for
10 min and midguts dissected and homogenized in 50 mM Tris
pH 8.0 at 4 ◦C. This material was further centrifuged at 17 226 g,
4 ◦C for 15 min, and the clear supernatant was aliquoted and frozen
at − 80 ◦C until further use.
Protease activity profiles
The protein content in the gut extract was estimated by the method
of Bradford [15]. Gut extracts (5 μg/lane) were diluted in a sample
buffer without 2-mercaptoethanol and resolved on SDS/12 %
PAGE pre-incorporated with 1 % gelatin. The gel was resolved at
120 V at 10 ◦C. After the run, the gel was incubated in a solution
containing 2.5 % Triton X-100 with gentle shaking for 3 h to
replace the SDS with Triton X-100. The gel was then incubated
in Hepes buffer (50 mM Hepes, pH 8.0, 1 % Triton X-100 and
5 mM cysteine) at 37 ◦C for 15 h. Finally, the gel was stained in
0.5 % Coomassie Brilliant Blue G-250 for 3 h and destained with
10 % acetic acid for several hours till the halo regions depicting
protease activity appeared.
In a separate protease assay, the resolved gut extracts
(5 μg/lane) were incubated with 20 ml of Tris buffer (50 mM Tris,
pH 8.0, and 20 mM CaCl2 ) containing 0.3 % solubilized Cry1Ac
protoxin for 30 min at 4 ◦C followed by 2 h at 25 ◦C. The gel was
washed three times thoroughly with sterile water and stained with
0.5 % Coomassie Brilliant Blue G-250 for 30 min. The activity
profile was obtained by destaining the blot for 3 h in 10 % acetic
acid to observe the white halos.
Toxin preparation
The recombinant Cry 1Ac protoxin was prepared as IB (inclusion
bodies) by the method reported in [13] from a plasmid pKK223-3
bearing the Cry1Ac gene (Bacillus Genetic Stock Centre, Ohio
State University, OH, U.S.A.). The amount of the toxin was
quantified densitometrically by resolving the IB on SDS/PAGE.
The inclusion bodies containing the toxin were solubilized in
50 mM sodium carbonate buffer, pH 10.4. This protein was
purified by anion exchange chromatography and eluted with a
linear gradient of 0 to 1000 mM NaCl gradient prepared in 50 mM
sodium carbonate buffer, pH 10.4.
Activation of Cry1Ac protoxin by midgut juices
Purified Cry1Ac protoxin (2.5 μg) was incubated with midgut
juices (500 μg/ml total protein) of naı̈ve and laboratory-reared
Cry1Ac-resistant H. armigera population in the F10 generation at
37 ◦C. The reaction was stopped by adding 4× SDS/PAGE sample
buffer containing 2-mercaptoethanol. The reaction products were
resolved in a SDS/7.5 % PAGE gel and stained with Coomassie
Blue R-250 for 30 min. The proteins were visualized by destaining
with 10 % acetic acid.
N-terminal sequencing
Purified Cry1Ac protoxin (2.5 μg) was incubated with 0.2 μl
(500 μg/ml of total protein) of midgut juices for 10 min at 37 ◦C.
The reaction was stopped by adding 4× SDS/PAGE sample
Improper protoxin activation leads to Cry1Ac resistance in H. armigera
buffer containing 2-mercaptoethanol. The reaction products were
resolved on a SDS/7.5 % PAGE gel and electroblotted to a
nylon membrane (Millipore) using CAPS (3-[cyclohexylamino]1-propanesulfonic acid) buffer. The blot was stained with Commassie Blue for 10 min and washed repeatedly with methanol.
The bands corresponding to 95 kDa and 68 kDa were cut and
subjected to N-terminal sequencing at Iowa State University
protein facility (Ames, IA, U.S.A.). The first ten amino acids
were read from the proteins.
Cloning of serine proteases from H. armigera
Sequences of midgut serine proteases of H. armigera were
retrieved from the GenBank® , analysed for multiple alignment
by CLUSTAL W and dendrogram analysis by the UPGMA (unweighted pair group method with arithmetic mean) method using
the MacVector software (version 7.0). A protease representing
each of the six groups was chosen, and gene-specific primers for
the forward and reverse directions were synthesized. Midguts
from 5th instar 1st day larvae were dissected in diethyl pyrocarbonate water and total RNA was extracted using TRIzol reagent
(Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol. RT (reverse transcription) with oligo dT
primer used 5 μg of total RNA as template and Superscript II
to generate the cDNA. Using the respective sets of end primers (the
following primer sets were employed: HaSP1For, 5 -ATGAACAAATGGGTGCTGACTGCTGCTCACTGCC-3 and HaSp1Rev,
5 -GTACTCAACAACGCGGAC3 to yield 852 bp; HaSP2For, 5 ATGGCTTCCTACGCTACCTTCAC-3 and HaSP2Rev, 5 -AACAGCGTTTTCAACGAT-3 to yield 768 bp; HaSP3For, 5 -ATGAAACTCTTGGCTGTGACTCTATTG-3 and HaSP3Rev,
5 -AAGAAGATTGTTGATCCAGCTGAT3 to yield 885 bp;
HaSP4For, 5 -ATGAAGGGTTTTGCTTTCGTTTTGT-3 and
HaSP4Rev, 5 -GTTGACGAAACGGACGGCTTC-3 to yield
768 bp; HaSP5For, 5 -ATGCGTATCCTTGCTTTGGTAGCTCT3 and HaSP5Rev, 5 -CGCGTTAGATGAAATCCAGGAAGTGTAGCGAGA-3 to yield 762 bp; HaSP6For, 5 -ATGCGCATTCTTGCAGTTCTCGCCCT-3 and HaSP6Rev, 5 -CGCGTTAGATGAAATCCAGGAAGTGTAGCGAGA-3 to yield
769 bp), the six different proteases were cloned from the
cDNA of the naı̈ve population of H. armigera by performing 30
cycles of PCR, each of 94 ◦C for 30 s, 55 ◦C for 30 s and 72 ◦C
for 1 min. The PCR products from each of these 6 reactions were
cloned in pGEM-T easy vector and sequenced for their full length
in both directions. Ten clones each for each of the six different
proteases were sequenced. Identical sequences were obtained
from all clones, thus confirming the fidelity of the PCR reactions.
Table 1
311
Median lethal doses of Cry1Ac in H. armigera neonates
LC50 and LC90 are expressed as μg/ml diet. Sr., serial number; FL, fiducial limits.
Sr.
Number
Generation
LC50 dose
(μg/ml)
LC90 dose
(μg/ml)
FL limit
LC50
Slope
χ2
(+
− S.E.M)
1
2
3
4
5
6
7
8
9
10
11
F1 (selected)
F2 (selected)
F3 (selected)
F4 (selected)
F5 (selected)
F6 (selected
F7 (selected)
F8 (selected)
F9 (selected)
F10 (selected)
Susceptible
0.47
0.74
0.98
0.96
1.13
1.66
1.42
2.90
3.19
3.63
0.05
5.66
4.61
5.9
7.41
4.88
10.83
14.89
24.19
23.38
33.51
0.29
0.31–0.72
0.54–1.00
0.71–1.34
0.65–1.41
0.87–1.48
1.23–2.22
0.99–2.06
1.68–3.48
2.27–4.51
2.46–5.32
0.036–0.072
1.19
1.61
1.46
1.44
2.02
1.57
1.25
1.27
1.48
1.32
1.69
0.31 (0.23)
3.56 (0.21)
0.40 (0.26)
0.65 (0.25]
1.53 (0.29)
8.9 (0.18)
1.86 (0.19)
2.32 (0.2)
1.11 (0.22)
0.6 (0.21)
0.22(0.29)
were the same as those used for cloning these proteases as
described above. Before loading on to 1 % agarose gels, the
RT–PCR products were treated with 1 μg of RNase (Qiagen)
at 37 ◦C for 10 min to eliminate template RNA, since it obliterates
the accurate estimation of the β-actin gene product in the gel. The
gels were photographed with Polaroid 667 black-and-white print
film and scanned for net intensity of each RT–PCR product using
the software Kodak 1D, version 2.0.
Expression and purification of HaSP2
The sequence of HaSP2 was analysed using various bioinformatic tools for the ORF (open reading frame), signal peptide
region and the pro-peptide region. The mature protease region
(after the pro-peptide region) was amplified by PCR using
the HaSP2 cloned in pGemT-easy as template, forward primer
5 -ATGTACCCCAGCACCGTCCAACTG-3 (position 90–110
nucleotides), reverse primer 5 -AACAGCGTTTTCAACGAT-3
(position 757–775 nucleotides) and the resultant PCR product
cloned in pGemT-easy. This mature protein region, cloned into
pGemT-easy, was digested with EcoR1, cloned at the corresponding site in the expression vector pET 32a and transformed into
E. coli strain BL21(DE3). Heterologous protein expression was
induced by adding 1 mM IPTG to the bacterial culture after
reaching an attenuance of 0.5 at 600 nm and allowed to grow
for a further 2 h. The expressed protein accumulated in the
soluble fraction of E. coli and was purified by Ni-NTA (Ni2+ nitrilotriacetate) affinity chromatography. The bound protein was
eluted with 150 mM imidazole and used for Cry1Ac protoxin
activation assays.
Quantitative RT–PCR
For comparing the relative transcript abundance of each of
the above six different proteases between the naı̈ve and laboratory-reared Cry1Ac-resistant H. armigera populations of the
F10 generation, they were subjected to quantitative RT–PCR
analysis. The amount of RNA in both the treatments was
normalized by amplifying the β-actin gene in each treatment to
equal intensity after 20 cycles of RT–PCR using the one step RT–
PCR kit (Qiagen GmbH, Hilden, Germany). The amplification
regimen was as follows: reverse transcription at 43 ◦C for 35 min,
an initial activation step at 95 ◦C for 15 min followed by 20 PCR
cycles of denaturation at 94 ◦C for 30 s, annealing at 54 ◦C for 30 s
and extension at 72 ◦C for 1 min. Using this normalized amount of
RNA as template, the transcripts of all the six different proteases
in the Cry1Ac-resistant and naı̈ve populations were probed by
amplifying their gene products. The primers used for amplification
RESULTS
Development of resistance by H. armigera to Cry1Ac
H. armigera naı̈ve 1st instar larvae were selected on an artificial
diet containing Cry1Ac protoxin. After ten generations, the larvae
were able to tolerate 72-fold more Cry1Ac protoxin (in terms
of their LC50 dose) than the susceptible larvae (Table 1). The
resistance dose is reflected more in the LC90 values than in the LC50
values, suggesting that the larvae become much more adapted
to survive an ever increasing dose of the selection pressure.
Ligand-blot analysis of BBMV (brush border membrane vesicle)
proteins from the Cry1Ac-resistant and susceptible population
with activated Cry1Ac did not show any differences between
them (Supplemental Figure S1 at http://www.BiochemJ.org/bj/
419/bj4190309add.htm).
c The Authors Journal compilation c 2009 Biochemical Society
312
R. Rajagopal and others
Figure 2 Delayed activation of Cry1Ac 135 kDa protoxin by H. armigera
midgut juice
Lane 1, The protein molecular-mass marker (BioRad, 161-0309), with molecular masses in kDa
to the left; lane 2, ion-exchange purified Cry1Ac protoxin (2.5 μg); lane 3, as a control, the
Cry1Ac protoxin was also activated with 100 ng of trypsin for 30 min; lane 4, lane 2 incubated for
5 min at 37 ◦C with 25 ng total protein of midgut juice from H. armigera population susceptible
to Cry1Ac; lane 5, lane 2 incubated for 5 min at 37 ◦C with 25 ng total protein of midgut juice
from H. armigera population resistant to Cry1Ac. Lanes 6 and 7, 2.5 μg of ion-exchange purified
Cry1Ac protoxin was incubated for 10 min at 37 ◦C with 100 ng total protein of gut juice from
H. armigera population resistant to Cry1Ac (lane 6) or with recombinantly expressed HaSP2
(lane 7). Reactions were stopped as described in the Experimental section. The gels were stained
with Coomassie Blue to visualize the protein bands. The bands obtained in each treatment are
numbered and explained in the Results section.
Figure 1 Protease activity profiles of midgut extracts from naı̈ve and
Cry1Ac-resistant Helicoverpa armigera larvae
Activity profiles with (A) gelatin as substrate, bands P1 to P4 are protease bands seen in
susceptible insects; and (B) with Cry1Ac 135 kDa protoxin as substrate. Lane 1, midgut extract
from naı̈ve larvae; and lane 2, midgut extract from Cry1Ac-resistant larvae. CP1 to CP4 are
Cry1Ac protease bands seen in susceptible insects and CP5 and CP6 are Cry1Ac protease
bands seen in resistant insects.
Protease activity profiles of H. armigera midgut
Proteins from midgut extracts of H. armigera 5th instar 2nd
day larvae of Cry1Ac-resistant (10th generation) and susceptible
population were assayed for their ability to hydrolyse gelatin.
Midgut proteins were resolved in SDS/12 % PAGE and the gel
was subjected to an activity blot using gelatin as substrate. In
all the protease activity profiles, care was taken to resolve equal
amounts of protein. Differences were observed in the activity
profiles between Cry1Ac-resistant and normal insects (Figure 1).
Four halo regions representing proteolytic dissolution of gelatin
corresponding to the range of 25–35 kDa were clearly detected in
susceptible insects. The expression and mobility profiles of these
proteolytic halos were reduced in the Cry1Ac-resistant insects.
Besides these, higher molecular-mass proteins with gelatinhydrolysing abilities were also detected. These high-molecularmass proteins were more abundant in Cry1Ac-resistant insects
(Figure 1A).
Similar activity blots, where polypeptide Cry1Ac was used as
the substrate, revealed that susceptible insects had four major
halo regions acting on Cry1Ac. The expression of two of the
faster-migrating proteases in resistant insects are totally abolished,
whereas the upper two that move slowly are greatly reduced
(Figure 1B, proteases marked by arrows). Interestingly, two
different halo regions that are induced in resistant insects are
not observed in susceptible insects (Figure 1B). Taken together,
these results clearly demonstrate that insects resistant to Cry1Ac
have an altered protease profile with respect to both Cry1Ac and
gelatin.
c The Authors Journal compilation c 2009 Biochemical Society
Preliminary characterization of the nature of the protease involved in gelatin and Cry1Ac degradation by specific protease
inhibitors, leupetin and PMSF tentatively classified them as serine
proteases (results not shown).
Activation of Cry1Ac protoxin by H. armigera midgut juices
Subsequent to observing an altered protease activity profile
between resistant and susceptible insects, we studied the ability
of these midgut proteases to process Cry1Ac protoxin (Figure 2,
lane 2, band 1) to an activated form. Commercial trypsin was
able to cleave the protoxin to a 65 kDa activated Cry1Ac toxin
(Figure 2, lane 3, band 2). Gut juice from the susceptible insect
population (25 ng total protein) could properly cleave and activate
Cry1Ac to its reported 65 kDa form (Figure 2, lane 4, band 3)
while a 68 kDa band is also seen (Figure 2, lane 4, band 4),
whereas the gut juices from resistant insects (25 ng total protein)
failed to appreciably cleave the protoxin (Figure 2, lane 5). On
incubating Cry1Ac protoxin with a higher concentration (100 ng
total protein) of gut juice from resistant insects for 10 min, a
95 kDa polypeptide (Figure 2, lane 6, band 5) was formed in
addition to the 68 kDa protein (Figure 2, lane 6, band 6), and a
fully activated toxin of 65 kDa is also visible, but at very low
amounts below the 68 kDa band (Figure 2, lane 6). On prolonged
incubation of Cry1Ac protoxin with the gut juice of resistant
insects (for 30 min or more), the proportion of the 95 kDa protein
is reduced, whereas the proportion of the 68 kDa protein increased
(results not shown).
Furthermore, to demarcate the origin of the 95 and 68 kDa
polypeptides, we analysed their respective N-terminal sequence.
The analysis revealed that the 95 and 68 kDa polypeptides were
not preteolytically processed from the N-terminus and retained
the sequence of the protoxin. A cartoon in Supplemental Figure S2 (http://www.BiochemJ.org/bj/419/bj4190309add.htm)
depicts these results.
Cloning of serine proteases from H. armigera
The above results suggested that serine proteases (similar
to trypsin and chymotrypsin) of sizes between 25–35 kDa are
Improper protoxin activation leads to Cry1Ac resistance in H. armigera
Figure 3
313
Comparison of different midgut protease genes from H. armigera
Bootstrap (1000 repeats) dendrogram tree using the UPGMA method was constructed by analysing the CLUSTAL-W alignment of the reported protease sequences from the larval midgut of H.
armigera . GenBank® accession numbers of the proteases are Y12286 for SR 111; Y12276 for SR 28; Y12275 for SR 24 and SR 40; Y12277 for SR 36; Y12269 for HaTC11, TC21, TS4 and SR 99;
Y12271 for Ha TS7; Y12278 for SR41p; Y12283 for SR66; Y12283 for HaTC16; Y12273 for SR 19 and SR 110; Y12279 for SR42p; Y12284 for SR84; Y12281 for SR58; Y12287 for SR117; Y12285
for SR106; Y12280 for SR55; Y12274 for SR21; Y12272 for SR15p; and Y12282 for SR 65. Branch lengths are arbitary. The bootstrap values detected are given before the branch. One candidate
gene from each of the six protease groups identified (underlined) was further studied in detail in the naı̈ve and Cry1Ac-resistant H. armigera populations.
involved in the improper processing and hence the reduced activity
of Cry1Ac towards H. armigera. Analysis of the GenBank®
database for these proteins from H. armigera showed the proteins
to cluster into six different groups (Figure 3). We selected one
protein from each of the groups, and cloned and sequenced
them from our population of H. armigera (Figure 3). The
sequences of the proteases, namely HaSP1 (EF104918), HaSP2
(EF104913), HaSP3 (EF104914), HaSP4 (EF104915),
HaSP5 (EF104916) and HaSP6 (EF104917) have been deposited
in GenBank® . Sequence analysis of these proteins suggest that
they are serine proteases (Figure 4) with > 95 % identity with the
earlier reported sequences [16] deposited in GenBank® .
Quantitative comparison by semi-quantitative RT–PCR on the
level of expression of these serine protease transcripts between
naı̈ve and Cry1Ac-resistant populations showed the total absence
of protease HaSP2 in resistant insects (Figure 5). The expression
levels of other proteases remained almost similar, with an
increased level of HaSP6 transcript in resistant insects. The
protease HaSP2 was then cloned in expression vector pET32(b)
in-frame with an N-terminal tag of 22 kDa [TRX-109 amino acids,
followed by His, Thrombin, S-Tag, enterokinase site and the MCS
(multiple cloning site) amounting to approx. 90 amino acids],
which resulted in the expression of the protein in the supernatant of host E. coli BL21(DE3) cells (Supplementary Figure S3A at http://www.BiochemJ.org/bj/419/bj4190309add.htm).
The expressed protein was purified and found to hydrolyse the
substrate βApNA (N-benzoyl-DL-arginine p-nitroanilide) with
a specific activity of 121.9 nmoles/mg per h. This expressed
protease was purified to near homogeneity by ion exchange and
affinity chromatography. This purified protease, upon incubation
with Cry1Ac protoxin, yielded a 65 kDa activated polypeptide
thus confirming its ability to hydrolyse Cry1Ac to an active core
polypeptide (Supplementary Figure S3B and Figure 2, lane 7,
band 7).
Response of Cry1Ac resistant and susceptible H. armigera to
Cry1Ac transgenic cotton plants (Event 531)
The response of Cry1Ac-susceptible and -resistant H. armigera
neonates were similar when assayed on non-transgenic cotton
Mech-162. However, marked differences were observed when the
larval populations were assayed against Cry1Ac transgenic Mech162 B. thuringiensis (BollGard I). The mean percentage mortality
in the B. thuringiensis-susceptible strain was 95.5 %, whereas it
was 42.2 % in the Cry1Ac-resistant population (Table 2). Thus
the laboratory-reared Cry1Ac-resistant H. armigera population
was able to withstand the dose of Cry1Ac expressed in Cry1Ac
transgenic cotton plants of Mech 162 B. thuringiensis (BollGard
I). Although the LC50 dose of the Cry1Ac-resistant population
increased 72-fold against Cry1Ac, the same population showed increased susceptibility against another bioactive insecticidal
protein, Cry2Ab, present in BollGard II (Table 2) and there was no
significant difference in their LC50 values with respect to Cry2Ab.
Furthermore, when Cry1Ac protoxin-resistant insects were fed
purified trypsin-activated toxin, the magnitude of resistance
came down from approx. 72-fold to almost complete loss of
the resistance phenotype. The results indicated that there is
insignificant difference in the toxicity of activated Cry1Ac in
the resistant and susceptible stain of H. armigera and the toxicity
is nearly the same.
DISCUSSION
Insecticides constitute a major ingredient in modern crop husbandry, especially in tropical regions. Chemical insecticides
constitute nearly 90 % of the total insecticide market, the rest
being biopesticides. Insecticidal proteins produced by strains of
B. thuringiensis are the most successful biopesticide presently
used in commercial agriculture, especially against lepidopteran
c The Authors Journal compilation c 2009 Biochemical Society
314
Figure 4
R. Rajagopal and others
Protein sequence alignment of different proteases from the larval midgut of H. armigera populations used in the present study
One candidate gene from each of the six protease groups identified in Figure 3 was cloned from the Indian population of H. armigera and sequenced full-length. The ORF of these genes were
translated to their respective protein sequences which were compared by multiple alignment analysis among themselves. The protein residues that are conserved are shaded. The gene sequences of
these protease genes have been deposited in the Genbank® as described in the Results section.
Table 2 Response of neonate H. armigera strains to B. thuringiensis and
non-B. thuringiensis cotton
Ten neonate larvae were released in a leaf disc of the respective treatment and each treatment was
replicated ten times. The mortality data were analysed by Student’s t test and values superscripted
with different letters are significantly different at 95 % confidence limits. *H. armigera naı̈ve
population maintained without selection pressure for 10 generations. **H. armigera maintained
following 10 generations of selection on Cry1Ac.
H. armigera mortality (%)
Figure 5 Differential expression of serine protease genes in the larval
midgut of H. armigera from a naı̈ve population and a population resistant to
Cry1Ac
The relative expression of these genes was established by relative RT–PCR and normalized
using their β-actin transcripts. The primers used for amplifying the different protease genes
are described in the Experimental section. The gel was photographed with a Polaroid 667
black-and-white film and scanned for its mean intensity for each of the RT–PCR products using
the software KODAK 1D, version 2.0. M, markers; R, Cry1Ac-resistant; S, Cry1Ac-sensitive.
insects. Following ingestion by susceptible larvae, insecticidal
proteins are solubilized in the alkaline gut environment and
subsequently proteolytically activated in releasing an active
core toxin polypeptide that binds to specific receptors, leading
to pore formation and the eventual death of the insect. The
c The Authors Journal compilation c 2009 Biochemical Society
Variety for bioassay
Cry1Ac-susceptible population*
Cry1Ac-resistant population**
MECH-162
MECH-162 BolGard I
Mech 162 BolGard II
5.55a
95.55b
97.6b
4.44a
42.22c
67.9d
Table 3
Median lethal doses of Cry1Ac in H. armigera neonates
*H. armigera naı̈ve population maintained without selection pressure for 10 generations. **H.
armigera maintained following 10 generations of selection on Cry1Ac.
H. armigera mortality
Cry1Ac-susceptible population*
Cry1Ac-resistant population**
LC50 to Cry2Ab
LC50 to trypsin-activated
Cry1Ac
128.1 ng/ml
0.18 ng/ml
135 ng/ml
0.22 ng/ml
cotton bollworm, H. armigera, is a cosmopolitan, polyphagus
agricultural pest and is effectively controlled by the ICP Cry1Ac.
Transgenic cotton plants bearing the 3.6 kb cry1Ac gene (Event
Improper protoxin activation leads to Cry1Ac resistance in H. armigera
531, Monsanto Inc.) have been very successful in preventing the
damage caused by this insect in cotton plants in India for
the last 4 years [17]. There have been a number of reports of
laboratory-reared populations of insect pests such as Heliothis
virescens, Manduca sexta, Lymantria dispar, Plutella xylostella
and Plodia interpunctella developing resistance to Cry1Ac [18].
Studies directed towards understanding the basis of this resistance
reveal changes in the expression profile of the cadherin protein
and reduced binding of the toxin to midgut epithelium. Mutations
in the cadherin gene resulting in shorter transcripts have been
reported from Heliothis virescens and Pectinophora gossypiella
[6,7]. Other studies have reported changes in the activity profiles
of midgut proteases between resistant and naı̈ve insects [19].
Development of resistance has been directly correlated with the
loss in function of a major gut protease in Plodia interpunctella
[9]. There have been two reports on the development of resistance
by H. armigera to Cry1Ac. One of them reports a mutation in the
cadherin gene, whereas the other reports changes in the activity
of midgut proteases belonging to the class esterase [8,20].
During the course of this study, we developed a laboratory
population of H. armigera showing 72-fold resistance to the
insecticidal protein Cry1Ac. The midgut protease profile between
the naı̈ve and resistant populations showed characteristic differences with the loss in activity of two major protease polypeptides
hydrolysing gelatin in resistant insects. When presented with
Cry 1Ac as substrate, the two populations revealed contrasting
protease profiles. In susceptible insects, there were four major
Cry1Ac-hydrolysing protease zones, whereas in resistant insects,
activity of two of these zones is abolished and two new
Cry1Ac-metabolising zones are recruited. The Cry1Ac protoxin
is proteolytically processed by the midgut trypsin proteases, both
from the N-terminus and the C-terminus, to yield a 65 kDa active
toxin [21]. The exact nature of this reaction is not fully understood
and there has been no report of a single trypsin protease being
implicated in this process. It is believed that the correct activation
of the protoxin is one of the most important and essential steps for
proper insecticidal activity. Precise cleavage of the first 28 amino
acids from the N-terminus is absolutely essential for optimum
biological activity. It has been observed that Cry1Ac protoxins
not protolytically cleaved at the N-terminus result in 25-fold
less toxicity than the properly activated toxins lacking the first
28 amino acids [22]. N-terminal analysis of the 95 and 68 kDa
polypeptides of the Cry1Ac protoxin after processing by resistant
H. armigera gut juices revealed that in both polypeptides the first
28 amino acids of the protoxin have not been cleaved. Hence
it appears that a quantitative change in N-terminal proteolytic
processing of Cry1Ac is a major contributing event for resistance.
Earlier studies have reported that activated Cry1Ac with an
intact N-terminus binds more tightly with the receptors in the
BBMV of insect guts as compared with the precisely activated
form [22]. Such preferential binding probably results in the
competing out of precisely processed active toxin from the viable
toxin-binding sites on the receptor molecule by the imprecisely
processed and less potent toxin molecules, thus manifesting the
resistance phenotype. Taken together, these results with the earlier
observation that resistant insects have a faster degradation of the
activated toxins than the naı̈ve population clearly establishes an
important role for the protoxin-activating step in the resistance
development process.
Analogous resistance development processes has been reported
earlier from Plodia interpunctella and Plutella xylostella resistant
to Cry1Ac [19,23]. Such resistant Plodia. interpunctella were
reported to lack a major proteinase that activates the B.
thuringiensis protoxin [9]. Cry1Ac-resistant Plutella xylostella
showed more than 15000-fold resistance to full-length Cry1Ac
315
solubilized protoxin, with only a 94-fold resistance to the activated
65 kDa toxin [23]. These results clearly argue for a defect in
toxin activation as a major resistance mechanism in lepidopteran
insects against Cry1Ac. Another study on H. armigera with
resistance to Cry1Ac reported that resistant insects displayed
higher levels of enzyme esterase [20]. These upregulated esterases
were also shown to bind tightly to Cry1Ac insecticidal proteins.
Thus changes in activity of midgut-associated serine proteases
appear to be common physiological mechanism in lepidopteran
insects developing resistance to the midgut-acting B. thuringiensis
insecticidal proteins.
Action of insecticidal proteins upon ingestion depends on two
major facilitating events, activation by a midgut protease and
recognition by the specific receptor for docking and eventual lysis
of the midgut epithilium. In this context, aberration of any of the
two events would result in reduced toxicity of the ingested protein.
Earlier experiences with chemical insecticides and gut protease
inhibitors have shown that the gut protease transcriptome and
proteome is not a static entity, but rather is able to modulate the
activity of various proteases both collectively and individually
under different conditions [24,25].
H. armigera has been extensively studied for its proteolytic
enzymes in the midgut and it has been reported that serine proteases, such as trypsin and chymotrypsin, contribute maximally
to the proteolytic activity [13]. The amino acid sequence of
proteases from the H. armigera midgut fall into six different
groups. Since there is very little divergence between sequences
of proteases within a group, it was not possible to design primers
that could clearly differentiate one from the other. Hence, one
protease was selected from each group and analysed for its transcript abundance by semi-quantitative RT–PCR. The transcript of
the protease HaSP2 was substantially reduced in the resistant
insect population, whereas those of the other five proteases
did not show significant changes. Earlier reports also indicate
that the protease HaSP2 shows altered expression in H.
armigera populations insensitive to the soybean trypsin inhibitor
[16].
Further studies to understand the specific role of this protease in
Cry1Ac activation were performed to determine its specific ability
to hydrolyse Cry1Ac protoxin (135 kDa) to the activated form.
The heterologously expressed HaSP2 cleaved Cry1Ac protoxin to
its activated 65 kDa core. Hence it is tempting to assign the function of proteolytic activation of the Cry1Ac protoxin in the larval
midgut to protease HaSP2, although we cannot rule out other
proteases also being implicated.
As already emphasized, the use of transgenic plants expressing
the Cry1Ac protein has revolutionized the field of insect pest
management for the last few years. This ICP is toxic to most
of the important lepidopteran insect pests of major agricultural
crops such as Heliothis virescens, H. armigera. Pectinophora
gossypiella, Earias vitella and Plutella xylostella. The Cry1Ac
protein expressed in these transgenic plants is in the form of
the 135 kDa protoxin [26]. The insect consumes this protoxin,
which is then proteolyticlly activated in the insect gut to the more
active 65 kDa toxin form. Several studies, including the present
study, have demonstrated that development of resistance to this
ICP is mediated by changes in the protease profile of resistant
insects leading to altered protoxin processing [9,19,20,22,23,27].
The major practice suggested and assidiously implemented in
growing transgenic B. thuringiensis crops is to grow a 20 % refuge
population of non-transgenic crop plant so that the chances of a
homozygous resistant population is greatly reduced. Furthermore,
much of this practice is modelled on the basis of a change in
the site of the receptor in the resistant insects. A recent report
has indicated that Helicoverpa zea has developed field-level
c The Authors Journal compilation c 2009 Biochemical Society
316
R. Rajagopal and others
resistance against Cry1Ac on transgenic crops in the U.S.A.
[28]. An alternate approach to manage possible resistance to
Cry1Ac in target insects is to deploy structurally divergent
insecticidal proteins with a different mechanism of action. A
number of studies have reported the ability of insects resistant
to Cry1Ac to acquire cross resistance to structurally similar
insecticidal proteins such as Cry1Aa, Cry1Ab, Cry1C etc. It
has also been shown that insecticidal proteins such as Cry2Ab,
Cry1F and Cry9 retain their ability to kill Cry1Ac-resistant
insects, although instances of insects developing resistance to both
Cry1Ac and Cry2Ab have been reported [29,30]. Importantly,
our results reveal that an H. armigera population showing 72fold resistance to Cry1Ac protoxin (which has reduced HaSP2
protease activity) remains susceptible to trypsin-activated Cry1Ac
and to Cry2Ab, indicating that activation of Cry2Ab is not affected
in Cry1Ac-resistant insects. This result gives further credence
to the earlier observation of improper activation of the Cry1Ac
protoxin as a reason for resistant phenotype and the effectiveness
of gene pyramiding (such as BollGard II) with two different
Cry proteins offering effective protection against development
of resistance to Cry1Ac.
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Biochem. J. (2009) 419, 309–316 (Printed in Great Britain)
doi:10.1042/BJ20081152
SUPPLEMENTARY ONLINE DATA
Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus
thuringiensis is due to improper processing of the protoxin
Raman RAJAGOPAL*, Naresh ARORA*, Swaminathan SIVAKUMAR*, Nagarjun G. V. RAO†, Sharad A. NIMBALKAR†
and Raj K. BHATNAGAR*1
*Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi 110 067, India, and †Division of Entomology,
Dr. Panajbrao Deshmukh Agricultural University (Dr. PDKV), Akola 444 104, India
Figure S1
to Cry1Ac
Ligand–blot analysis of Cry1Ac-resistant and -susceptible BBMV
Membrane preparations (10 μg of protein) from Cry1Ac-susceptible and -resistant H. armigera
5th instar larvae were subjected to SDS/7.5 % PAGE and transferred on to a nitrocellulose
membrane. After blocking the membranes in 3 % BSA contained in PBS, they were incubated
with 200 ng/ml activated Cry1Ac toxin for 1 h. The blot was then washed three times with PBS
and overlaid with Cry1Ac rabbit polyclonal antibodies for 1 h. After three washes with PBS, they
were overlaid with alkaline phosphatase-conjugated anti-rabbit secondary antibodies. The
blot was developed with Nitro Blue Tetrazolium after washing three times with PBS. Lane 1,
Cry1Ac-susceptible insect; lane 2, Cry1Ac-resistant insect; lane 3, Bio-Rad pre-stained marker
with molecular masses to the right in kDa.
Figure S3 Heterologus expression of midgut serine protease HaSP2 and
its ability to activate Cry1Ac protoxin
(A) The gene encoding HaSP2 was cloned in-frame to an N-terminal fusion protein in the vector
pET32(b) and transformed into BL21(DE3) E. coli cells. Such transformed cells were induced
with IPTG for 3 h, which resulted in the expression of HaSP2 protein in the soluble fraction of
E. coli cells, shown in lane 1. Lane 2, uninduced cells; lane 3, protein molecular-mass marker
(Bio-Rad low-molecular-mass); lane 4, Ni-NTA affinity chromatography purified protein. (B) The
heterologusly expressed HaSP2 protein was purified by affinity chromatography using Ni-NTA
resin and 50 μg of this protein was incubated with 10 μg of Cry1Ac protoxin overnight at
37 ◦C. The reaction was stopped by adding 4× SDS/PAGE sample buffer and the samples were
resolved by SDS/7.5 % PAGE. The protein bands were visualisd by staining with Coomassie
Blue. Lane 1, BioRad broad range pre-stained marker; lane 2, HaSP2 purified protein; lane 3,
Cry1Ac protoxin incubated with the purified protease; lane 4, Cry1Ac protoxin. The activated
form of Cry1Ac (65 kDa) is indicated by an arrow. Molecular masses are shown to the left in
kDa.
Figure S2 Imprecise processing of Cry1Ac protoxin by H. armigera larval
midgut proteases
The 95 and 68 kDa proteins resolved in Figure 2(B) were transferred to nylon membrane
and sequenced for the first 10 amino acids from the N-terminus. The 95 and 68 kDa
polypeptides retained the N-terminal sequence of the protoxin whereas the 68 kDa protein
from the Cry1Ac-susceptible insect has the activated toxin form starting from the 28th amino
acid.
Received 5 June 2008/19 December 2008; accepted 16 January 2009
Published as BJ Immediate Publication 16 January 2009, doi:10.1042/BJ20081152
1
To whom correspondence should be addressed (email [email protected]).
The nucleotide sequence data reported for HaSP1, HaSP2, HaSP3, HaSP4, HaSP5 and HaSP6 will appear in the DDBJ, EMBL, GenBank® and GSDB
Nucleotide Sequence Databases under the accession numbers EF104918, EF104913, EF104914, EF104915, EF104916 and EF104917 respectively.
c The Authors Journal compilation c 2009 Biochemical Society