Bacillus thuringiensis Protein Cry6B (BGSC ID 4D8) is Toxicto Larvae of Hypera postica

Anil Sharma • Suresh Kumar • Raj K. Bhatnagar

Received: 24 February 2010 / Accepted: 10 August 2010

� Springer Science+Business Media, LLC 2010

Abstract Insecticidal proteins produced by strains of

Bacillus thuringenesis are specific toward target pests. One

of the Bt proteins, Cry 1Ac has been used successfully for

controlling crop predation by polyphagous pests Heli-

coverpa armigera. Structurally, Bt proteins consist of three

domains; domain I and III are fairly homologous in various

Bt proteins while domain II is hypervariable. The hyper-

variable domain II is believed to be responsible for speci-

ficity toward target pest. Successful deployment of Bt

proteins requires knowledge of its specificity toward the

insect. Various Bt proteins have been characterized for

activity against coleopteran pests. Some Bt proteins of

class Cry6 have been found to be active against potato

weevil. We have evaluated the activity of Cry6B protein

(BGSC-4D8) against lucerne weevil, Hypera postica,

which is a major pest of forage crop Medicago sativa.

Results revealed that the purified Cry6B protein is signif-

icantly active against the coleopteran pest with LC50 value

280 ng/ll. The leaves coated with the purified Cry6 toxin

were three times less damaged as compared with the neg-

ative control.

Introduction

Medicago sativa L. (lucerne or alfalfa) is one of the most

important forage legumes used as cattle feed [14]. Alfalfa

is well known for its nutritionally rich biomass production.

The record average yield of alfalfa is 10 tons/acre without

irrigation, while 24 tons/acre with irrigation (United States

Department of Agriculture, 1998). It is highly palatable and

nutritious to the cattle as it is enriched with protein, cal-

cium, phosphorus, and vitamins (A, B, and D). It is the

third important forage crop in India following Sorghum

(Sorghum bicolor) and Berseem (Trifolium alexandrinum).

One of the rampant problems in lucerne cultivation is the

damage caused by lucerne weevil (Hypera postica Gyll.,

Coleoptera), particularly in the Northern India. Larva of

weevil ravages phyto-phagously, reducing productivity of

the crop. Larvae cause skeletonization of leaves, stunted

plant growth and up to 20–30% reduction in green fodder

yield [10]. Severe infestation may even ruin entire stand of

the crop. No usable source of the insect resistance in

M. sativa gene pool is available so far. Screening of the

germplasm of Medicago led to identification of a few

Medicago species showing resistance to lucerne weevil [4].

Inter-specific incompatibility among the Medicago species

is the major limitation for conventional breeding methods.

In the absence of conventional breeding, genetic transfor-

mation of lucerne plants with Bt gene is a promising

approach to control the damage caused by lucerne weevil.

Bacillus thuringiensis Serovar. Kurstaki is a Gram-

positive bacterium, which produces proteinaceous inclu-

sions during sporulation. The inclusions are composed of

proteins known as ICPs, Cry proteins, or d-endotoxins,

which are encoded by cry genes. Cry proteins are highly

toxic to a wide variety of important agricultural and health

related insect pests as well as other invertebrates. Due to

A. Sharma � R. K. Bhatnagar (&)

Insect Resistance Group, International Center for Genetic

Engineering and Biotechnology, Aruna Asaf Ali Marg,

New Delhi 110067, India

e-mail: raj@icgeb.res.in

S. Kumar

Division of Crop Improvement, Indian Grassland and Fodder

Research Institute, Jhansi 284003, India

123

Curr Microbiol

DOI 10.1007/s00284-010-9749-4

their high specificity and their safety for the environment,

ICPs are valuable alternative to chemical pesticides for

control of insect pests in agriculture and forestry, and in the

home. It is believed that the rational application of

B. thuringiensis toxins will provide a variety of alternatives

for insect control and for coping with the problem of insect

resistance to pesticides.

Cry toxins have specific activities against species of the

orders Lepidoptera (Moth and butterflies), Diptera (Flies

and Mosquitoes) and Coleoptera (Beetles and Weevils).

Several Bt proteins (Cry6A, Cry3, and that from many

other strains of Bt) have been identified to be effective

against Coleopteran insects, in particular against lucerne

weevil [9]. Here, we report expression, purification, and

toxicity of codon-optimized Cry6B insecticidal protein.

The cry6b gene with codon bias for expression in Medi-

cago sativa was expressed in E. coli and screened for

activity against lucerne weevil, Hypera postica. The puri-

fied protein was active against the larvae of the weevil at

LC50 280 ng/ll.

Materials and Methods

Codon Optimization of cry6b According to M. sativa

Sequence of a delta-endotoxin cry6b gene (GenBank:

L07024.1) from Bacillus thuringiensis was optimized

according to codon usage of Medicago sativa and the gene

was synthesized commercially from GENEART (Regens-

berg) with BamH1 and Sac1 restriction endonuclease sites

attached at the 50 and 30 end of open reading frame. Codon

selection and % frequency of occurrence of a codon are

given in Table 1.

Cloning and Expression of the cry6b gene in Bacteria

The cry6b gene was cloned into pQE30 expression vector

at BamH1 and Sac1 sites down stream the T5 promoter and

was transformed into competent E. coli M15 cells by heat-

shock method. Expression of the Cry6B protein was

induced by 1 mM IPTG. The expression of the recombi-

nant Cry6B was checked by SDS-PAGE and western blot

analysis using anti-His antibodies. The Cry6B was

expressed in both soluble as well as in aggregated forms.

The recombinant Cry6B protein containing 6-histidine

residue-tag was purified from E. coli M15 cells using

affinity chromatography on Ni-NTA column (Qiagen). For

the purpose, the supernatant was passed through the Ni-

NTA resin at pH 8.0 to allow the ‘‘His’’ tag of the protein

to bind to the resin. The resin was washed with the wash

buffer (50 mM tris, 300 mM NaCl and 30 mM Immidaz-

ole) to elute out non-specific proteins. ‘‘His’’ containing

recombinant Cry6B was eluted with elution buffer con-

taining 50 mM tris buffer pH 8.0, 300 mM NaCl and

300 mM Immidazole. Different eluted fractions were

checked on SDS-PAGE gels for the purified Cry6B.

Cry6B-positive eluted fractions were pooled, dialyzed

overnight against 10 mM Tris–Cl (pH 8.0) and concen-

trated using 10 kDa centricon membrane. The quantity of

the concentrated protein was estimated using Bradford’s

reagent [2].

Table 1 Codon optimization of gene encoding Bacillus thuringiensis insecticidal protein cry6b as per codon usage of Medicago sativa

Codon Amino acid Freq. Codon Amino acid Freq. Codon Amino acid Freq. Codon Amino cid Freq.

UUU F 7 UCU S 10 UAU Y 11 UGU C 3

UUC F 5 UCC S 5 UAC Y 9 UGC C 2

UUA L 5 UCA S 9 UAA * UGA *

UUG L 11 UCG S 2 UAG * UGG W 8

CUU L 12 CCU P 5 CAU H 5 CGU R 2

CUC L 6 CCC P 2 CAC H 4 CGC R 1

CUA L 4 CCA P 4 CAA Q 20 CGA R 1

CUG L 4 CCG P 1 CAG Q 11 CGG R 1

AUU I 21 ACU T 10 AAU N 25 AGU S 7

AUC I 11 ACC T 6 AAC N 19 AGC S 5

AUA I 9 ACA T 9 AAA K 18 AGA R 3

AUG M 8 ACG T 2 AAG K 19 AGG R 2

GUU V 9 GCU A 12 GAU D 26 GGU G 7

GUC V 3 GCC A 4 GAC D 10 GGC G 3

GUA V 3 GCA A 10 GAA E 17 GGA G 7

GUG V 4 GCG A 2 GAG E 12 GGG G 2

GC content: 43.7%

A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

Raising of Polyclonal Antisera

For raising polyclonal antisera against the purified cry6B

toxin, 1 mg/ml of protein was mixed with equal volume of

complete Freund’s adjuvant, emulsified and injected 100 ll

each intra-dermally at 10 different places of a New Zealand

rabbit [11]. The rabbit was boosted with Cry6B plus

incomplete Fruend’s adjuvant after 21 days of injection.

Serum was collected after 12 days of first booster. Anti-

Cry6B antibody titer in rabbit blood was measured by

western blotting against purified Cry6B protein using serial

dilutions of the antibodies ranging from 1:1,000 to

1:20,000.

Bioassay Against Weevil on Leaves Coated with ICPs

Purified Cry6B insecticidal crystal protein (ICP) was used

for insect bioassay, along with controls using neonate lar-

vae of the lucerne weevil (during January–March, 2009) to

assess the bio-efficacy of the insecticidal protein. For the

purpose, the Cry6B protein was expressed in E. coli and the

protein was isolated from the bacterial culture using protein

extraction and purification kit (Medox-Bio) followed by

washing with crystal wash buffers. The wet paste of the

250 ng/ll of Cry6B protein with 0.6% of gelatin was

coated on both the sides of trifoliate leaves of lucerne. The

average area of a normal M. sativa leaf is nearly

12.37 sq cm. The leaf was placed on a moist Whatman

filter paper and five neonate larvae were released on each

trifoliate leaf coated with the protein in a 90 mm petri

plate. The petriplate was covered with its lid to restrict run-

away of the larvae. Weight of the leaf material was taken

before and after 2 days of setting up the bioassays to

estimate the loss incurred to the leaves. Similarly, the

insects were also weighed before and after 2 days of the

application of the ICP on leaf to estimate the growth rate of

the larvae. Mortality of the larvae was recorded after

5 days of incubation. The leaf damage, larval growth, and

mortality were compared with corresponding Cry1Ac and

uncoated leaves as negative control.

For calculation of lethal concentrations (LC50) values, a

range of purified Cry6B toxin from 100 to 1,000 ng was

prepared and was coated on trifoliate leaf of M. sativa. Five

larvae were released on each leaf as described above.

Mortality was recorded on fifth day of the release of larvae.

Data of all the independent experiments were pooled to

calculate the LC50 value using Probit analysis. Each bio-

assay experiment was independently conducted thrice in

three replications. Further to plot the survival ship curves,

the mortality of the larvae were daily recorded till ninth

day of start of experiment till all the Cry6B treated insects

were dead and control larvae started pupating.

Statistical Analysis

The data obtained from the bioassay were found to be nor-

mally distributed. The normally distributed data were ana-

lyzed using one-way analysis of variance (ANOVA) and

HSD Tukey test to check the significance of the differences

in the mean values of mortality, growth rate, and leaf damage

using VassarStats: Statistical Computation Website—

http://faculty.vassar.edu/lowry/VassarStats.html. The sur-

vivalship curves were plotted using Microsoft Excel and the

significance in the difference is calculated using Log Rank

Test on http://bioinf.wehi.edu.au/software/russell/logrank/

index.html site. LC50 and LC90 values were manually cal-

culated using Log Probit analysis.

Results

Codon Optimization of cry6b gene According

to M. sativa

The codon of 1.5 kb cry6b gene was optimized according

to the codon usage table of M. sativa. For the choice of

codon used in M. sativa, care was taken to distribute pre-

dominately used codons evenly. The overall codon usage

and their relative abundance are described in Table 1. The

synthesized gene corresponds to 52 kDa insecticidal pro-

tein. The sequence of the modified cry6b gene is given in

Fig. 1.

Cloning and Expression of the cry6b gene in E. coli

and Evaluation of Activity Against Lucerne Weevil

The codon-optimized cry6b was cloned into expression

vector, pQE30, and transformed into E.coli M15 cells. The

transformed cells were induced with IPTG to express the

protein. The recombinant Cry6B protein was accumulated

in soluble and insoluble fractions of the cells (Fig. 2a,b).

Since recombinant Cry6B was expressed with a 6xHis tag,

the latter was used to purify the protein using affinity

chromatography on Ni-NTA columns (Fig. 2c). The puri-

fied protein was dialyzed and concentrated. Polyclonal

antiserum was raised against the purified protein in New

Zealand rabbit.

The purified protein was employed for toxicity against

lucerne weevil. Different concentrations of purified Cry6B,

ranging from 100 to 1,000 ng/ll were coated on M. sativa

leaves and five neonate larvae were released on each tri-

foliate leaf. The bioassay results indicated significant dif-

ference in leaf damage and insect mortality (Tables 2, 3,

Figs. 3, 4) caused by the insect. The LC50 and LC90 values

of the Cry6B were calculated to be *280 and 630 ng/ll,

respectively (Table 4). Significantly less insect mortality

A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

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A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

and consequently high foliage damage was observed in

leaves coated with Bt protein, Cry1Ac, or buffer control.

Based on visual observations, it was estimated that

uncoated leaves and Cry1Ac coated leaves were four to

five times more damaged than those coated with Cry6B

protein (ANOVA; P [ 0001). Similarly, significant dif-

ference between treatment and control group was observed

in growth rate and of larval mortality (Table 3). The

growth of the larvae that were forced to feed on leaves

coated with Cry6B toxin was one-third as compared to the

larvae that were fed upon Cry1Ac coated or uncoated

leaves (ANOVA; P [ 001). On fifth day of experiment,

nearly 5-folds more larvae were dead on leaves that were

pre-coated with Cry6B as compared with the larvae on

leaves coated with Cry1Ac. Whereas, the mortality on cry6B

coated leaves was[9-fold more as compared with the larvae

on uncoated leaves (ANOVA; P [ 0.0001). The survival

ship curves show significant difference in the survival of the

weevil larvae upon treatment with Cry6B protein as com-

pared with the control Cry1Ac treatment (Fig. 4).

Discussion

Leading bio-rational pesticide, Bacillus thuringiensis, is a

ubiquitous Gram-positive, spore-forming bacterium that

forms a parasporal crystal during the stationary phase of its

growth cycle. B. thuringiensis was initially characterized as

an insect pathogen, and its insecticidal activity was

attributed to the parasporal crystals. Bt strains are spe-

cialized in killing certain target insects rather than

becoming generalist killers of all classes of insect. This

observation led to the development of bio-insecticides

based on B. thuringiensis for the control of certain insect

Vector pQE30 + Cry6alone Unind Ind

A

53kDa

Suptt PelletUnind Ind Uunind Ind

B

53kDa

Flow Wash ElutionThrough 1 2 1 2 4 6 8 10

C

53kDa

Fig. 2 Expression and

purification of Cry6B protein.

The Cry6B protein was cloned

and expressed using pQE30

vector and E. coli M15 host

cells. The protein can be seen as

53 kDa polypeptide band on

SDS-PAGE Gel (a) and on

western blot developed by anti-

His antibodies (b). The

insecticidal protein was purified

using Ni-NTA column

chromatography (c)

A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

species among the orders Lepidoptera, Diptera, Coleoptera,

Hymenoptera, Homoptera, Orthoptera, and Mallophaga

and against nematodes, mites, and protozoa [1, 5, 6].

B. thuringiensis is already a useful alternative or supple-

ment to synthetic chemical pesticide application in com-

mercial agriculture, forest management, and mosquito

control. It is also a key source of genes for transgenic

expression to provide pest resistance in plants.

Bacillus thuringiensis Cry6 subfamily contains two

proteins, Cry6A and Cry6B. These are nearly 50% identi-

cal to each other, but they are unrelated by protein

sequence to the main family of Bt crystal toxins. Delta-

endotoxin proteins Cry6A and Cry6B were identified as

anti-nematocidal agent, of which anti-nematocidal activity

of Cry6B was not found to be reproducible [7, 15]. Both

Cry6 family toxins have been described in patent on

Table 2 Leaf damage caused by the lucerne weevil during insect bioassay using ICPs

Treatment Replication Weight of trifoliate leaf (mg) Damage (%)

Initial Wt. Wt. 2 days after

inoculation

No ICP I 2,938 2,262 676 (23.01) 717.66 ± 40.1

(23.66)II 3,004 2,283 721 (24.00)

III 3,153 2,397 756 (23.98)

Cry1Ac I 2,689 2,286 403 (14.98) 533 ± 115.3

(15.33)II 3,825 3,252 573 (14.98)

III 3,888 3,265 623 (16.02)

Cry6 I 2,701 2,545 156 (5.77) 134 ± 35.6

(4.27)II 3,100 3,007 93 (3.00)

III 3,810 3,657 153 (4.02)

ANOVA summary for loss of larval weight

Source Sum of squares Degree of freedom Mean of squares F value P value

Treatment (Between Groups) 533969.556 2 266984.778 49.53 0.00019

Error 32342 6 5390.444

Ss/Bl

Total 566312.222 8

HSD Tukey test

HSD [0.05]=184.14; HSD [0.01]=268.03

No ICP vs. Cry1Ac P \ 0.5

No ICP vs. Cry6 P \ 0.01

Cry1Ac vs. Cry6 P \ 0.01

Each leaf was coated with 250 ng/ll of Bt protein as indicated. Each replication contains data from 6 plates and the experiment was repeated

three times

Fig. 3 Effect of Cry6B protein

on leaf damage in Medicagosativa caused by lucerne weevil.

Five neonate larvae were

released on each trifoliate leaf

coated with 250 ng/ll of

insecticidal protein and the

damage was recorded after

2 days of release

A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

nematocidal Bt toxins, but only sparse data were presented

[13]. Mark et al. [9] described activity of truncated Cry6

toxin against coleopterans. In the present study, full Cry6B

gene was recombinantly expressed in E.coli and was found

to be active against coleopteran lucerne weevil with nearly

5- and 9-fold mortality than that of Cry1Ac and negative

control, respectively. The Cry6B protein is found to have

LC50 and LC90 values *280 and 630 ng/ll, respectively.

These results clearly identified lucerne weevil to be sen-

sitive to Cry6B protein. Besides killing the pests, the

presence of Cry6B mitigates the leaf damage as evidenced

by nearly three times less damaged Cry6B coated leaves as

compared with the uncoated or Cry1Ac coated leaves.

Minor injury (feeding) on the Cry6B coated leaves

Table 3 Growth and mortality of larvae during insect bioassay using Bt protein

Treatment Replication Weight of 5 larvae (mg) Growth rate (%) Mortality (%) (N = 15 per replication)

Initial Wt. Wt. 2 days

after inoculation

No ICP I 62.0 80.5 18.5 (29.84) 21.8 ± 2.8 (32.83) 1 (06.60) 1.33 ± 0.58 (8.8)

II 67.8 91.5 23.7 (34.95) 2 (13.20)

III 68.5 91.6 23.1 (33.72) 1 (06.60)

Cry1Ac I 81.6 102.1 20.5 (25.12) 22.5 ± 3.6 (25.26) 4 (26.60) 2.33 ± 1.52 (15.46)

II 85.8 106.1 20.3 (23.66) 2 (13.20)

III 98.5 125.1 26.6 (27.00) 1 (06.60)

Cry6 I 61.0 67.1 6.1 (10.00) 7.2 ± 1.6 (10.59) 13 (93.20) 12.67 ± 1.52 (84.33)

II 65.3 71.8 6.5 (9.95) 11 (73.20)

III 77.0 86.1 9.1 (11.82) 14 (86.60)

ANOVA summary for mortality

Source Sum of squares Degree of freedom Mean of squares F value P value

Treatment (Between groups) 78740741 2 39.37037 70.87 \0.0001

Error 13.333 24 0.5556

Ss/Bl

Total 92.074 26

HSD Tukey test

HSD [0.05] = 3.24; HSD [0.01] = 4.71

No ICP vs. Cry1Ac Non-significant

No ICP vs. Cry6 P \ 0.01

Cry1Ac vs. Cry6 P \ 0.01

ANOVA summary for loss of larval weight

Source Sum of squares Degree of freedom Mean of squares F value P value

Treatment (Between groups) 443.7622 2 221.8811 28.24 \0.001

Error 47.14 6 7.8567

Ss/Bl

Total 490.9022 8

HSD Tukey test

HSD [0.05] = 7.03; HSD [0.01] = 10.23

No ICP vs. Cry1ac Non-significant

No ICP vs. Cry6 P \ 0.01

Cry1ac vs. Cry6 P \ 0.01

Each leaf was coated with 250 ng/ll of insecticidal protein. Difference in the body weight of surviving larvae before and after 2 days of

treatment was taken as indicative of their growth rate. Mortality in the larvae was recorded after 5 days of incubation

A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

indicates that larvae do start feeding on the coated leaves

but subsequently fails to do so. Bt toxins are known to

induce the paralysis of larval midgut, followed by the death

of the larvae due to starvation and massive septiemia [3].

Feeding on Cry toxins is known to retard the growth of

larvae of various insects [8, 16]. Larvae with retarded

growth are more prone to other infections since, B. thur-

ingiensis and other bacteria are known to be more virulent

on weakened insect larvae [12].

Conclusion

The study describes identification of toxicity range of the

Cry6B protein. The E. coli-expressed Cry6B was found to be

toxic to lucerne weevil. High mortality in Cry6B fed insects,

their less growth, and less damage of leaves suggests a

possible bio-pesticidal control molecule to minimize the

damage caused by lucerne weevil. Transgenesis of M. sativa

with Cry6B gene, thus, seems to be an answer to the problem

imposed upon lucerne crop by the lucerne weevil.

Acknowledgments Financial assistance from the Department of

Biotechnology, Ministry of Science and Technology, Government of

India, New Delhi, is gratefully acknowledged.

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Table 4 Bioassay for calculation of lethal concentration values

Bt Toxin Conc. (ng/ll) Mortality (N: 15 9 3 = 45)

I II III Total Percent

Cry6 0 0 0 0 0 0

125 2 1 1 4 9

250 7 7 8 22 49

500 10 12 11 33 75.5

750 14 14 13 41 91

1000 15 15 14 44 98

Cry1Ac 0 0 0 0 0 0

125 0 0 0 0 0

250 1 1 1 3 7

500 2 1 2 5 11

750 2 2 1 5 11

1000 3 2 2 6 13

The leaves were coated with purified Cry6B in the range 100 to

1,000 ng/ll. Five lucerne larvae were released per leaf in three such

replicates and mortlity of the larvae were calculated on fifth day of

release of larvae. Each experiment was independently carried out thrice

Lethal concentrations LC50 and LC90 values are 280 and 630 ng/ll,

respectively

Cry1Ac vs Cry6B

Call: survdiff(formula = Surv(time, censor) ~ group)

N Obs Exp (O-E)^2/E (O-E)^2/V Cry1Ac 300 65 235 123 425 Cry6B 300 300 130 220 425

Chisq= 425 on 1 degrees of freedom, p= 0

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9

Days

Mo

rtal

ity

Cry1Ac Cry6

Fig. 4 Survivalship curve of lucerne larvae allowed to feed on

Cry1Ac and Cry6B toxins coated leaves. Bt toxins were applied on

the leaves at concentration slightly higher than the LC50 value (i.e.,

375 ng/ll). Mortality was monitored per day till ninth day of setting

up the bioassay. No mortality, but yellowing of the larvae, was

observed with in 2 days of inoculation. All the larvae fed upon Cry6B

coated leaves were found dead on ninth day, while the larvae on

Cry1Ac coated leaves started pupating

A. Sharma et al.: Cry6B is Toxic to Hypera postica

123

13. Schnepf EH, Schwab GE, Payne J et al. (1998) U.S. Patent

5,753,492—Genes encoding nematode-active toxins from Bacil-lus thuringiensis strains

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16. Wu X, Leonard BR, Zhu YC et al (2009) Susceptibility of

Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera:

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Pathol 100:29–34

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