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Journal of Biotechnology 171 (2014) 71– 75

Contents lists available at ScienceDirect

Journal of Biotechnology

jo u r n al homep age: www.elsev ier .com/ locate / jb io tec

ynthetic fusion-protein containing domains of Bt Cry1Ac and Alliumativum lectin (ASAL) conferred enhanced insecticidal activity againstajor lepidopteran pests

unita Tajne, Dayakar Boddupally, Vijayakumar Sadumpati, Dashavantha Reddy Vudem,enkateswara Rao Khareedu ∗

entre for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India

r t i c l e i n f o

rticle history:eceived 22 October 2013eceived in revised form6 November 2013ccepted 30 November 2013vailable online 17 December 2013

a b s t r a c t

Different transgenic crop plants, developed with �-endotoxins of Bacillus thuringiensis (Bt) and mannose-specific plant lectins, exhibited significant protection against chewing and sucking insects. In the presentstudy, a synthetic gene (cry-asal) encoding the fusion-protein having 488 amino acids, comprising DIand DII domains from Bt Cry1Ac and Allium sativum agglutinin (ASAL), was cloned and expressed inEscherichia coli. Ligand blot analysis disclosed that the fusion-protein could bind to more number ofreceptors of brush border membrane vesicle (BBMV) proteins of Helicoverpa armigera. Artificial diet bioas-

eywords:acillus thuringiensisusion-proteinarlic lectin

nsecticidal activity

says revealed that 0.025 �g/g and 0.50 �g/g of fusion-protein were sufficient to cause 100% mortality inPectinophora gossypiella and H. armigera insects, respectively. As compared to Cry1Ac, the fusion-proteinshowed enhanced (8-fold and 30-fold) insecticidal activity against two major lepidopteran pests. Bindingof fusion-protein to the additional receptors in the midgut cells of insects is attributable to its enhancedentomotoxic effect. The synthetic gene, first of its kind, appears promising and might serve as a potential

crop

igand blot candidate for engineering

. Introduction

Expression of �-endotoxins of Bt and plant-derived defense pro-eins in diverse crop plants conveyed ample protection againstifferent insect pests (Bravo et al., 2011; Sharma et al., 2004).ransgenic plants expressing Bt toxins have been used success-ully to confer resistance against major insect pests for more thanfteen years (Kaur, 2000). Bt endotoxins exhibited narrow spec-rum of activity due to their mode of action, which is usuallyeceptor-specific and lethal to lepidoptera, diptera, coleoptera andrthoptera groups of insects (Ferre and Van Rie, 2002). The exten-ive use of single Cry toxin contributed to the evolution of resistanttrains of insects (Tabashnik et al., 2005; Wan et al., 2012). The mostrequent mechanism of insects’ resistance to Cry toxin involveslterations in the receptor binding sites present in the midgut cellsf insects (Tiewsiri and Wang, 2011). Attempts have been made toxpress two different Cry toxins or fusions of different Cry proteins

or enhanced toxic effects against various insects (Ho et al., 2006;onee et al., 1990). Among different strategies, the domain swap-ing is considered as a promising approach which could enhance

∗ Corresponding author at: Centre for Plant Molecular Biology, Osmania Univer-ity, Hyderabad 500 007, A.P., India. Tel.: +91 40 27098087; fax: +91 40 27096170.

E-mail address: rao kv1@rediffmail.com (V.R. Khareedu).

168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jbiotec.2013.11.029

plants against major insect pests.© 2013 Elsevier B.V. All rights reserved.

the insecticidal activity as well as broaden the insecticidal spec-trum (Tajne et al., 2012). Hybrid Cry proteins, such as Cry1Ab-Cry1Band Cry1Ac-Cry1Ab, were synthesized and expressed in differentplants for high-level resistance and enhanced toxicities against tar-get insects (Tang et al., 2012). Furthermore, different Cry1 toxins,viz., Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ea, exhibiting low or no tox-icity against Spodoptera exigua, became active when their domainIII is replaced by Cry1Ca domain III (de Maagd et al., 2000).

Although transgenic plants expressing Bt �-endotoxins pro-vided ample protection to lepidopteran and coleopteran pests butare not useful against sap-sucking homopterans (Rao et al., 1998).Therefore, it is desirable that the transgenic plants should con-tain �-endotoxins along with another toxin for effective controlof different insect groups. Plants are known to serve as sources ofnon-Bt insecticidal proteins such as lectins and protease inhibitors(Sharma et al., 2004). The 3D-crystal structure of different plantlectins have revealed the presence of a beta-prism-II fold com-prising three antiparallel four-stranded beta-sheets arranged astwelve-stranded beta-barrel (Barre et al., 2001). It was reportedthat the DIII domain of Cry1A protein contained a lectin-like fold(Burton et al., 1999). Among the plant derived lectins, garlic lectin

(ASAL) proved to be more toxic to sap-sucking pests (Yarasi et al.,2008; Saha et al., 2006) and inhibitory to the growth of certainlepidopteran pests, viz., Helicoverpa armigera and Spodoptera litura(Sadeghi et al., 2007). ASAL protein exists as a dimer and each

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ubunit contains three highly conserved mannose-binding siteshich bind to several proteins in the midgut cells of insects

Upadhyay and Singh, 2012).Earlier, we reported in silico analysis of the fusion-protein (Bt

ry1Ac::ASAL) comprising domains I and II of Cry1Ac and ASAL, asell as its binding ability towards APN-receptor of Manduca sexta

Tajne et al., 2012). Docking of Cry1Ac-ASAL fusion-protein withPN-receptor revealed higher binding affinity and contact patternith the insect receptor when compared to Cry1Ac protein. Fur-

hermore, it was proposed that the fusion-protein comprising an-terminal Bt Cry1Ac and C-terminal ASAL would provide a uniqueinding domain that interacts with more number of insect recep-ors compared to Cry1Ac (Tajne et al., 2012).

The present study deals with the synthesis, purification andnsecticidal activity of recombinant fusion-protein (Cry-ASAL) con-aining domains I and II of Cry1Ac fused to ASAL domain. This report,hich is first of its kind, clearly demonstrates that the fusion-rotein is more toxic to Pectinophora gossypiella and H. armigerahen compared to Cry1Ac.

. Materials and methods

.1. Cloning of synthetic (cry-asal) and cry1Ac genes

The 1089 bp of Cry1Ac (268–1356 bp) coding for DI and DIIomains (Ramesh et al., 2004) and 375 bp of asal (55–429 bp)ncoding carbohydrate binding domain (Yarasi et al., 2008)ere amplified by PCR using pfu polymerase, employing for-ard and reverse primers, viz., 5′-GAATTCGAGTTCGCCAGGAACAG-3′ and 5′-GGATCCGATGATGCTCACGGAACTG-3′, and 5′-GATCCGCTATTCTAACCAT ACTG-3′ and 5′-GAGCTCACCCACCTTCTTCTGTAGG-3′, respectively. The underlined regions in therimers depict EcoRI, BamHI and SacI restriction sites. Amplifiedragments of 1089 bp and 375 bp were independently cloned atmaI site of pBluescript KS (+) (Stratagene, USA). Later, 375 bp frag-ent was excised and cloned at the 3′ end of 1089 bp sequence in

ry1Ac-pBSK (+) plasmid. The recombinant clones were confirmedhrough restriction analysis and the synthetic gene was sequencedsing automated DNA sequencer, and designated as cry-asal. Later,he synthetic gene (KC782833) of ∼1.4 kb, excised with EcoRI andacI, and 1.86 kb fragment of full-length cry1Ac gene, restrictedith BamHI and EcoRI from pSB11-bar-Ubi-cry1Ac vector (Ramesh

t al., 2004), were independently cloned down-stream to T7 pro-oter in frame with 6X His-tag of pET28a. Both the recombinant

lones were independently transformed into BL21 (DE3) cells of. coli for protein expression studies (Sambrook and Russell, 2001).

.2. Expression, purification and analysis of recombinant proteins

E. coli cells, harboring cry-asal/cry1Ac, were grown in LBedium containing 50 �g/ml of kanamycin at 37 ◦C and 200 rpm

ntil the cell density reached O.D 0.6–0.8. Isopropyl �-d-1-hiogalactopyranoside (IPTG) was added to the final concentrationf 500 �M and cultures were grown at 30 ◦C. After 4–6 h of incuba-ion, cells were harvested by centrifugation at 8000 × g for 15 mint 4 ◦C. The cells were lysed and the expressed proteins were puri-ed according to Kamarthapu et al. (2008). The concentration ofurified proteins of Cry1Ac, and Cry-ASAL were determined by theethod of Bradford (Bradford, 1976).

.3. Western blot analysis of recombinant proteins

Purified proteins of Cry-ASAL, Cry1Ac and ASAL (Yarasi et al.,008) were subjected to western blot analysis. Protein sam-les (∼2 �g) were subjected to 15% SDS PAGE gel (Laemmli,970). Following electrophoresis, the proteins were transferred

nology 171 (2014) 71– 75

onto nitrocellulose P membrane (Amersham) by electro blotting(Towbin et al., 1979). After protein transfer, the membrane wasblocked by incubating in PBS solution containing 10% nonfat driedmilk and 0.1% Tween 20 for 2 h at room temperature, and was incu-bated with polyclonal rabbit anti-ASAL/anti-Cry1Ac serum. Later,the membrane was incubated with goat anti-rabbit IgG Alkalinephosphatase conjugate as a secondary antibody (1:10,000 dilution)for 2 h. The membrane was washed and analyzed with saturatedbenzidine solution containing 20% ammonium chloride and 0.1%H2O2.

2.4. Isolation of brush border membrane vesicles (BBMV) fromthe midgut of H. armigera for ligand blot assay

Midguts were isolated and prepared as described by English andReaddy (English and Readdy, 1989). The BBMV proteins were quan-tified using the Bradford assay (Bradford, 1976). For ligand blotanalysis, ∼30 �g of BBMV proteins were subjected to 10% SDS PAGE(Laemmli, 1970) and were transferred onto nitrocellulose P mem-brane (Amersham) by electro blotting (Towbin et al., 1979). Themembrane was blocked by incubating in PBS solution containing5% nonfat dried milk and 0.1% Tween 20 for 2 h at room tempera-ture. Later, membrane was washed with PBST and cut into stripsand were incubated with purified proteins of Cry1Ac, ASAL andCry-ASAL (0.5 �g/ml) for 4 h at 4 ◦C. ASAL/Cry1Ac polyclonal anti-bodies raised in rabbit were used as primary antibodies (1:3000dilution) and incubated for 4 h at room temperature. Later, themembrane was incubated with goat anti-rabbit IgG horse-radishperoxidase conjugate as a secondary antibody (1:10,000 dilution)for 2 h. Strips were washed and analyzed with saturated benzidinesolution containing 20% ammonium chloride and 0.1% H2O2.

2.5. Insect bioassays

Insect bioassays were carried out with neonatal larvae of H.armigera and P. gossypiella by feeding them on artificial diet incor-porated with purified proteins of ASAL/Cry1Ac/Cry-ASAL. Neonatesof H. armigera and P. gossypiella used for bioassays were main-tained in the laboratory on artificial diet at constant temperatureof 26 ± 2 ◦C with 80% relative humidity. Molten diet mixture wascooled to 40 ◦C and different concentrations (0.005, 0.010, 0.015,0.025, 0.075, 0.1, 0.2, 1.0 and 1.5 �g/g) of ASAL/Cry1Ac/Cry-ASALwere mixed with 1 g of diet, and used for bioassays. After solidi-fying the diet, it was cut into small (2 cm × 2 cm) pieces and keptin small plastic boxes. Neonate larvae of H. armigera and P. gossyp-iella were released into each box and bioassays were carried outfor a period of 10 days and data was recorded. Ten individual lar-vae were used for each concentration. All the bioassay experimentswere repeated three times. Data was analyzed using the sigma plotsoftware, version 12.0, for windows (SPSS, Richmond, CA, USA).Differences between the mean values were subjected to unpairedt-test.

3. Results

3.1. Cloning and expression of synthetic (cry-asal) and cry1Acgenes

The synthetic gene (KC782833) contained 1464 bp sequence(1089 bp of cry and 375 bp of asal) that codes for a polypeptideof 488 amino acids. Restriction analysis of recombinant pET28a(+) clones carrying cry-asal/cry1Ac genes disclosed the presence

of ∼1.4 kb/∼1.86 kb bands corresponding to synthetic and nativegenes, respectively (Fig. 1A and B). E. coli BL21 (DE3) cells har-boring synthetic/cry1Ac genes downstream to T7 promoter, wheninduced with 500 �M IPTG, exhibited ∼60 kDa/∼70 kDa induced

S. Tajne et al. / Journal of Biotechnology 171 (2014) 71– 75 73

Fig. 1. Restriction analysis and expression of recombinant clones containing fragments of Cry1Ac (DI & DII) and ASAL domains. (A) Recombinant pET28a (+) harboringfusion-gene. Lane M: 1 kb DNA marker; Lane 1: ∼1.4 kb insert of fusion-gene upon digestion with EcoRI & SacI; Lane 2: 1089 bp of cry1Ac and 375 bp of asal with BamHI &SacI restriction enzymes and ∼5.4 kb pET28a (+). (B) Recombinant pET28a (+) containing cry1Ac. Lane M: 1 kb DNA marker; Lanes 1 & 2: ∼1.8 kb insert of cry1Ac gene and∼5.4 kb of pET28a (+) vector. (C) Expression of Cry1Ac and fusion-proteins. Lanes 1 & 2: IPTG induced cell lysates of E. coli BL21 (DE3) Cry1Ac; Lanes 3 & 4: IPTG induced celllysates of E. coli BL21 (DE3) fusion-protein. (D) Schematic diagram of cry1Ac under T7 promoter. (E) Schematic diagram of cry-asal fusion-gene under T7 promoter.

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ig. 2. Protein purification and western blot analyses of Cry1Ac and fusion-proteins.arker; Lane 1: ∼60 kDa purified fusion-protein. (C) Lane C: Control protein treate

ntibodies; Lane 2: Fusion-protein treated with anti-ASAL antibodies.

rotein bands (Fig. 1C). The purified Cry1Ac/fusion proteins on SDS-AGE showed ∼70 kDa and ∼60 kDa bands (Fig. 2A and B). Westernlot analysis of purified proteins with anti-ASAL/anti-Cry1Ac anti-odies showed ∼60 kDa and 70 kDa bands (Fig. 2C). Furthermore,he purified ASAL, Cry1Ac and fusion proteins were confirmed byLISA using ASAL/Cry1Ac antibodies (data not shown).

.2. Ligand blot analysis

Ligand blot analysis was performed to investigate the interac-ion profile of Cry1Ac, ASAL and Cry-ASAL with the BBMV proteins

f H. armigera. The fusion-protein was bound to four different pro-eins, viz., ∼55 kDa, ∼80 kDa, ∼120 kDa and ∼250 kDa, and Cry1Acould bind to ∼80 kDa, ∼120 kDa and ∼220 kDa proteins, whileSAL was bound only to ∼80 kDa protein (Fig. 3A).

ne M: Protein marker; Lane 1: ∼70 kDa purified Cry1Ac protein. (B) Lane M: proteinh anti-Cry and anti-ASAL antibodies; Lane 1: Cry1Ac protein treated with anti-Cry

3.3. Insect bioassays against P. gossypiella and H. armigera

Bioassays were carried out, using the diet containing the ASAL,Cry1Ac and fusion-protein to assess their insecticidal activity ontarget insects, viz., P. gossypiella and H. armigera. Both neonates fedon control medium as well as medium supplemented with ASALprotein showed 100% survival with normal growth, while, mediumsupplemented with fusion and Cry1Ac proteins exhibited variedlevels of mortality (Fig. 3B and C). Bioassays using P. gossypiellarevealed that 0.015 �g/g and 0.025 �g/g of the fusion-protein wassufficient to cause 50% and 100% mortality, respectively, within 6days (Fig. 4A). Whereas with Cry1Ac protein, it was observed that

an amount of 0.1 �g/g and 0.2 �g/g was required to cause 50% and∼100% mortality of P. gossypiella larvae (Fig. 4A).

In the case of H. armigera, 0.05 �g/g, 0.075 �g/g and 0.5 �g/gof fusion-protein were found to cause 20%, 50% and 100% larval

74 S. Tajne et al. / Journal of Biotechnology 171 (2014) 71– 75

Fig. 3. Ligand blot analyses and entomotoxic effects of ASAL, Cry1Ac and fusion proteins against target insects. (A) Ligand blot analysis of Cry1Ac, ASAL and fusion-protein withBBMV proteins of H. armigera. Lane M: protein marker; Lane A: incubated with Cry1Ac protein and probed with Cry1Ac antibodies; Lane B: incubated with ASAL protein andprobed with ASAL antibodies; Lane C: incubated with Cry-ASAL fusion-protein and probed with Cry1Ac/ASAL antibodies. (B). Neonates of P. gossypiella on diets supplementedwith control, ASAL, Cry1Ac and fusion-proteins. (i) Larvae showing normal growth on control diet; (ii) larvae showing normal growth on diet supplemented with ASALprotein; (iii) larvae showing reduced growth on diet supplemented with Cry1Ac protein (0.025 �g/g); and (iv) larvae showing complete mortality on diet supplemented withfusion-protein (0.025 �g/g). (C). Neonates of H. armigera on diets supplemented with control, ASAL, Cry1Ac and fusion-proteins. (i) Larvae showing normal growth on controldiet; (ii) larvae showing normal growth on diet supplemented with ASAL protein; (iii) larvae showing reduced growth on diet supplemented with Cry1Ac protein (0.5 �g/g);and (iv) larvae showing complete mortality on diet supplemented with fusion-protein (0.5 �g/g).

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oncentrations of fusion and Cry1Ac proteins. Data were recorded from three repleans were significant at p value 0.0001 (unpaired t test).

ortality, respectively, within 6 days (Fig. 4B). On the other hand,igher concentration of Cry1Ac (1.5 �g/g) caused only 20% mortal-

ty of the larvae under identical conditions (Fig. 4B).

. Discussion

Development of resistance in pests against transgenic Bt cropsoses major threat to their sustainable use in agriculture. Varioustrategies, such as combinations of multiple transgenes, differentusion-genes constructs and adoption of various deployment tac-ics, have been employed to ensure the long-term usefulness ofransgenic crops (Honee et al., 1990; Maqbool et al., 2001; Naimovt al., 2003; Mehlo et al., 2005). Earlier, based on in silico analy-is we proposed that the fusion-protein containing Cry DI–DII andSAL domains could exhibit higher insecticidal activity than thatf Cry1Ac protein (Tajne et al., 2012). Our primary objective foresigning the fusion-protein was to develop a more potent insec-icide with broader activity than that of Cry toxins. In the presenttudy, we have cloned and expressed cry1Ac and synthetic genecry-asal) in E. coli (Fig. 1A and B) to assess the efficacy of fusion toxingainst target insects. The purified recombinant proteins when sub-ected to western blot analysis showed positive signals confirming

hat these proteins are Cry1Ac and Cry-ASAL (Fig. 2C).

Ligand blot analysis revealed the differential binding patternf Cry1Ac, ASAL and fusion-protein with the BBMV proteins of. armigera. The fusion-protein could recognize more proteins of

ra (B) neonates. Ten neonates were released on diets supplemented with differentafter 6–10 days of feeding on the diet. Bars indicate mean ± SE. The differences in

BBMVs than Cry1Ac and ASAL (Fig. 3A). The altered binding patternof the fusion-protein could be attributed to the presence of ASALdomain. In S. exigua, it was reported that the Cry1Ac could bind to∼120 kDa, ∼140 kDa and ∼250 kDa proteins, while the hybrid pro-tein containing domains I and II of CryIC and domain III of Cry1Acwas also able to bind to the same proteins (de Maagd et al., 1996).However, the Cry1Ac could bind to only 154 kDa and 220 kDa BBMVproteins of Ostrinia nubilalis (Hua et al., 2001). Recently, it wasreported that when ASAL and Cry1Ac were used in combination,the toxicity of Cry protein increased against H. armigera. Further-more, the two toxins interacted and increased the binding abilityof each other to the insect receptors (Upadhyay et al., 2012).

Variable mortality of P. gossypiella and H. armigera wereobserved when the larvae of these insects were fed on diets contain-ing different concentrations of Cry1Ac and fusion-proteins (Fig. 3Band C). The fusion-protein at 0.025 �g/g concentration caused 100%mortality, while 0.20 �g/g of Cry1Ac is required to cause ∼100%larval mortality of P. gossypiella (Fig. 4A), indicating that the fusion-protein has 8-fold higher insecticidal activity than that of Cry1Ac(Fig. 4A). In the case of H. armigera, the fusion-protein caused 20%,50% and 100% mortality at 0.05 �g/g, 0.075 �g/g and 0.50 �g/g,while only 20% mortality was recorded with 1.5 �g/g of Cry1Ac

(Fig. 4B), suggesting that the fusion-protein has 30-fold increasedinsecticidal activity.

Earlier, it was reported that the hybrid protein consisting DI andDII domains of Cry1Ab and DIII domain of Cry1C showed LC50 of

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.66 �g/g compared to ∼100 �g/g and 11.0 �g/g for Cry1Ab andry1C parental proteins against S. exigua (de Maagd et al., 1996,000). Furthermore, the replacement of domain DIII of Cry1Aa withhat of Cry1Ac resulted in about 300-fold and 10-fold increase in theoxicity of fusion-protein against Heliothis virescens and Trichoplu-ia ni as compared to Cry1Aa (Ge et al., 1991). Similarly, when theybrid protein, comprising DI and DII domains of Cry1Ia and DIIIomain of Cry1Ba, exhibited LC50 of 22.4 �g/g compared to 33.7nd 142 �g/g of Cry1Ia and Cry1Ba parental proteins against Col-rado potato beetle larvae (Naimov et al., 2001). The results of theresent study demonstrate that the fusion-protein at lower con-entrations is more toxic to both the insects when compared tory1Ac.

In this investigation, a synthetic gene encoding the fusion-rotein containing domains of Bt Cry1Ac and ASAL was constructednd expressed in E. coli. An overview of the results indicate themportance of ASAL in combination with DI–DII of Cry1Ac for thencreased toxic effects against P. gossypiella and H. armigera insectsompared to that of Cry1Ac. The enhanced entomotoxic effect ofusion-protein (Cry-ASAL) is attributable to its ability to bind to

ore number of receptor proteins of the insect. The recombinantrotein holds promise in providing broad-based resistance as wells in delaying the development of resistance in the insects. As such,he developed fusion-protein might serve as a potent toxin againstifferent pests of major crop plants.

cknowledgements

We thank Dr. Ashok Varma, ACTREC (Mumbai) and Dr. N.ariprasad Rao, M/S Nuziveedu Seeds Pvt. Ltd. (Hyderabad) for

heir help in protein purification and insect bioassays. BD and SVKre thankful to the University Grants Commission and Council ofcientific and Industrial Research, New Delhi, for the award of fel-owships.

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