Development of a hybrid d-endotoxin and its expression in tobacco

and cotton for control of a polyphagous pest Spodoptera litura

P.K. Singh, Mithilesh Kumar, C.P. Chaturvedi, Dinesh Yadav & Rakesh Tuli*National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India

Received 2 September 2003; accepted 9 March 2004

Key words: Bacillus thuringiensis, hybrid d-endotoxin, insect resistance, protein engineering, Spodopteracontrol, transgenic cotton

Abstract

A hybrid d-endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodopteralitura, which is tolerant to most of the known d-endotoxins. The hybrid d-endotoxin was created byreplacing amino acid residues 530–587 in a poorly active natural Cry1Ea protein, with a highly homolo-gous 70 amino acid region of Cry1Ca in domain III. The truncated d-endotoxins Cry1Ea, Cry1Ca andthe hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilisedCry1EC made from E. coli was 4- fold more toxic to the larvae of S. litura than Cry1Ca, the bestknown d-endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. colicaused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilisedfrom inclusion bodies caused mortality of S. litura with LC50 513 ng/ml semi synthetic diet. A syntheticgene coding for the hybrid d-endotoxin Cry1EC was designed for high level expression in plants, takinginto consideration several features found in the highly expressed plant genes. Transgenic, single copyplants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1–0.7% ofsoluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in allstages of larval development. Hence, unlike in E. coli, the hybrid d-endotoxin folded into a functionallyactive conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobaccoleaves was about 8-fold more toxic (LC50 58 ng/ml diet) compared to expression in E. coli.

Introduction

The d-endotoxin proteins of the soil bacteriumBacillus thuringiensis show toxicity to the larvaeof a number of insect species and have thereforebeen deployed in agricultural crops to developtransgenic cultivars, improved for pest resistance.However, the success of a given d-endotoxin intransgenic cultivars is limited in a crop environ-ment because crops are damaged by a number ofinsect species while the d-endotoxin proteins showhigh toxicity to a rather narrow range of insectpests. For example, transgenic cotton cultivars

expressing Cry1Ac have been released for com-mercial cultivation worldwide. The Cry1Acd-endotoxin is highly toxic to Pectinophora gos-sypiela and Heliothis virescens, moderately toxicto Helicoverpa zea but poorly effective againstSpodoptera exigua and S. frugiperda (Perlak et al.,2001). Therefore, despite a large number of reportscovering at least 22 classes of d-endotoxins(Crickmore et al., 1998), several laboratories havescreened B. thuringiensis strains for d-endotoxinswith new host specificities and mechanism ofaction. The d-endotoxins have been engineered inseveral cases to modify their receptor recognition(Honee et al., 1991; Bosch et al., 1994; Aronsonet al., 1995. Rajamohan et al., 1996; de Maagdet al., 1996), and broaden the host range (Honee

*Author for correspondenceE-mail: rakeshtuli@hotmail.com

Transgenic Research 13: 397–410, 2004.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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et al., 1990; Honee et al., 1991; Naimov et al.,2001) or enhance their membrane interacting orpore forming ability (Chandra et al., 1999). A fewhybrid or engineered proteins with toxicity toS. exigua (de Maagd et al., 1996), H. armigera(Chandra et al., 1999), Plutella xylostella (Ballesteret al., 1999), Leptinotarsa decemlineata (Naimovet al., 2001) and insect cell lines (Rang et al.,1999), and improved over the parental toxinshave been reported. Stable expression of the firsthybrid toxin in plants was reported recently(Naimov et al., 2003) to control a broader spec-trum of target pests in transgenic potato.

The genus Spodoptera contains five primaryspecies (litura, littoralis, exigua, frugiperda andexempta), that occur worldwide. These are thepests of cotton, tobacco, castor, groundnut, anumber of grain legumes and vegetable crops.The primary natural d-endotoxin reported toshow significant toxicity to S. exigua (Sanchiset al., 1988; Visser et al., 1990) and S. littoralis(Honee et al., 1991) is Cry1C. Its variantsCry9Ca (Lambert et al., 1996), Cry1Da and 1Fa(Chambers et al., 1991; de Maagd et al., 2000)also show toxicity to Spodoptera sp., though athigher LC50 values than Cry1Ca. The young lar-vae of S. exigua (Bai et al., 1993; Mazier et al.,1997) are susceptible to Cry1C, but larvaebeyond the 2nd instar display considerable toler-ance (Keller et al., 1996). Plants expressingCry1Ca at a high level were reported to causemortality and effectively give protection againstSpodoptera sp. (Strizhov et al., 1996; Mazieret al., 1997). Not much data has been publishedon the toxicity of the d-endotoxins to S. liturawhich is a common pest in warm and humid cli-mates. We describe the development of a novelhybrid toxin between Cry1Ea and Cry1Caagainst S. litura. These two proteins were selectedfor constructing the hybrid, because the former isknown to have receptor specificity different fromthat of Cry1C (Bosch et al., 1994). The hybridd-endotoxin Cry1EC developed in this studyshowed higher toxicity than the parental toxinsagainst S. litura. A synthetic gene coding for thehybrid d-endotoxin was deployed in transgenictobacco and cotton. In both cases, complete pro-tection against the target pest was observed. Thestudy also suggests differences in functional activ-ity of the d-endotoxins expressed in E. coli andplants.

Materials and methods

Designing and chemical synthesisof the chimeric cry1EC gene

A 1.9 kbp double stranded DNA was theoreti-cally designed (Figure 1) by modifying thesequence of natural cry1Ea gene, to encode thechimeric d-endotoxin Cry1EC. Plant-preferredtranslation initiation context (Sawant et al.,2001) and codons were used in designing thegene. The putative transcription termination sig-nals (AAUAAA and its variants), mRNA insta-bility elements (ATTTA) and potential splicesites were eliminated and long hairpin loops wereavoided. The whole sequence was synthesised as58 overlapping oligonucleotides. The oligonucleo-tides were synthesised on Gene Assembler SpecialDNA synthesiser (Pharmacia Biotech, Sweden),purified on urea – polyacrylamide gel andassembled in four parts (Figure 2) by assemblypolymerase chain reaction (Singh et al., 1996).Eight oligonucleotides were assembled into aBamHI – XhoI fragment (340 bp long), 12 intoa XhoI – AccI fragment (511 bp long), 24 into aAccI – ApaI fragment (680 bp long) and 14 intoa ApaI – EcoRI fragment (519 bp long). Thesewere cloned in pBluescript SK+ cloning vector.At least four clones were sequenced in each caseto locate the errors in synthesis. Sequencing wasdone on model 373 automatic DNA sequencerusing fluorescent – dye termination cyclesequencing kit (Applied Biosystem Inc., USA).The errors were corrected by exchanging theregions containing mutations with those fromcorrect clones. Finally, the error-free DNA frag-ments were stepwise ligated to give about1.9 kbp full length gene (Figure 1).

Expression of the d-endotoxin coding genesin E. coli

The sequence-verified 1.9 kbp DNA coding forthe chimeric cry1EC was placed downstream ofT7 promoter in E. coli expression vector pET19b (Novagen, Madison, WI). The resultant plas-mid, pPK206 was introduced in E. coli BL21DE3to prepare the chimeric protein for the toxicityexperiments. The DNA encoding Cry1Ca (Honeeet al., 1988) and Cry1Ea (Bosse et al., 1990) pro-toxins and truncated toxins was amplified from

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plasmids kindly supplied by Luke Masson (Bio-technology Research Institute, N.R.C. of Can-ada), using suitable primers that created NcoIand BamHI restriction sites at the 50 and 30 ends,respectively. These were also cloned in pET19bto obtain high expression in E.coli. The E. colistrains, growing in log phase were induced in the

presence of 1mM IPTG for 2 h at 15�C. The cellswere harvested, lysed with lysozyme and soni-cated. The inclusion bodies were collected bycentrifugation, solubilised in 50 mM bicarbonatebuffer (pH 9.5) at 28�C and quantified on SDS-PAGE by densitometric scanning (Fluor S MultiImager, Bio-Rad). The protoxins were trypsinised

Figure 1. Designing of the gene coding for chimeric d-endotoxin Cry1EC. Nucleotide sequence of the native components of the chi-meric gene is shown as the upper strand (codons). The modifications introduced in the sequence are indicated underneath. Positionsindicating substitution of the sequence from codons 530 to 587 in Cry1Ea (see Figure 3) with those for codons 533–602 in Cry1Caare indicated.

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to obtained activated toxins. The quantificationwas done on SDS-PAGE, using dilution series ofbovine serum albumin as standard.

Genetic transformation of tobacco and cotton

For the purpose of expressing the cry1EC genein plant leaves, it was cloned downstream ofCaMV35S duplicated enhancer promoter (Kayet al., 1987). The nos transcription terminator(amplified from pBI 101.1 with MfeI and EcoRIrestriction sites at the ends) was placed down-stream. The expression cassette with theenhanced CaMV 35S promoter – cry1EC – nosterminator was cloned in pBI 101.1 (Clontech)by replacing the BamHI–EcoRI gusA–Tnos frag-ment. The binary vector containing cry1EC wasnamed pPK202 (Figure 2). Agrobacterium tum-efaciens LBA4404 containing the helper plasmidpAL4404 was transformed with pPK202 by elec-troporation and used for plant transformation.Tobacco (Nicotiana tabacum cv. Petit Havana)was transformed using the leaf disc method(Horsch et al., 1984) and cotton (Gossypiumhirsutum L. cv Coker 312), by modifying an ear-lier protocol (Sunilkumar and Rathore, 2001).

Larval toxicity tests on semi synthetic dietand leaves of transgenic plants

Larval toxicity tests were conducted using crudeleaf extract or d-endotoxins purified partiallyfrom E. coli. The three truncated d-endotoxins

solubilised from the inclusion bodies expressed inE. coli were used to conduct insect bioassays.The Cry1Ea and Cry1Ca full length protoxinswere first treated with trypsin (5 lg trypsin/mgprotein for 2 h at 37�C) to obtain the activatedtoxins for insect bioassays. The d-endotoxinexpressed in plants was tested by preparing leafextracts. Leaf tissue (1 g) from 60 day old trans-genic plants was crushed in 10 ml buffer (carbon-ate buffer 50 mM pH 9.6 containing 2 mMEDTA and 0.5% (w/v) insoluble PVP). Serialdilutions of the d-endotoxin preparation weremixed in insect diet. Control E. coli pelletor plant leaf, handled in similar manner, wasused as control in the diet. First instar larvaeof a S. litura colony (maintained on castorleaves through the last 2 years in our laboratory)were used to compare relative toxicity of thed-endotoxins. Fifteen larvae were released ontothe diet cake in 100 ml beaker. Each experimentwas repeated six times. The diet was changed onalternate days. The LC50 or EC50 were deter-mined as the concentration of d-endotoxinrequired in diet to cause 50% killing or growthretardation, respectively. It was calculated byprobit analysis, using SAS version 6 (SAS Insti-tute Inc., NC).

In case of leaf bioassays, leaves from the 4thto 6th node from the base of two month oldtransgenic tobacco or cotton were fed to the lar-vae. The bioassays were conducted on 1st (3 dayold), 2nd (5 day old), 3rd (7 day old) and 5th(12 day old) instar larvae. The leaf was cut into15 cm2 discs, placed in a beaker onto a wet filter

(A)

(B)

(C)

Figure 2. Construction of cry1EC for plant transformation. (A) the four fragments assembled from the oligonucleotides; (B)complete gene and (C) the plant transformation cassette cloned in pPK202 are shown. The arrows in C indicate positions of theprimers used to obtain probe for Southern hybridisation.

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paper and 10 larvae were released per leaf disc.In case of the 3rd or 5th instar larvae, intact leafwas held in a cotton plug over a 250 ml flaskcontaining 1/2 MS salt solution. Five insect lar-vae were released on a leaf. In case of bioassayswith the 3rd and 5th instar larvae, the leaveswere changed after 16 and 8 h, respectively, asthe larvae consumed the leaf completely by thistime in case of non-transgenic control. The larvalweight and mortality were scored on alternatedays.

Some experiments were also conducted onneonatal larvae of Helicoverpa armigera, fed onleaves of transgenic or control plants. The larvaeof H. armigera were maintained individually inthe laboratory on semi synthetic diet. Pairedmatings within a colony were done up to 3rdgeneration before progeny from a given pair wasused to conduct a toxicity experiment.

Detection of d-endotoxin expression in plants

Leaf tissue (200 mg) from 60 day old control andtransgenic plants was crushed in 1 ml carbonatebuffer pH 9.6 containing 0.1% (v/v) TritonX-100, 100 lM PMSF, 1% (w/v) insoluble PVP,0.05% (v/v) Tween-20 and 0.02% (w/v) Na-azide.The insoluble plant material was pelleted by cen-trifugation for 10 min at 10,000 rpm. Total solu-ble protein was estimated by the dye-bindingassay (Bio-Rad). A 96-well microtitre plate wascoated with 100 ll of the protein extract of con-trol or transgenic plant lines. Two more controlswere included in the experiment, one was extrac-tion buffer coated directly on wells (buffer con-trol) and the other was standard Cry1EC proteinpurified from E. coli and dissolved in extractionbuffer. The plates were incubated in dark at 37�Cfor 2 h and processed by standard ELISA meth-ods (Harlow and Lane, 1999). The free bindingsites were blocked with 0.5% BSA in physiologi-cal saline containing 0.05% Tween-20 (PBS-Tbuffer). Between any two steps of addingreagents, the plates were emptied and wellsflooded with PBS-T for 3 min. Wash and soakoperation was repeated three times in each step.After blocking, the anti Cry1EC antibody (pre-pared in rabbit by injecting purified Cry1ECd-endotoxin made from E. coli) was added to allsample wells. The plates were incubated for 2 hat 37�C. Subsequently, alkaline phosphatase

labeled goat anti rabbit IgG (secondary anti-body), 30,000-fold diluted in PBS-T-BSA wasadded to the sample wells. After 2 h incubationat 37�C, 200 ll freshly prepared substrate pNPPin an alkaline phase (0.1 M diethanolamine pH9.8) was added to the wells. After incubating for1 h at 37�C, the reaction was terminated by add-ing 50 ll 3 M NaOH to each well. The plate wasread on ab ELISA reader. Dilution series of stan-dard protein purified from E. coli was used toplot a standard curve and determine Cry1EC intransgenic leaves.

Molecular analysis of transgenic plants

Integration of the cry genes in the plant genomewas examined by Southern hybridisation andtheir expression by western immunoblotting byusing standard methods (Sambrook et al., 1989).Total genomic DNA was isolated from freshleaves by DNeasy Plant Maxi kit (Qiagen). About15 lg of genomic DNA was digested with HindIIIand separated by electrophoresis on 0.8% agarosegel in 1 � TAE buffer at 4 v/cm. The gel wasplaced in 0.25 N HCl for depurination and thenneutralised in 0.4 M NaOH. The DNA was trans-ferred to +vely charged nylon membrane (Hy-bond N+, Amersham Life Sciences) with 0.4 MNaOH following downward capillary transfer.After 12 h of transfer, the membrane was washedbriefly with 2 � SSPE buffer, pre-hybridised andhybridised as recommended by the manufacturer.A fragment of 800 bp was amplified from the 50

side of the cry1EC gene, using pPK202 as thetemplate. The position of the probe is marked inFigure 2C. The PCR product was radiolabelledwith a32P-dCTP by random priming method. Theradiolabelled probe was purified, denatured andused for hybridisation. After washing, the mem-brane was wrapped in cling film, exposed to Fujiscreen for 24 h and scanned on phosphorimager(Molecular Imager FX, Bio-Rad). Since there isno Hind III cleavage site within cry1EC, the num-ber of fragments hybridizing in Southernhybridisation, indicated the number of insertionevents.

For western blotting, the leaf extract, pre-pared as for ELISA was used. It was mixed withsample loading buffer (25 mM Tris–Cl, pH 6.8,2% SDS, 200 mM DTT, 20% glycerol and 0.05%BPB) in 1:1 ratio, gently vortexed and

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immediately boiled in a bath for 5 min. Thesamples were centrifuged at 13,000 rpm. Thesupernatant was electrophoresed in 10% SDS-PAGE and transferred to PVDF membrane (Bio-Rad) in blotting buffer (25 mM Tris base,192 mM glycine and 20% methanol). The pri-mary antibody was used at 10,000-fold dilutionand the blot was developed with alkaline phos-phatase colour developer kit (Bio-Rad).

Results

Designing of the chimeric d-endotoxin

The amino acid sequences of the truncatedd-endotoxins Cry1Ea and Cry1Ca show about60% homology (data not included). In an earlierstudy (Bosch et al., 1994), following in vivorecombinations between cry1C and cry1E inE. coli, domain III of the former was reported toenhance the toxicity of Cry1E, whose native formis inactive against the target pests. A specificregion within domain III, with certain aminoacid deletions and substitutions in an otherwisehomologous background between the two d-en-dotoxins was identified for the exchange in thisstudy. Figure 3 shows the amino acid sequenceof domain III of Cry1Ea d-endotoxin (Massonet al., 1992) aligned against a highly homologousdomain III of Cry1Ca (Honee et al., 1988). Thepeptide domain of Cry1Ea from amino acid 530

to 587 was replaced by that of amino acid resi-dues 533–602 of Cry1Ca. The amino acid resi-dues within this region show differences at 31positions while the immediate flanking residuesare highly conserved. Hence, the novel chimericd-endotoxin comprised residues 1–529 of Cry1Ea,530–599 of Cry1Ca and 600–641 of Cry1Ea. Inaddition to the above exchange, an optimaltranslation initiation TAAACCATGGCT wasintroduced at the 50 end of the chimeric gene. Ithas been identified (Sawant et al., 2001) as a pre-ferred sequence for efficient translational initia-tion in plants. It also provides an alanine(instead of glutamate in the native Cry1Ea pro-tein) residue after the initiator methionine. Asreported earlier, a met- ala- at N-terminalenhances stability of proteins in plant leaves (Sa-want et al., 2001).

Toxicity of the chimeric d-endotoxinexpressed in E. coli

The chimeric gene cry1EC and its natural par-ents, cry1Ea and cry1Ca were expressed from T7promoter in E. coli. In spite of the induction byIPTG at low temperature, inclusion bodies wereformed in all the three cases. The solubilisedinclusion bodies were mixed in semi-syntheticdiet and fed to the 1st instar larvae of S. litura.Though no larval mortality was seen, severegrowth retardation was observed in the bioas-says. As shown in Table 1, the synthetic Cry1EC

Figure 3. Alignment of amino acid residues in Domain III of Cry1Ca and Cry1Ea proteins. The domain III is fairly conservedamong the two proteins. The region 530 (I) to 587 (G) of Cry1Ea (underlined) was replaced by the domain 533 (V) to 602 (S) ofCry1Ca to obtain the chimeric d-endotoxin Cry1EC.

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was nearly 4-fold more toxic than Cry1Ca. Theparent toxin Cry1Ea failed to cause any growthretardation. The results show that a nearly inac-tive protein Cry1Ea could be converted into abiologically active toxin by substituting a smallpart of domain III from Cry1Ca. The hybridprotein was more toxic to S. litura than the twoparent proteins.

The Cry1EC, Cry1Ca and Cry1Ea coding con-structs expressed the toxins of molecular mass�69–70.5 kDa in E. coli. The constructs codingfor the protoxins Cry1Ea and Cry1Ca expressedproteins of �130 kDa (Figure 4). On treatmentwith trypsin, the protoxins were hydrolysed to�65 kDa activated toxins (Figure 4). The trypsin-activated Cry1Ca was moderately toxic to S. lituraand gave LC50 of 513 ng/ml (FL50 120–1420 ng)diet (Table 1). The results suggested that the trun-cated 70 kDa Cry1Ca toxin does not fold well inE. coli. However, the full length 130 kDa Cry1Caprotoxin folds differently in E. coli and is con-verted into a functionally active 65 kDa toxin,following trypsinisation.

Toxicity of transgenic tobacco lines expressingthe chimeric d-endotoxin

Encouraged by a higher toxicity of truncatedCry1EC toxin expressed in E. coli, as comparedto Cry1Ca, we decided to express the former intobacco, since it is amenable to efficient transfor-mation. The novel cry1EC gene was placeddownstream of the CaMV 35S promoter andintroduced into tobacco. Leaves from 54 inde-pendent T0 transgenic plants were tested for tox-icity to the 1st instar larvae of S. litura. As given

in Table 2, the independently generated T0

tobacco plants showed variable extent of larvaltoxicity-ranging from group 1 comprising 22plants that caused larval mortality to the extentof 100%, to group 6 that caused only growthretardation. The T0 plants were selfed to obtainT1 seeds. Segregation of kanamycin resistance ina ratio of 3:1 in T1 seeds suggested single copyinsertions in the case of transgenic plants # 6, 8and 19. These were subjected to detailed molecu-lar analysis. The results (Figure 5 Ia) of Southernhybridisation indicated single DNA fragments of22.9, 16.2 and 2.7 kbp in transgenic lines 19, 8and 6 respectively, thus establishing single inser-tion events in the three lines. Following westernblotting, an immunoreactive protein of �70 kDawas detected in the leaves (Figure 5 IIa). The T1

kanamycin resistant seedlings from plants show-ing single copy insertion were grown to obtain T1

population. The T1 lines were grown to maturityto collect T2 seeds. A few T2 seeds were germi-nated on Hoagland solution in the presence of100 lg/ml kanamycin. The lines that gave 100%kanamycin resistant seedlings were considered ashomozygous at the transgenic locus. The hetero-zygous single copy transgenic lines showed segre-gation of kanamycin resistance. Seeds from thelines that were homozygous at the transgeniclocus were germinated to get T2 plants forrepeating the insect toxicity assays. Results fromsuch T2 plants are given in Table 3. The selectedsingle copy homozygous plants showed a highlevel of toxicity to all stages of larval growth.The younger larvae were killed within one to twodays while the 5th instar larvae took upto 5 daysto show complete mortality. The results show

Table 1. Sensitivity of Spodoptera litura 1st instar larvae to the d-endotoxins expressed in E. coli and tobacco leaves

d-endotoxin EC50 or LC50 (lg/ml diet)a Fiducial Limits 95% (lg/ml diet)

I. Truncated toxins expressed in E. coli

Cry1Ea truncated >108

Cry1Ca truncated 23.9 (17.0–30.6)

Cry1EC hybrid 5.8 (0.02–11.8)

II. Full length protoxins expressed in E. coli

Cry1Ea trypsinised >10

Cry1Ca trypsinised 0.513 (0.12–1.42)

III. Truncated toxin expressed in tobacco leaf

Cry1EC truncated 0.058 (0.016–0.150)

a The truncated toxins expressed in E. coli caused growth retardation only. Hence, in case of I, EC50 and in II and III, LC50 values aregiven.

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that the chimeric d-endotoxin Cry1EC made inthe leaves of tobacco is highly toxic to S. litura.The selected T2 transgenic tobacco plantsexpressed the d-endotoxin at upto 0.19% of totalsoluble leaf protein. In contrast to LC50 for thetrypsinised Cry1Ca (LC50 ¼ 513 ng) obtainedfrom E. coli, the Cry1EC expressed in tobaccoleaves (LC50 ¼ 58 ng) was 8-fold more potent(Table 1).

Comparison of the larval toxicity resultsbetween the d-endotoxin Cry1EC expressed intobacco and E. coli, shows that the protein foldsinto functionally different forms in tobacco leavesand E. coli. The truncated d-endotoxins dissolvedfrom the inclusion bodies made in E. coli causegrowth retardation only, suggesting folding ofthe solubilised inclusion bodies into a function-ally less active form. However, higher toxicity of

Figure 4. Various types and forms of d-endotoxins used in the larval bioassays. The inclusion bodies made in E. coli were solubi-lised and subjected to SDS-PAGE. The gels stained with BPB show, (a) 130 kDa Cry1Ca protoxin (lane 2) in E. coli crude extractand 65 kDa trypsinised purified toxin (lane 3); (b) Cry1Ea protoxin (lane 2) and trypsinised toxin (lane 3); (c) trypsinized Cry1Ca(lane 7), truncated 70 kDa Cry1Ca (lane 8), truncated 70.5 kDa Cry1EC (lane 9) and truncated 69 kDa Cry1Ea (lane 10). In a andb, lane 1 shows the position of molecular mass markers. The positions of the d-endotoxins are indicated by arrows. In c, lanes 1–6show dilution series of BSA used as standard for quantification of the d-endotoxins.

Table 2. Toxicity of T0 tobacco plants to 1st instar larvae of Spodoptera litura

T0 transgenic plants of tobacco Larval mortality (%)

Group number No. of plants Day 2 Day 4 Day 6 Day 8

1 22 100 – – –

2 9 20–36 38–57 48–76 80–100

3 7 12–22 16–35 31–49 60–80

4 7 9–24 11–28 19–42 42–60

5 6 0 5–16 9–21 25–40

6 3 0 0 0 0

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the Cry1EC as compared to Cry1Ca made inE. coli, may be suggestive of the former beingmore toxic or being more amenable to function-ally active folding in E. coli. Since we have notprepared transgenic plants expressing truncatedCry1Ca, it is not possible to make an assessmentof relative toxicity of the Cry1Ca and Cry1ECtoxins made in plant leaves.

Toxicity of transgenic cotton lines expressingthe chimeric d-endotoxin

The cry1EC was also transformed into Coker 312cotton. The results on selected T2 lines of Cokerare given in Table 4. The highest expressing trans-genic cotton line T2-24 expressed the toxin at0.67% of total soluble leaf protein. Southern

hybridisation of the cotton lines is shown in Fig-ure 5 Ib. The hybridisation of single Hind III frag-ments of 9.9 and 5 kbp in case of T39 and T35,respectively and two fragments of about 7.4 kbpin case of T-24 with the cry1EC probe, establishsingle copy insertions in the former and two in thelatter. However, segregation analysis showed thatthe two insertion events in T-24 were tightlylinked. Western immunoblotting (Figure 5 IIb)indicated high level expression of the d-endotoxinin leaves. Molecular mass of the leaf-expressed d-endotoxin Cry1EC was �70 kDa, as in the case oftobacco. The molecular mass of the proteinsexpressed in different cases, is as expected fromthe design of the genes. Like transgenic tobacco,the T2 cotton lines also caused complete mortalityof S. litura larvae in all stages of growth. The

Figure 5. Molecular analysis of d-endotoxin expression in transgenic lines of tobacco and cotton. (I) Southern hybridisation show-ing integration events in different lines and (II) western immunoblotting showing expression of the hybrid d-endotoxin Cry1EC inT-19, T-8 and T-6 tobacco (a) and T-39, T-35 and T-24 cotton (b) transgenic T1 lines (l 1–3) described in the text. In IIb, lane 1shows trypsinized Cry1Ca protoxin purified from E. coli and run with the cotton leaf samples (l 2–4) for comparison of mol mass.The trypsinised protein resolves into the 65 kDa activated toxin and smaller minor peptides. The truncated Cry1EC expressed inleaves is �70.5 kDa in both plants.

Table 3. Toxicity of selected single copy, homozygous T2 tobacco lines to Spodoptera litura at different stages of larval growth

T2 tobacco line % mortality of S. litura, 5 days post feeding Cry1EC expression (% leaf protein)

1st instar 5th instar

Untransformed plant 0 0 –

T2-6 100 100 0.15 � 0.013

T2-8 100 100 0.16 � 0.018

T2-19 100 100 0.19 � 0.021

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fourth instar larvae took 3–5 days to show com-plete mortality, in contrast to the 1st instar larvaethat were killed within 2 days of their release ontransgenic leaves. The cotton plants expressedCry1EC at 0.18–0.67% of soluble leaf protein andcaused 100% mortality of 2nd instar larvae after48 h of feeding.

Selected homozygous, single copy T2 trans-genic lines of tobacco and cotton were examinedfor toxicity to the 1st instar larvae of another com-mon polyphagous pest, Helicoverpa armigera. TheCry1EC expressing plants showed a reduction infeeding within 24 h of the larval release. Thelarvae showed substantial growth retardation,following feeding on both transgenic tobacco andcotton (Table 5). Inspite of the expression beinghigher in cotton leaves, the larval weight loss wasgreater in the case of tobacco. This is presumablydue to secondary metabolites or other factors intobacco leaf that may act synergistically with thed-endotoxin in causing larval growth retardation.In fact, the rate of weight gain on control cottonleaves is also more than that on tobacco leaves,suggesting the presence of such natural inhibitorsin tobacco.

Discussion

The deployment of d-endotoxins for the protec-tion of crops against insect pests requires the

availability of proteins that cause high toxicity totarget pests at low level of expression. It is alsoimportant to develop novel toxins that bind todifferent receptors and can therefore be valuablein pyramiding for durable pest resistant. Sincecrops are damaged by a variety of herbivores, itis desirable to identify d-endotoxins that cancause mortality or growth inhibition of differenttarget pest species. In this respect, the develop-ment of Cry1EC in this work is important sinceboth Spodoptera and Helicoverpa are commonagricultural pests and serious bollworms of cot-ton. Several proteins, especially Cry1Ac causevery high toxicity to Helicoverpa sp. Other pro-teins with satisfactory activity against Heli-coverpa are Cry1Ab, Cry1Ia5, Cry2Ab2 etc.(Chambers et al., 1991; von Tersch et al., 1991;Lambert et al., 1996; Selvapandiyan et al., 1998;Perlak et al., 2001). The Cry1C has beenreported to show significant toxicity to Spodopterasp. (Honee et al., 1988; Hofte and Whiteley,1989; Gill et al., 1992; Kalman et al., 1993) atreasonably low level in larval diet. Within thesubgroup of Cry1 d-endotoxins, a few other pro-teins that resemble Cry1C (Visser et al., 1990;Kalman et al., 1993) and Cry1F (Chamberset al., 1991) have been reported to show toxicityto Spodoptera sp., though at relatively higherLC50 values. The Cry1E resembles the Cry1Csubgroup to the extent of about 70%. It does notcause significant toxicity to S. exigua (Visseret al., 1990; Masson et al., 1992; Bosch et al.,1994) but appears to bind to receptors differentfrom Cry1C. Therefore, modification of Cry1Eand search for proteins with high toxicity to Spo-doptera sp. have been objectives in several earlierstudies.

The crystal structures of two d-endotoxins,Cry1Aa and Cry3Aa have been reported as topo-logically conserved. They comprise three distinctstructural domains connected by peptide linkers.The domain I at N-terminal consists of seven

Table 4. Toxicity of selected T2 cotton lines to the 2nd instarlarvae of Spodoptera litura

T2 cotton plant % mortality of

S. litura,

3 days post feeding

d-endotoxin

expression

(% leaf protein)

Untransformed

plant

0 –

T2-24 100 0.67 � 0.051

T2-35 100 0.2 � 0.021

T2-39 100 0.18 � 0.01

Table 5. Toxicity of CryIEC expressing T2 tobacco and cotton lines to the 1st instar larvae of two different lepidopteran pests

Transgenic line Level of expression in leaf

(% of soluble protein)

Mortality or weight loss of 1st instar larvae

S. litura H. armigera

Tobacco T2-19 0.19 100% mortality 46 � 2.4% wt. loss

Cotton T2-24 0.67 100% mortality 21 � 1.6% wt. loss

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a-helical structures. Domain II comprises threeanti parallel b-sheets and two short a-helices. TheC-terminal domain III is a b-sandwich of two antiparallel highly twisted b-sheets (Li et al., 1991;Grochulski et al., 1995). The proteins used in thisstudy, as also other proteins of Cry1C-type aremore than 65% homologous to Cry1Aa (Cham-bers et al., 1991; Crickmore et al., 1998) andtherefore, expected to resemble it structurally.The region that determines receptor recognition,and therefore, host specificity has been mappedprimarily in the hypervariable domain II by anumber of mutational studies (Rajamohan et al.,1996) and exchange of domains between geneswith different specificities (reviewed in Grochulskiet al., 1995; Rang et al., 1999). The pore formingrole is primarily assigned to domain I (Aronsonet al., 1995). Mutations in domain III indicatemultiple roles of this region, including channelformation (Chen et al., 1995; Wolfersbergeret al., 1996) and receptor binding (Bosch et al.,1994; Aronson et al., 1995; de Maagd et al.,2000). The region further downstream stabilisesprotein folding (Nishimoto et al., 1994). Foldingof the d-endotoxins is determined by complexinteractions within and among the three domains.In several cases, incompatible combinations givemisfolded hybrid proteins that show no or verylittle functional activity. Such hybrids are ofteninsoluble in E. coli and sensitive to proteolyticenzymes (de Maagd et al., 2000; de Maagd et al.,2003). Our results suggest that some of the toxinsthat may not fold correctly in E. coli, can foldinto biologically highly toxic form in plants.

Studies on soluble, functionally active hybridproteins made in E. coli in earlier studies, demon-strated that overall toxicity is determined by mul-tiple interactions among the three domains. Forinstance, though substitutions with domain III ofCry1C converted five Cry1 type d-endotoxins intoactive toxins against S. exigua, a 40-fold differenceamong the hybrids was noticed. This suggested thecontribution of domains I and II in determiningfunctional folding of the d-endotoxins and there-fore, final toxicity (de Maagd et al., 2000).Though domain III may be important and suffi-cient for binding certain receptors, this may nothold true for other receptors and toxins, asreported for S. exigua (de Maagd et al., 1999) andP. xylostella (Ballester et al., 1999). Honee et al.(1991) generated eleven hybrids between two

Cry1Ab and Cry1C proteins. The former is highlytoxic to Helicoverpa sp. while the latter shows hightoxicity to Spodoptera sp. Only one hybrid, com-prising a large C-terminal domain (residues 258–646) of Cry1C joined to the N-terminal of Cry1Abshowed significant though reduced, toxicity toSpodoptera. The results suggested the presence ofinsect specificity region in the C-terminal part ofCry1C. Bosch et al. (1994) screened a large num-ber of random combinations between Cry1C andCry1E generated by in vivo recombination inE. coli. Most of these were insoluble, unstable,poorly toxic or non-toxic. However, in one casethe transfer of a large fragment comprising aminoacid residues 473 (close to the junction of domainsII and III) to 630 of Cry1C to Cry1E, gave ahybrid protein (G27) that was insecticidal likeCry1C and had receptor specificity resemblingCry1E. The chimeric recombinant comprised ofthe native domains I and II of Cry1E and there-fore the receptor specificity was suggested to residein domains I and II of Cry1E. Domain substitu-tions resulting in hybrids that were more toxicthan either of the parental proteins have beenreported in a few cases. For example, the exchangecomprising the first two domains of Cry1Ab andthe third domain of Cry1C gave a hybrid HO4that was more toxic than the preferred parentCry1Ab against Plutella xylostella (Ballester et al.,1999) and Cry1C against S. exigua (de Maagdet al., 2000). Similarly, substitutions betweenCry1Ba and Cry1Ia gave a hybrid with enhancedtoxicity against Leptinotarsa decemlineata (Nai-mov et al., 2001). Rang et al. (1999) alsoexchanged regions coding for either domain I orIII among Cry1Ab, Cry1Ac, Cry1Ca and Cry1Ea.A chimeric toxin consisting of domains I and II ofCry1Ca and domain III of Cry1Ab was more effi-cient in pore formation on Sf 9 insect cell linesthan the parental toxins. They suggested that thedomain II from an active toxin was necessary,though not sufficient for converting Cry1E into anactive toxin.

Domain III of Cry1E and Cry1C is highlyhomologous, except certain amino acid substitu-tions and the two peptide blocks with deletions(Figure 3). A natural variant of Cry1Ca, calledCry1Cb, has earlier been reported to be 5-foldless toxic than Cry1Ca (Kalman et al., 1993) toS. exigua though they are 92% identical in thefirst two domains. The domain III of these

407

proteins shows only 48% homology. This is alsoindicative of the role of domain III in toxicity ofCry1Ca protein. We exchanged a rather smallbut variant part of domain III of Cry1Ea thatshows two deletions, with the correspondingregion of Cry1Ca to obtain the chimeric toxinCry1EC. The resultant protein, inspite of beingpredominantly (571 residues) Cry1Ea and havingonly 70 residues (530–599) of Cry1Ca was notonly toxic to S. litura (unlike Cry1Ea) but alsoseveral fold more toxic than the Cry1Ca. Theresults are at variance with Bosch et al. (1994)who identified the residues 473–494 of Cry1Cand Rang et al., (1999) who identified domain IIas most critical to converting Cry1E into a func-tionally active chimera. We find, a relativelymore variable region spanning from V533 to S602of Cry1Ca contains sufficient architectural infor-mation for converting Cry1Ea into a highly toxicprotein against Spodoptera sp. The results suggesta variety of different exchanges between twod-endotoxins can lead to the formation of newactive conformations, depending upon otherinteractions in the polypeptide backbone.

The derivative H8 (Bosch et al., 1994; deMaagd et al., 1999) of the hybrid G27 (Ballesteret al., 1999) is closest to the hybrid Cry1EC madein our study. The H8 involves an exchange ofdomain III from the 494th residue till the end ofthe protoxin i.e. 1785th residue of Cry1C. The H8is 10-fold less active than G27 against S. exigua,though the two proteins differ in the former havingonly three additional amino acids in (473–494thregion) of Cry1C rather than Cry1E. The hybridtoxin we report here involves the exchange ofCry1C residues downstream of 533 but only till602 into Cry1E and yet gives a toxin with severalfold higher activity than the two parental proteins.The exchanged region includes the two blocks thathave 7 and 5 residue deletions (Figure 3) in nativeCry1E as compared to Cry1C. Our study suggeststhat the three amino acid differences in region473–494 may not be critical to constructing a func-tionally active toxin against Spodoptera sp., if theremaining domain III backbone provides thedesired molecular environment. Hence, the threedimensional structure improved for functionaltoxicity against a target pest can be achieved by avariety of mutations or domain substitutions ind-endotoxins. A knowledge-based approach fortargeted engineering of the d-endotoxins however

requires establishing the relationship betweens thestructure and function of such chimeric proteins.The expression of a protein in heterologous envi-ronment such as plant cells, may further fine tuneits folding and thus, influences its function. Ourresults suggest that the folding of d-endotoxins ina plant cell milieu is different and can facilitatefunctionally more relevant conformation than thefolding in E. coli.

The gene encoding Cry1EC expressed at ahigh level in leaves of both tobacco and cottoncaused complete mortality of S. litura larvae. Inearlier reports, the native cry1C (Mazier et al.,1997) has been reported to give some protectionagainst early developmental stages of S. littoralis.However, Strizhov et al. (1996) deployed a syn-thetic cry1C and reported a high level of protec-tion against S. exigua and S. littoralis in alfalfaand tobacco. The deployment of a chimericd-endotoxin in transgenic plants for insect resis-tance has been reported earlier only by Naimovet al. (2003). Thus the development of transgeniccotton against S. litura and the use of a hybridprotein with Cry1E backbone in a transgeniccrop are useful developments. In addition ofcomplete protection against this bollworm, thenovel d-endotoxin causes growth retardation inH. armigera. Since the growth retarded larvaemay be compromised reproductively or fail tocomplete metamorphosis or become more sensi-tive to a complimenting d-endotoxin, such genesmay also make a useful component of resistancemanagement strategies.

Acknowledgements

The authors thank The Council of Scientific andIndustrial Research and The Department of Bio-technology, Government of India for providingfinancial support for the work reported in themanuscript.

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