Transgenic Research (2006) 15:349357 DOI 10.

1007/s11248-006-0007-2

Springer 2006

Spider venom toxin protects plants from insect attack

Sher Afzal Khan, Yusuf Zafar, Rob W. Briddon, Kauser Abdulla Malik & Zahid Mukhtar*,
Plant Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P.O. Box 577, Faisalabad, Pakistan
Received 17 August 2005; accepted 31 January 2006

Key words: insect, Hadronyche versuta, resistance, spider, toxin, transgenic

Abstract Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specic or are only mildly eective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specically to kill insects. Here we show that the x-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the puried toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco eectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and eective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture.

Introduction Heterologous expression of insecticidal toxins in plants to protect them from attack by herbivorous insects has proven an eective means of reducing farmings reliance on environmentally harmful chemical alternatives. Resistance to insects has been demonstrated in transgenic plants expressing genes for d-endotoxins from Bacillus thuringiensis (Bt) (Mehlo et al., 2005), protease inhibitors, enzymes and plant lectins (Abdeen et al., 2005). Most of the plant derived genes produce chronic rather than toxic eects and some insect pests are not sensitive to some of these factors. However, the range of toxins in common use has remained narrow, almost exclusively derived from the soil bacterium B. thuringiensis (Schnepf et al., 1998).
*Author for correspondence E-mail: zahid@nibge.org The authors Sher Afzal Khan and Zahid Mukhtar contributed equally to this work.

The venoms of insectivorous spiders are a complex mixture of compounds that have remained largely untapped for biotechnology application (Fletcher et al., 1997; Atkinson et al., 1998; Tedford et al., 2004a). These toxins have a wide range of mechanisms of action, can have a very narrow spectrum of organisms that are susceptible and are thus ideal candidates for use in the engineering of resistance to herbivorous insects (Grishin, 1999). The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif common to a large group of polypeptides with diverse sequences and bioactivities. Although found in dierent phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. The ICK structural motif consists of a cystine knot with a triple-stranded beta-sheet (Norton and Pallaghy, 1998; Grishin, 1999; Mouhat et al., 2004). A number of ion channel toxins contain the ICK motif, including the x-ACTX-Hv1a toxin (Hvt) found in the venom of the Australian funnel

350 web spider (Hadronyche versuta) (Norton and Pallaghy, 1998). Hvt is a 37 amino acid, insectspecic calcium channel antagonist. The peptide is toxic to a range of agriculturally important arthropods in the orders Coleoptera, Lepidoptera and Diptera but has been reported to have no eects on a number of mammals (Norton and Pallaghy, 1998; Tamaoki et al., 1998; Tedford et al., 2001; Tedford et al., 2004b). We have investigated the potential usefulness of expressing a spider venom toxin in plants to provide protection from herbivorous insects. Our results demonstrate that, when expressed in a heterologous system, the Hvt peptide retains its biological activity and toxicity to insects. Hvt thus shows promise for the transgenic protection of crops and should be further investigated. described previously (Studier et al., 1990; Stewart et al., 1998). The fusion protein was puried from claried bacterial lysate by anity chromatography on His Bind Resin (Novagen). A polyclonal antiserum was prepared in rabbits against Hvt fused with thioredoxin. Biological toxicity assays Larvae of H. armigera and S. littoralis were collected from cotton elds in Faisalabad District, Pakistan. The larvae were caged on cotton leaves in petri plates lined with moist lter paper. The leaves were replaced with fresh leaves every 2 days until pupation. The emerged adults were transferred to plastic jars lined with muslin cloth. Eggs laid on the muslin were collected from the jars and placed in vials containing an articial insect diet and maintained at a temperature of 252C.

Materials and methods Expression constructs The nucleotide sequence of the gene encoding Hvt was deduced, with codon optimization for expression in plants, from the published amino acid sequence of the protein (Fletcher et al., 1997), synthesized commercially (Medigenomix, Germany) and cloned in the vector pCR3.2 (Invitrogen). The gene was excised as a HindIII-EcoRI fragment and cloned in the expression vector pJIT60 (Guerineau and Mullineaux, 1993) to produce plasmid SAK-I (Figure 1(a)). The pSAK-I expression cassette was excised as a KpnI-XhoI fragment and ligated into the binary vector pGreen0029 (Hellens et al., 2000) to produce pSAK-II (Figure 1(b)). pSAK-II was transformed into Agrobacterium tumefaciens LBA4404 containing the helper plasmid pSoup (Hellens et al., 2000). For expression in E. coli the HindIII-XhoI fragment of pSAK-I was ligated in pET-32a(+) (Novagen) to yield pSAK-III (Figure 1(c)). Protein expression in E. coli and antiserum production pSAK-III was transformed into E. coli strain BL21(DE)pLysS (Novagen). Large-scale E. coli cultures for protein purication were produced as

Figure 1. Constructs for the expression of Hvt. The 131 bp synthetic Hvt gene was cloned as a HindIII-EcoRI fragment in pJIT60 (Guerineau and Mullineaux, 1993) and the E. coli expression vector pET-32a(+) (Novagen) to yield pSAK-I and pSAK-III respectively. Expression from pET-32a(+) was as a fusion with thioredxin (trxA) and the His tag. The pJIT60 expression cassette, consisting of a double Cauliower mosaic virus (CaMV) 35S promoter, the Hvt gene and a CaMV terminator, was then sub-cloned as a KpnI-XhoI fragment into the binary vector pGreen0029 (Hellens et al., 2000) to produce pSAK-II for plant transformation.

351 The Hvt-thioredoxin fusion protein in elution buer (1 M imidazole, 0.5 M NaCl, 20 mM Tris HCl [pH 7.9]) was applied in droplet form (2 ll) to the thoracic region of 2ND instar H. armigera and S. littoralis larvae. The LD50 was determined by applying various doses (0.02, 0.06, 0.12, 0.25, 0.5 or 1.0, 2.0, 4.0, 8.0, or 16.0 pmol) of the fusion protein to the larvae. Control larvae were treated with 2 ll of thioredoxin protein (without Hvt fusion). Twenty larvae were treated with each protein amount. Larvae were monitored at regular intervals for signs of toxicity. LD50 values were calculated at 12 h post-application. Detached leaf bioassays were used to assess the eectiveness of transgenically expressed Hvt protein. Small, mature Nicotiana tabacum leaves from four selected transgenic lines and non-transformed plants were placed in triplicate on moist lter paper in petri dishes and second instar S. littoralis larvae or rst and second instar H. armigera larvae were caged on these. Approximately one larva per 20 cm2 leaf area was used. The plates were kept at a temperature of 252C and relative humidity of 5070%. Mortality was recorded 24, 48 and 72 h after release of the insects. Larval weight was determined before release of the insects and after 72 h. Bioassay data was arranged in a completely randomized design (CRD) and subjected to analysis of variance. The means were compared using Duncans multiple range test (DMRT). The extent of leaf damage (surface area), by S. littorallis larvae, was calculated 5 days after release of the insects by printing digital images of the leaves onto graph paper. Whole plant bioassays were conducted with S. littorallis larvae. Thirty-two second instar larvae were placed on a single plant of transgenic line T21 and a non-transformed N. tabacum plant. Plants were maintained at 252C and relative humidity of 5070%. Plant transformation and analysis Transformation of N. tabacum L. cv. Samsun was conducted as described by Horsch et al. (1984). A total of 25 lines, resulting from independent transformation events, were selected for analysis. Transgenic lines were initially screened for the presence of the toxin expression construct by PCR with primers designed to the 35S promoter and the C-terminal end of the toxin gene (forward primer 5-GGTGGCTCCTACAAATGCC-3 and reverse primer 5-TGGGCAGGGCTGACCGGAAG-3 respectively). Southern blot analysis was carried out following standard protocols (Sambrook et al., 1989). Approximately 10 lg of total genomic DNA, extracted from T0 transformed and non-transformed plants, was digested with KpnI (for which there is only one recognition site in the expression cassette). Blots were probed with an approx. 831 bp, radioactively labelled, HindIIIXhoI fragment derived from pSAK-II. Transgenic tobacco plants were analyzed for protein expression by SDS PAGE. Leaf tissue was frozen in liquid nitrogen, ground to a ne powder and then the total soluble protein was extracted using the Trizole reagent (Life technologies). The protein concentration of the supernatant was determined by the Bradford assay (Bradford, 1976). Protein from both transformed and control plants (approx. 50 lg per lane) was electrophoresed on 15% SDS-PAGE gels. Gels were either stained with Coomassie blue or used for Western blotting and immunological detection. HPLC analysis HPLC analysis for Hvt was performed as described previously (Fletcher et al., 1997). Total soluble protein was analysed by rpHPLC on a Techsphere C18 analytical column (4.63 250 mm, 5-mm pore size). Samples were run at a ow rate of 1 ml per min using an initial linear gradient of 520% acetonitrile/0.1%TFA over a period of 35 min.

Results Hvt expression in E. coli To investigate the toxicity of Hvt to insects when expressed in a heterologous system, a codon optimized (for expression in plants) gene encoding the toxin (EMBL accession no. AJ938032), deduced from the published amino acid sequence of the protein, was produced (Fletcher et al., 1997; Norton and Pallaghy, 1998; Tedford et al., 2004a). The Hvt gene was initially expressed as a fusion with thioredoxin (to enhance the stability and solubility of the peptide) and with a His tag (for

352 anity column purication) in E. coli (Figure 1c). High levels of expression of the fusion protein (more than 30% of the total soluble protein) were achieved without apparent toxic eects on the growth of the bacterium (Figure 2a). Following anity purication, several bands were evident on polyacrylamide gels, with the major band being above the expected size for the fusion (25 kDa rather than the expected size of 18 kDa; Figure 2a, lane 2). The reason for this is unclear but it may be due to strong secondary structure. The puried fusion protein, when digested with enterokinase (to release the thioredoxin tag) and run on an SDS-PAGE gel, showed the correct migration of the thioredoxin tag (14 kDa) (Figure 2a, lane 1). This indicates that the anomalous migration of the fusion protein is due to Hvt. Hvt has high intrinsic folding capacity, which may not be aected by denaturation procedures. However this property, as well as its resistance to proteolytic degradation, makes it an attractive peptide for expression in plants. The puried fusion product was used to produce a polyclonal antiserum in a rabbit. The antiserum labelled a protein band of approximately 25 kDa in western blots of lysates of bacteria transformed with pSAK-III (Figure 2b). Hvt expressed in E. coli retains biological activity To investigate the biological activity of the puried fusion protein, this was applied externally to the thoracic region of H. armigera and S. littoralis larvae. The minimum toxic concentration, for both H. armigera and S. littoralis, was shown to be approx 1.0 pmol. With this dose the larvae ceased feeding and showed the behavioural symptoms typical of this group of neurotoxins (initially a lack of coordination and head shaking followed by paralysis), within 2 h of application. The LD50 at 12 h after application was determined to be approximately 4 and 2 pmol of toxin per gram body weight of H. armigera and S. littoralis larvae, respectively. All insects died within 24 h. No

c Figure 2. Analysis of heterologously expressed Hvt protein. (a) Coomassie stained polyacrylamide gel (10%). Samples in lanes 1 and 3 were claried bacterial lysates of BL21 E. coli cells induced for expression of Hvt-thioredoxin-His tag fusion protein (pSAK-III). The sample in lane 1 was treated with enterokinase prior to loading. Puried fusion protein was loaded in lane 2. A molecular weight marker was run in lane M. (b). Western blot probed with an antiserum raised against the Hvt-thioredoxin fusion protein. Samples were claried bacterial lysates of E. coli BL21 cells induced for expression of Hvt-thioredoxin-His tag fusion protein (pSAK-III) (lane 1), and total soluble proteins extracted from N. tabacum; a nontransformed, control plant (lane 2) and transgenic lines T21 (lane 3), T10 (lane 4) and T01 (lane 5). A pre-stained molecular weight marker (SM0441, MBI Fermentas) was run in lane M. (c) HPLC trace of puried Hvt (upper trace) and total soluble proteins extracted from Hvt transformed N. tabacum plants (lower trace). The peak corresponding to Hvt is marked with an arrow for each trace.

353 aected larvae recovered from the toxic eect of Hvt during the experiments. In control experiments, larvae receiving an equal amount of thioredoxin were unaected, developed normally to pupation and emergence of adults. These results show that Hvt, expressed in a heterologous system as a fusion with thioredoxin, remains biologically active against two agriculturally important insect pests. Constitutive expression of Hvt in Nicotiana tabacum N. tabacum cv Samsun was transformed with the Hvt gene under the control of the Cauliower mosaic virus 35S promoter (pSAK-II; Figure 1b) so as to constitutively express the toxin in all plants tissues. Initial transformants were selected on kanamycin and a total of 25 independently transformed lines were obtained. Presence of the intact expression cassette was conrmed in all lines by PCR-mediated amplication. Southern blot analysis of total genomic DNA extracted from transformed plants indicated that the majority of transgenic lines had the expression construct integrated at two distinct loci (Figure 3). Only line T35 exhibited only a single band, suggestive of a single locus insertion. The expression level of Hvt in the transformed plants was estimated to be between 0.1 and 0.25% of total soluble protein. All resulting transformed lines were morphologically normal and fertile, with no detectable eect of toxin expression. The transgenic lines with the highest protein expression level (T01, T10 and T21) were chosen for further analysis. A western blot of total soluble protein extracted from these transgenic lines probed with the anti-Hvt antiserum is shown in Figure 2a. The highest level of Hvt expression was observed in line T21 (Figure 2). A signicant proportion of the Hvt protein detected migrated with an apparent molecular weight of 18 kDa, well above the expected size of 4 kDa. The reason for this is unclear. Possibly this may be due to a strong secondary structure or post-translational modication of Hvt in a plant background. By HPLC analysis the protein expressed in E. coli (protease treated to remove the thioredoxin fusion partner) and in transformed plants eluted as a single peak with identical retention times (Figure 2). N. tabacum constitutively expressing Hvt is protected from insect attack H. armigera larvae were caged on detached, mature leaves from either transformed or nontransformed, control N. tabacum plants. Within 24 h of being placed on leaves of transformed plants the insects showed the eects of toxin ingestion consisting of cessation of feeding, uncontrolled movement followed by paralysis. Typically the larvae died within 48 h when placed on detached leaves of transgenic line T21, while the insects on leaves from non-transformed plants continued feeding throughout the experiment, eventually consuming the whole leaf (Figure 4). H. armigera larvae feeding on leaves of the transgenic lines showed signicantly higher mortality rates compared to controls (Table 1). The highest levels of mortality were with line T21. Larval weight, determined 72 h after release onto detached leaves, was signicantly lower for all the insects feeding on the transgenic lines in comparison to the control. Detached leaf assays conducted with S. littoralis larvae showed a similar trend (Figure 4, Table 2). For the detached leaf assays using S. littoralis larvae, the extent of leaf damage was calculated 5 days after releasing the insects on the detached leaves of transgenic line

Figure 3. Southern blot analysis of transgenic tobacco lines. Total genomic DNA (approx. 10 lg) was digested with KpnI (a restriction endonuclease that restricts pSak-II only once) and electrophoresed on a 0.8% agarose gel. Samples were extracted from a non-transformed tobacco plant (lane 1), from transformed lines T1, T10, T21, T26, T27 and T35 (lanes 27 respectively). HindIII-XhoI digested pSAK-I was run as a marker (lane 1). The blot was probed with a 32P-labelled 831 bp HindIII-XhoI fragment of pSAK-I.

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Figure 4. Detached leaf toxicity assays. Insect larvae were caged on detached tobacco leaves of transformed (a, c, e) and control, non-transformed (b, d, f) plants. First instar (a, b) and second instar (c, d) H. armigera larvae and second instar S. littoralis larvae (e, f) were released on the leaves and photographed after 24 h.

Table 1. Mortality of second instar H. armigera larvae on detached leaves of Hvt transformed and non-transformed N. tabacum plants Plant line Larvae weight (mg)* Time after introduction (h) 0 NT T1 T10 T15 T21 0.3200.006 0.3090.004 0.3080.005 0.3080.004 0.3090.005 72 3.5060.085 0.2450.008 0.2340.006 0.2300.005 0.2210.006 Mean a b b b b Mortality (%) Time after introduction (h) 24 0 0 6.7 0 6.7 2.68 c 48 0 33.3 33.3 13.3 46.7 25.3 b 72 0 86.7 66.7 53.3 100.0 61.3 a Mean 0.00 d 40.0 b 35.6 b 22.2 c 51.1 a

*Mean of three replicates; each replicate having ve insects. Means followed by same letter are not signicantly dierent at p=0.05.

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Table 2. Mortality of second instar S. littoralis larvae on detached leaves of Hvt transformed and non-transformed N. tabacum plants Plant line Larvae weight (mg)* Time after introduction (h) 0 NT T1 T10 T15 T21 0.2020.0025 0.2070.0268 0.2390.0041 0.2330.0044 0.2320.0034 72 1.6670.0307 0.1050.0055 0.1480.0090 0.1850.0186 0.1070.0082 Mean a c bc b bc Mortality (%) Time after introduction (h) 24 0 0 7.7 0 57.1 13.0 c 48 0 60 38.5 30.8 100 45.7 b 72 0 100 92.3 76.9 67.3 a Mean 0.00 53.33 46.11 35.56 78.55 d b b c a

*Mean of three replicates; each replicate having ve insects. Means followed by same letter are not signicantly dierent at p=0.05.

T21. Signicantly less leaf area was consumed by the larvae feeding on leaves of line T21 (an average of 137.60 mm2 in three replicates; total area consumed 412.9 mm2) than on the leaves of nontransformed plants (an average of 1604 mm2 in three replicates; total area consumed 4812.9 mm2). The visual dierence in damage caused by S. littoralis larvae feeding on whole, transformed and non-transformed plants is shown in Figure 5.

Discussion We have investigated the toxicity of an arachnidderived protein when expressed in a heterologous system and its potential use for engineering resistance in plants to agriculturally important insect species. The properties of the H. versuta toxin make it an ideal candidate for this purpose. It is a small, stable peptide, has a mode of action distinct

Figure 5. Whole plant toxicity assay. Second instar S. littorallis larvae, 32 per plant, were released on (a) transformed (line T21) and (b) non-transformed tobacco plants. The photograph was taken 5 days after release of the insects.

356 to those of the toxin genes already in use in transgenic crops and is reported to have exceptional phylogenetic specicity; aecting only certain classes of insects, many of which are of agricultural signicance (Fletcher et al., 1997). Recent research shows the x-atracotoxin-2 family of toxins to exhibit at least a 10,000-fold preference for insect over vertebrate voltage-gated calcium channels (Wang et al., 2001). When expressed heterologously, either in E. coli or in N. tabacum, the Hvt toxin retains its neurological toxicity to at least two insect species. The symptoms induced by the transgenically expressed toxin are identical to those described for the native, H. versuta venom-isolated peptide, consisting of a lack of coordination, disorientation and uncontrolled movement. The mode of action of the heterologously expressed and native peptides are thus likely the same, acting as an antagonist of the insect-specic calcium channels. In both systems there was no apparent deleterious eect of transgene expression on growth, development and fertility; highly desirable traits for any gene to be expressed for plant protection/improvement. When applied topically in relatively large doses, Hvt provided a rapid (in a few minutes) cessation of feeding in the larvae of the two insect species tested. However, even for the much smaller doses ingested by larvae feeding on transgenic N. tabacum, the eects of the toxin were suciently rapid (2448 h) to prevent signicant foliar damage, indicating that even before toxin-induced behavioural changes in the insect are evident, the larvae were exhibiting a reduced consumption of foliage. Crops with engineered resistance to herbivorous insects are being used extensively in many countries (James, 2002). Since its introduction in 1996, for example, the area planted with Bttransformed crops increased to 22.4 million hectares worldwide in 2004 (Lawrence, 2005). These crops provide not only an economic benet to farmers, particularly in resource poor developing countries where the reduced requirements for expensive insecticides are particularly desirable, but also environmental benets. However, the range of insecticidal genes available in transgenic crops remains low, raising concerns over the potential for resistance to develop in the targeted insect populations. Although the potential for resistance to Bt developing in insect populations has been demonstrated experimentally (Liu et al., 2001) this has not so far occurred in the eld. The availability of a larger range of genes for introduction into crop plants could signicantly reduce the impact of resistance arising to a single group of toxins and, if multiple genes are introduced into a single crop variety (so called pyramiding or stacking), would greatly reduce the likelihood of resistance developing (Zhao et al., 1997; Tabashnik et al., 2002). Based on the results presented here, Hvt (and possibly other spider venom-derived toxins) makes an attractive candidate for the transgenic protection of crop species from herbivorous insects. Further studies, addressing possible non-target toxicity to, in particular, benecial insects, browsing mammals and birds need to be conducted. Our own eorts are now aimed at determining the eects of Hvt expression on plant growth and potential toxicity to non-target organisms. In the rst instance the gene will be introduced into a non-food crop, such as cotton, for which numerous herbivorous insect species (including H. armigera and S. littoralis) are a problem throughout the world, to allow further investigation of its suitability for transgenic application in agriculture. Acknowledgements We would like to thank Farooq Latif for HPLC analysis. This work was supported in part by grants from ACIAR, BSCV-MINFAL to YZ. We also thank Mazhar Hussain for nancial assistance.

References
Abdeen A, Virgos A, Olivella E, Villanueva J, Aviles X, Gabarra R et al. (2005) Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors. Plant Mol Biol 57: 189202. Atkinson RK, Howden MEH, Tyler MI and Vonarx EJ (1998) Insecticidal toxins derived from funnel web (Atrax or Hadronyche) spiders. US Patent No. 5,763,568, Zeneca Limited, USA. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254. Fletcher JI, Smith R, ODonoghue SI, Nilges M, Connor M, Howden ME et al. (1997) The structure of a novel insecticidal neurotoxin x-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nat Struct Biol 4: 559566. Grishin E (1999) Polypeptide neurotoxins from spider venoms. Eur J Biochem 264: 276280.

357
Guerineau F and Mullineaux P (1993) Plant transformation and expression vectors. In: Croy RRD (ed), Plant Molecular Biology Labfax (pp. 121147) Bios Scientic Publishers, Oxford, UK. Hellens RP, Edwards EA, Leyland NR, Bean S and Mullineaux PM (2000) pGreen: a versatile and exible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819832. Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A and Homan H (1984) Inheritance of functional genes in plants. Science 223: 496498. James C (2002) Preview, Global Status of Commercialized Transgenic Crops: 2002. ISAAA Briefs No. 27. ISAAA, Ithaca, New York. Lawrence S (2005) Agbio keeps on growing. Nat Biotechnol 23: 281. Liu Y, Tabashnik B, Meyer S, Carriere Y and Bartlett A (2001) Genetics of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac. J Econ Entomol 94: 248252. Mehlo L, Gahakwa D, Nghia PT, Loc NT, Capell T, Gatehouse JA et al. (2005) An alternate strategy for sustainable pest resistance in genetically enhanced crops. Proc Natl Acad Sci (USA) 102: 78127816. Mouhat S, Jouirou B, Mosbah A, De Waard M and Sabatier JM (2004) Diversity of folds in animal toxins acting on ion channels. Biochem J 378: 717726. Norton RS and Pallaghy PK (1998) The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 36: 15731583. Sambrook J, Fritsch ER and Maniatis T (1989) Molecular Cloning: a Laboratory Manual Cold Spring Harbor Laboratory Press, Woodbury, New York. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J et al. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62: 775806. Stewart EJ, Aslund F and Beckwith J (1998) Disulde bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J 17: 55435550. Studier FW, Rosenberg AH, Dunn JJ and Dubendor JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 6089. Tabashnik BE, Dennehy T, Sims MA and Larkin K (2002) Control of resistant Pink Bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin cry2Ab. Appl Environ Microbiol 68: 37903794. Tamaoki H, Miura R, Kusunoki M, Kyogoku Y, Kobayashi Y and Moroder L (1998) Folding motifs induced and stabilized by distinct cystine frameworks. Protein Eng 11: 649659. Tedford HW, Fletcher JI and King GF (2001) Functional signicance of the b-hairpin in the insecticidal neurotoxin x-atracotoxin-Hv1a. J Biol Chem 276: 2656826576. Tedford HW, Gilles N, Menez A, Doering CJ, Zamponi GW and King GF (2004a) Scanning mutagenesis of x-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels. J Biol Chem 279: 4413344140. Tedford HW, Sollod BL, Maggio F and King GF (2004b) Australian funnel-web spiders: master insecticide chemists. Toxicon 43: 601618. Wang X, Connor M, Wilson D, Wilson H, Nicholson G, Smith R et al. (2001) Discovery and structure of a potent and highly specic blocker of insect calcium channels. J Biol Chem 276: 4030640312. Zhao JZ, Fan XL, Shi XP, Zhao RM and Fan YL (1997) Gene pyramiding: an eective strategy of resistance management for Helicoverpa armigera and Bacillus thuringiensis. Resist Pest Manage Newslett 9: 1921.