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Chimeric δ-endotoxins of Bacillus thuringiensis with increased activity against Helicoverpa armigera

Published online by Cambridge University Press:  01 June 2011

Anuradha Chelliah ,
Gorakh Prasad Gupta ,
Sasikumar Karuppiah  and
Polumetla Ananda Kumar
Anuradha Chelliah*
Affiliation:
National Research Centre for Banana, Thogamalai Road, Thayanur Post, Tiruchirapalli620 102, Tamil Nadu, India
Gorakh Prasad Gupta
Affiliation:
Division of Entomology, Indian Agricultural Research Institute, New Delhi110 012, India
Sasikumar Karuppiah
Affiliation:
H.H. Raja's College, Pudukottai622 001, Tamil Nadu, India
Polumetla Ananda Kumar
Affiliation:
National Research Centre on Plant Biotechnology, New Delhi110 012, India
*
*E-mail: anuradha.chelliah@gmail.com
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Abstract

We selected 50 crystal (Cry) toxins specific to lepidopteran insects. Phylogenetic relationships of whole toxins as well as individual domains revealed that Cry1Jb and Cry1Ac are distantly placed but related to reduced cross-resistance in Helicoverpa armigera. Multiple alignments of Cry1Jb and Cry1Ac amino acid sequences showed significant differences in the composition and length of the loops of domains II and III. This was further confirmed by the superpositioning of 3D structures. cry1Jb and cry1Ac genes cloned in expression vectors were overexpressed in Escherichia coli Castellani & Chalmers, and proteins were harvested. Insect bioassays revealed that the wild-type Cry1Jb and Cry1Ac proteins showed differential specificity to H. armigera (Hübner), Spodoptera litura (Fab.) and Earias vittella F. Chimeric genes were constructed by exchanging the domains between cry1Jb and cry1Ac and overexpressed in BL21(DE3). Substitution of domain III of Cry1Jb with that of Cry1Ac enhanced its activity against H. armigera 7.8-fold. Bioassay of the parental and chimeric toxins against S. litura revealed no activity even at the discriminative dose of 100 μg/ml, but a significant difference in growth was observed. The present results, along with previous domain swapping experiments, suggest that protein engineering not only reveals the mechanism by which endotoxins work, but also generates novel toxins with enhanced toxicity/broader specificity.

Keywords

chimeric toxinsprotein engineeringdomain swappingCry1JbCry1AcLC50Helicoverpa armigeraSpodoptera lituraEarias vittella
Type
Research Paper
Information
International Journal of Tropical Insect Science , Volume 31 , Issue 1-2 , June 2011 , pp. 59 - 68
Copyright
Copyright © ICIPE 2011

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References

Avilla, C., Osunav, E., Cabrera, G. J., Ferre, J. and Zamoraj, E. (2005) Toxicity of several Bacillus thuringiensis delta-endotoxins against Helicoverpa armigera from Spain. Journal of Invertebrate Pathology 90, 51–54.CrossRefGoogle ScholarPubMed
Avisar, D., Keller, M., Gazit, E., Prudovsky, E., Sneh, B. and Zilberstein, A. (2004) The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells. Journal of Biological Chemistry 279, 15779–15786.CrossRefGoogle ScholarPubMed
Bohorova, N., Cabrera, M., Abarca, C., Quintero, R., Maciel, A. M., Brito, R. M., Hoisington, D. and Bravo, A. (1997) Susceptibility of four tropical lepidopteran maize pests to Bacillus thuringiensis CryI-type insecticidal toxins. Journal of Economic Entomology 90, 412–415.CrossRefGoogle Scholar
Bosch, D., Schipper, B., van der Kleij, H., de Maagd, R. A. and Stiekema, W. J. (1994) Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/Technology 12, 915–918.Google ScholarPubMed
Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle Scholar
Bravo, A., Gill, S. S. and Soberon, M. (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423–435.CrossRefGoogle ScholarPubMed
Bravo, A., Gomez, I., Conde, J., Munoz-Garay, C., Sanchez, J., Zhuang, M., Gill, S. S. and Soberon, M. (2004) Oligomerization triggers differential binding of a pore-forming toxin to a different receptor leading to efficient interaction with membrane microdomains. Biochimica et Biophysica Acta 1667, 38–46.CrossRefGoogle Scholar
Burton, S. L., Ellar, D. J., Li, J. and Derbyshire, D. J. (1999) N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognized by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin. Journal of Molecular Biology 287, 1101–1122.CrossRefGoogle ScholarPubMed
Caramori, T. A., Albertiai, A. M. and Galizzi, A. (1991) In-vivo generation of hybrids between two Bacillus thuringiensis insect toxin encoding genes. Gene 98, 37–44.CrossRefGoogle ScholarPubMed
Chakrabarti, S. K., Mandaokar, A., Kumar, P. A. and Sharma, R. P. (1998) Efficacy of lepidopteran specific δ-endotoxins of Bacillus thuringiensis against Helicoverpa armigera. Journal of Invertebrate Pathology 72, 336–337.CrossRefGoogle ScholarPubMed
Chen, X. J., Lee, M. K. and Dean, D. H. (1993) Site-directed mutagenesis in a highly conserved region of Bacillus thuringiensis δ-endotoxin affects inhibition of short circuit current across Bombyx mori midguts. Proceedings of the National Academy of Sciences of the USA 90, 9041–9045.CrossRefGoogle Scholar
de Maagd, R. A., Bakker, P., Masson, L., Adang, M. J., Sangadala, S., Stiekema, W. and Bosch, D. (1999) Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N. Molecular Microbiology 31, 463–471.CrossRefGoogle ScholarPubMed
de Maagd, R. A., Bravo, A. and Crickmore, N. (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics 4, 193–199.CrossRefGoogle Scholar
de Maagd, R. A., Kwa, M. S. G., van der Klei, H., Yamamoto, T., Schipper, B., Vlak, J. M., Stiekema, W. J. and Bosch, D. (1996) Domain III substitution in Bacillus thuringiensis CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Applied and Environmental Microbiology 62, 1537–1543.CrossRefGoogle ScholarPubMed
de Maagd, R. A., Weemen-Hendriks, M., Stiekema, W. and Bosch, D. (2000) Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids. Applied and Environmental Microbiology 66, 1559–1563.CrossRefGoogle Scholar
Gatehouse, J. A. (2008) Biotechnological prospects for engineering insect-resistant plants. Plant Physiology 146, 881–887.CrossRefGoogle ScholarPubMed
Ge, A. Z., Rivers, D., Milne, R. and Dean, D. H. (1991) Functional domains of Bacillus thuringiensis insecticidal crystal proteins. Journal of Biological Chemistry 266, 17954–17958.CrossRefGoogle ScholarPubMed
Ge, A. Z., Shivarova, N. I. and Dean, D. H. (1990) Hyper expression of a Bacillus thuringiensis delta-endotoxin encoding gene in Escherichia coli: properties of the product. Gene 93, 49–54.CrossRefGoogle Scholar
Grochulski, P., Masson, L., Borisova, S., Pusztai-Carey, M., Schwartz, J. L., Brousseau, R. and Cygler, M. (1995) Bacillus thuringiensis Cry-IA(a) insecticidal toxin: crystal structure and channel formation. Journal of Molecular Biology 254, 447–464.CrossRefGoogle Scholar
Gupta, G. P., Birah, A. and Rani, S. (2004) Development of artificial diet for mass rearing of American bollworm, Helicoverpa armigera. Indian Journal of Agricultural Science 74, 548–551.Google Scholar
Herrero, S., Gonzalez-Cabrera, J., Ferre, J., Bakker, P. L. and de Maagd, R. A. (2004) Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrated the role of domain II and III in the specificity towards Spodoptera exigua larvae. Biochemical Journal 384, 507–513.CrossRefGoogle ScholarPubMed
Jenkins, J. L., Lee, M. K., Valaitis, A. P., Curtiss, A. and Dean, D. H. (2000) Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor. Journal of Biological Chemistry 275, 14423–14431.CrossRefGoogle ScholarPubMed
Jurat-Fuentes, J. L. and Adang, M. J. (2001) Importance of cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.). Applied and Environmental Microbiology 67, 323–329.CrossRefGoogle ScholarPubMed
Karlova, R., Weemen-Hendriks, M., Naimov, S., Ceron, J., Dukiandjiev, S. and de Maagd, R. A. (2005) Bacillus thuringiensis δ-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids. Journal of Invertebrate Pathology 88, 169–172.CrossRefGoogle Scholar
King, A. B. S. (1994) Heliothis/Helicoverpa (Lepidoptera: Noctuidae), pp. 39–106. In Insect Pests of Cotton (edited by Mathews, G. A. and Turnstall, J. P.). CAB International, Wallingford.Google Scholar
Lee, M. K., You, T. H., Gould, F. L. and Dean, D. H. (1999) Identification of residues in domain III of Bacillus thuringiensis Cry1Ac toxin that affect binding and toxicity. Applied and Environmental Microbiology 65, 4513–4520.CrossRefGoogle ScholarPubMed
Luo, K., McLachlin, J. R., Brown, M. R. and Adang, M. J. (1999) Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells. Protein Expression and Purification 17, 113–122.CrossRefGoogle ScholarPubMed
Naimov, S., Hendriks, M. W., Dukiandjiev, S. and De Maagd, R. A. (2001) Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Applied and Environmental Microbiology 67, 5328–5330.CrossRefGoogle ScholarPubMed
Pardo, C. A., Cabreral, R., Parla, Y. F. and Rodriguez, P. T. (2006) Increased activity of a hybrid Bt toxin against Spodoptera frugiperda larvae from a maize field in Cuba. Biotecnologia Aplicada 23, 236–239.Google Scholar
Rang, C., Vachon, V., Coux, F., Carret, C., Moar, W. J., Brousseau, R., Schwartz, J. L., Laprade, R. and Frutos, R. (2001) Exchange of domain I from Bacilllus thuringiensis Cry1 toxins influences protoxin stability and crystal formation. Current Microbiology 43, 1–6.CrossRefGoogle Scholar
Rang, C., Vachon, V., de Maagd, R. A., Villalon, M., Schwartz, J. L., Bosch, D., Frutos, R. and Laprade, R. (1999) Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins. Applied and Environmental Microbiology 65, 2918–2925.CrossRefGoogle ScholarPubMed
Saraswathy, N. and Kumar, P. A. (2004) Protein engineering of δ-endotoxins of Bacillus thuringiensis. Electronic Journal of Biotechnology. doi:10.2225/vol7-issue2-fulltext-3.Google Scholar
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R. and Dean, D. H. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews 62, 775–806.CrossRefGoogle ScholarPubMed
Schnepf, H. E., Tomczak, J. P., Ortega, J. P. and Whiteley, H. R. (1990) Specificity determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis. Journal of Biological Chemistry 265, 20923–20930.CrossRefGoogle ScholarPubMed
Schwartz, J. L., Potvin, L., Chen, X. J., Brousseau, R., Laprade, R. and Dean, D. H. (1997) Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin. Applied and Environmental Microbiology 63, 3978–3984.CrossRefGoogle ScholarPubMed
Shanower, T. G. and Romeis, J. (1999) Insect pests of pigeon pea and their management. Annual Review of Entomology 44, 77–96.CrossRefGoogle Scholar
Sharma, H. C. (2005) Heliothis/Helicoverpa Management: The Emerging Trends and Strategies for Future Research. Oxford and IBH Publishing Co. Pvt. Ltd, New Delhi. 482 pp.CrossRefGoogle Scholar
Van Rie, J., Jansens, S., Höfte, H., Degheele, D. and Van Mellaert, H. (1989) Specificity of Bacillus thuringiensis delta-endotoxins of specific receptors on brush border membrane of the midgut of target insects. European Journal of Biochemistry 189, 239–247.CrossRefGoogle Scholar
Van Rie, J., Jansens, S., Höfte, H., Degheele, D. and Van Mellaert, H. (1990) Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxin. Applied and Environmental Microbiology 56, 1378–1385.CrossRefGoogle Scholar
Wolfersberger, M. G., Chen, X. J. and Dean, D. H. (1996) Site-directed mutations in the third domain of Bacillus thuringiensis δ-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles. Applied and Environmental Microbiology 62, 279–282.CrossRefGoogle ScholarPubMed