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. 1996 Oct;62(10):3581–3586. doi: 10.1128/aem.62.10.3581-3586.1996

Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

A Regev 1, M Keller 1, N Strizhov 1, B Sneh 1, E Prudovsky 1, I Chet 1, I Ginzberg 1, Z Koncz-Kalman 1, C Koncz 1, J Schell 1, A Zilberstein 1
PMCID: PMC168163  PMID: 8837413

Abstract

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.

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Selected References

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  1. Arantes O., Lereclus D. Construction of cloning vectors for Bacillus thuringiensis. Gene. 1991 Dec 1;108(1):115–119. doi: 10.1016/0378-1119(91)90495-w. [DOI] [PubMed] [Google Scholar]
  2. Aronson A. I. The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol Microbiol. 1993 Feb;7(4):489–496. doi: 10.1111/j.1365-2958.1993.tb01139.x. [DOI] [PubMed] [Google Scholar]
  3. Aronson A. I., Wu D., Zhang C. Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene. J Bacteriol. 1995 Jul;177(14):4059–4065. doi: 10.1128/jb.177.14.4059-4065.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chak K. F., Tseng M. Y., Yamamoto T. Expression of the crystal protein gene under the control of the alpha-amylase promoter in Bacillus thuringiensis strains. Appl Environ Microbiol. 1994 Jul;60(7):2304–2310. doi: 10.1128/aem.60.7.2304-2310.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen X. J., Curtiss A., Alcantara E., Dean D. H. Mutations in domain I of Bacillus thuringiensis delta-endotoxin CryIAb reduce the irreversible binding of toxin to manduca sexta brush border membrane vesicles. J Biol Chem. 1995 Mar 17;270(11):6412–6419. doi: 10.1074/jbc.270.11.6412. [DOI] [PubMed] [Google Scholar]
  6. Derksen A. C., Granados R. R. Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity. Virology. 1988 Nov;167(1):242–250. doi: 10.1016/0042-6822(88)90074-8. [DOI] [PubMed] [Google Scholar]
  7. Gill S. S., Cowles E. A., Pietrantonio P. V. The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol. 1992;37:615–636. doi: 10.1146/annurev.en.37.010192.003151. [DOI] [PubMed] [Google Scholar]
  8. Grochulski P., Masson L., Borisova S., Pusztai-Carey M., Schwartz J. L., Brousseau R., Cygler M. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation. J Mol Biol. 1995 Dec 1;254(3):447–464. doi: 10.1006/jmbi.1995.0630. [DOI] [PubMed] [Google Scholar]
  9. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., van Montagu M., Zabeau M. Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem. 1986 Dec 1;161(2):273–280. doi: 10.1111/j.1432-1033.1986.tb10443.x. [DOI] [PubMed] [Google Scholar]
  11. Keller M., Sneh B., Strizhov N., Prudovsky E., Regev A., Koncz C., Schell J., Zilberstein A. Digestion of delta-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC. Insect Biochem Mol Biol. 1996 Apr;26(4):365–373. doi: 10.1016/0965-1748(95)00102-6. [DOI] [PubMed] [Google Scholar]
  12. Li J. D., Carroll J., Ellar D. J. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature. 1991 Oct 31;353(6347):815–821. doi: 10.1038/353815a0. [DOI] [PubMed] [Google Scholar]
  13. Lorence A., Darszon A., Díaz C., Liévano A., Quintero R., Bravo A. Delta-endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers. FEBS Lett. 1995 Mar 6;360(3):217–222. doi: 10.1016/0014-5793(95)00092-n. [DOI] [PubMed] [Google Scholar]
  14. Martens J. W., Visser B., Vlak J. M., Bosch D. Mapping and characterization of the entomocidal domain of the Bacillus thuringiensis CryIA(b) protoxin. Mol Gen Genet. 1995 May 20;247(4):482–487. doi: 10.1007/BF00293150. [DOI] [PubMed] [Google Scholar]
  15. McBride K. E., Svab Z., Schaaf D. J., Hogan P. S., Stalker D. M., Maliga P. Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology (N Y) 1995 Apr;13(4):362–365. doi: 10.1038/nbt0495-362. [DOI] [PubMed] [Google Scholar]
  16. Murray E. E., Rocheleau T., Eberle M., Stock C., Sekar V., Adang M. Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts. Plant Mol Biol. 1991 Jun;16(6):1035–1050. doi: 10.1007/BF00016075. [DOI] [PubMed] [Google Scholar]
  17. Obukowicz M. G., Perlak F. J., Kusano-Kretzmer K., Mayer E. J., Watrud L. S. Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5. Gene. 1986;45(3):327–331. doi: 10.1016/0378-1119(86)90031-4. [DOI] [PubMed] [Google Scholar]
  18. Perlak F. J., Fuchs R. L., Dean D. A., McPherson S. L., Fischhoff D. A. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3324–3328. doi: 10.1073/pnas.88.8.3324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanchis V., Lereclus D., Menou G., Chaufaux J., Guo S., Lecadet M. M. Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-active delta-endotoxin gene of Bacillus thuringiensis aizawai 7.29. Mol Microbiol. 1989 Feb;3(2):229–238. doi: 10.1111/j.1365-2958.1989.tb01812.x. [DOI] [PubMed] [Google Scholar]
  20. Sanchis V., Lereclus D., Menou G., Chaufaux J., Lecadet M. M. Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29. Mol Microbiol. 1988 May;2(3):393–404. doi: 10.1111/j.1365-2958.1988.tb00044.x. [DOI] [PubMed] [Google Scholar]
  21. Sangadala S., Walters F. S., English L. H., Adang M. J. A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro. J Biol Chem. 1994 Apr 1;269(13):10088–10092. [PubMed] [Google Scholar]
  22. Smith G. P., Ellar D. J. Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC delta-endotoxin affects insecticidal specificity. Biochem J. 1994 Sep 1;302(Pt 2):611–616. doi: 10.1042/bj3020611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith G. P., Merrick J. D., Bone E. J., Ellar D. J. Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai. Appl Environ Microbiol. 1996 Feb;62(2):680–684. doi: 10.1128/aem.62.2.680-684.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stahly D. P., Dingman D. W., Bulla L. A., Jr, Aronson A. I. Possible origin and function of the parasporal crystal in Bacillus thuringiensis. Biochem Biophys Res Commun. 1978 Oct 16;84(3):581–588. doi: 10.1016/0006-291x(78)90745-3. [DOI] [PubMed] [Google Scholar]
  25. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  26. Tabashnik B. E., Finson N., Groeters F. R., Moar W. J., Johnson M. W., Luo K., Adang M. J. Reversal of resistance to Bacillus thuringiensis in Plutella xylostella. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4120–4124. doi: 10.1073/pnas.91.10.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tabashnik B. E., Finson N., Johnson M. W., Moar W. J. Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki Causes Minimal Cross-Resistance to B. thuringiensis subsp. aizawai in the Diamondback Moth (Lepidoptera: Plutellidae). Appl Environ Microbiol. 1993 May;59(5):1332–1335. doi: 10.1128/aem.59.5.1332-1335.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van Rie J., Jansens S., Höfte H., Degheele D., Van Mellaert H. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl Environ Microbiol. 1990 May;56(5):1378–1385. doi: 10.1128/aem.56.5.1378-1385.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Visser B., Munsterman E., Stoker A., Dirkse W. G. A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein. J Bacteriol. 1990 Dec;172(12):6783–6788. doi: 10.1128/jb.172.12.6783-6788.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Widner W. R., Whiteley H. R. Location of the dipteran specificity region in a lepidopteran-dipteran crystal protein from Bacillus thuringiensis. J Bacteriol. 1990 Jun;172(6):2826–2832. doi: 10.1128/jb.172.6.2826-2832.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wu D., Aronson A. I. Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity. J Biol Chem. 1992 Feb 5;267(4):2311–2317. [PubMed] [Google Scholar]
  32. Wu S. J., Dean D. H. Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA delta-endotoxin. J Mol Biol. 1996 Feb 2;255(4):628–640. doi: 10.1006/jmbi.1996.0052. [DOI] [PubMed] [Google Scholar]
  33. Wünn J., Klöti A., Burkhardt P. K., Biswas G. C., Launis K., Iglesias V. A., Potrykus I. Transgenic Indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control. Biotechnology (N Y) 1996 Feb;14(2):171–176. doi: 10.1038/nbt0296-171. [DOI] [PubMed] [Google Scholar]

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