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Fusing the vegetative insecticidal protein Vip3Aa7 and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvae

Abstract

Bacillus thuringiensis insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) have been widely used as a kind of safe bio-insecticides. A problem that has been of concern worldwide is how to improve their insecticidal activities. In this study, to determine the synergism between VIPs and ICPs effect on insecticidal activity, a construct that produces a chimeric protein of the Vip3Aa7 and the N terminus ofCry9Ca, named V3AC9C, was expressed in Escherichia coli BL21 cells. In additional experiments, the V3AC9C chimeric protein, the single Vip3Aa7, and the single N terminus of Cry9Ca were treated with trypsin. SDS–PAGE showed that the V3AC9C could be processed into two single toxins. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the chimeric protein was markedly better than either of the single toxins. Interestingly, the toxicity of the chimeric protein was 3.2-fold higher than a mixture of the Vip3Aa7 and Cry9Ca toxins (mass ratio of 1:1). The synergism factor (SF) of chimeric protein containing Vip3Aa7 and Cry9Ca was calculated to be 4.79. The SF in mixture of toxins is only 1.46. Hence, the effect was more than the sum of the Vip3Aa7 and Cry9C activities. Analysis of the protein’s solubility showed that the Vip3Aa7 helped the N terminus of Cry9Ca to dissolve in an alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly resulted from this increase in solubility. These results lay a foundation for the development of a new generation of bio-insecticides and multi-gene transgenic plants.

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References

  1. Boonserm P, Davis P, Ellar DJ, Li J (2005) Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J Mol Biol 348:363–382

    Article  CAS  Google Scholar 

  2. Boonserm P, Mo M, Angsuthanasombat C, Lescar J (2006) Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution. J Bacteriol 188:3391

    Article  CAS  Google Scholar 

  3. Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, Zhuang M, Gill SS, Soberon M (2004) Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta 1667:38–46

    Article  CAS  Google Scholar 

  4. Bravo A, Gill SS, Soberon M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49:423–435

    Article  CAS  Google Scholar 

  5. Cai Q, Liu Z, Sun M, Wei F, Yu Z (2002) The analysis of Bacillus thuringiensis vegetative insecticidal protein gene cloning and expression. Sheng Wu Gong Cheng Xue Bao—Chin J Biotechnol 18:578

    CAS  Google Scholar 

  6. Chen J, Yu J, Tang L, Tang M, Shi Y, Pang Y (2003) Comparison of the expression of Bacillus thuringiensis full–length and N–terminally truncated vip3A gene in Escherichia coli. J Appl Microbiol 95:310–316

    Article  CAS  Google Scholar 

  7. Crickmore N, Zeigler D, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean D (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807

    CAS  Google Scholar 

  8. de Barjac H, Burgerjon A, Bonnefoi A (1966) The production of heat-stable toxin by nine serotypes of Bacillus thuringiensis. J Invertebr Pathol 8:537

    Article  Google Scholar 

  9. Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci U S A 93:5389–5394

    Article  CAS  Google Scholar 

  10. Estruch JJ, Yu CG, Warren GW, Desai NM, Koziel MG, Nye GJ (2000) Class of proteins for the control of plant pests (World Intellectual Property Organization Patent WO)

  11. Fahnert B, Lilie H, Neubauer P (2004) Inclusion bodies: formation and utilisation. Physiological Stress Responses in Bioprocesses 93-142

  12. Fang J, Xu X, Wang P, Zhao JZ, Shelton AM, Cheng J, Feng MG, Shen Z (2007) Characterization of chimeric Bacillus thuringiensis Vip3 toxins. Appl Environ Microbiol 73:956–961

    Article  CAS  Google Scholar 

  13. Federici B, Park HW, Sakano Y (2006) Insecticidal protein crystals of Bacillus thuringiensis. Inclusions in Prokaryotes. Microbiol Monogr 1:195–236

    Article  Google Scholar 

  14. Gahan LJ, Gould F, Heckel DG (2001) Identification of a gene associated with Bt resistance in Heliothis virescens. Science 293:857–860

    Article  CAS  Google Scholar 

  15. Galitsky N, Cody V, Wojtczak A, Ghosh D, Luft JR, Pangborn W, English L (2001) Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis. Acta Crystallogr D Biol Crystallogr 57:1101–1109

    Article  CAS  Google Scholar 

  16. Hofte H, Whiteley H (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Mol Biol Rev 53:242

    CAS  Google Scholar 

  17. Jurat-Fuentes JL, Adang MJ (2006) Cry toxin mode of action in susceptible and resistant Heliothis virescens larvae. J Invertebr Pathol 92:166–171

    Article  CAS  Google Scholar 

  18. Lambert B, Buysse L, Decock C, Jansens S, Piens C, Saey B, Seurinck J, Van Audenhove K, Van Rie J, Van Vliet A (1996) A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae. Appl Environ Microbiol 62:80–86

    CAS  Google Scholar 

  19. Lee MK, Walters FS, Hart H, Palekar N, Chen JS (2003) The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab δ-endotoxin. Appl Environ Microbiol 69:4648–4657

    Article  CAS  Google Scholar 

  20. Lee MK, Miles P, Chen JS (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 339:1043–1047

    Article  CAS  Google Scholar 

  21. Li JD, Carroll J, Ellar DJ (1991) Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature 353:815–821

    Article  CAS  Google Scholar 

  22. Morin S, Biggs RW, Sisterson MS, Shriver L, Ellers-Kirk C, Higginson D, Holley D, Gahan LJ, Heckel DG, Carriere Y, Dennehy TJ, Brown JK, Tabashnik BE (2003) Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm. Proc Natl Acad Sci U S A 100:5004–5009

    Article  CAS  Google Scholar 

  23. Peng D, Xu X, Ruan L, Yu Z, Sun M (2010) Enhancing Cry1Ac toxicity by expression of the Helicoverpa armigera cadherin fragment in Bacillus thuringiensis. Res Microbiol 161:383–389

    Article  CAS  Google Scholar 

  24. Pigott CR, Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol Mol Biol Rev 71:255

    Article  CAS  Google Scholar 

  25. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    CAS  Google Scholar 

  26. Singh G, Sachdev B, Sharma N, Seth R, Bhatnagar RK (2010) Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda. Appl Environ Microbiol 76:7202

    Article  CAS  Google Scholar 

  27. Song R, Peng D, Yu Z, Sun M (2008) Carboxy-terminal half of Cry1C can help vegetative insecticidal protein to form inclusion bodies in the mother cell of Bacillus thuringiensis. Appl Microbiol Biotechnol 80:647–654

    Article  CAS  Google Scholar 

  28. Stewart S, Adamczyk J, Knighten K, Davis F (2001) Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid (Lepidoptera) larvae. J Econ Entomol 94:752–760

    Article  CAS  Google Scholar 

  29. Tabashnik BE (1992) Evaluation of synergism among Bacillus thuringiensis toxins. Appl Environ Microbiol 58:3343

    CAS  Google Scholar 

  30. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533

    CAS  Google Scholar 

  31. Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci 95:13959

    Article  CAS  Google Scholar 

  32. Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ (1997) The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. Appl Environ Microbiol 63:532–536

    CAS  Google Scholar 

  33. Zhang X, Candas M, Griko NB, Taussig R, Bulla LA (2006) A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proc Natl Acad Sci 103:9897

    Article  CAS  Google Scholar 

  34. Zhu C, Ruan L, Peng D, Yu Z, Sun M (2006) Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua. Lett Appl Microbiol 42:109–114

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (NO. u1170303), the Genetically Modified Organisms Breeding Major Projects of China (2009zx08005-009b), and the Genetically Modified Complex Transgenic Insect-resistant Rice Breeding Foundation of China (2011zx08001-001).

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Correspondence to Ziduo Liu.

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Dong, F., Shi, R., Zhang, S. et al. Fusing the vegetative insecticidal protein Vip3Aa7 and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvae. Appl Microbiol Biotechnol 96, 921–929 (2012). https://doi.org/10.1007/s00253-012-4213-y

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Keywords

  • Bacillus thuringiensis
  • Vegetative insecticidal proteins
  • Insecticidal crystal proteins
  • Fusing expression
  • Synergism factor