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A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells

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Abstract

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL50 of 19.49 ng/ml for 2nd instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

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Literature Cited

  1. Bietlot HPL, Vishnubhatla I, Carey PR, Pozsgay M, Kaplan H (1990) Characterization of the cysteine residues and disulfide link ages in the protein crystal of Bacillus thuringiensis. J Biochem 267:309–315

    CAS  Google Scholar 

  2. Bobrowski VL, Pasquali G, Zanettini MHB, Fiuza LM (2001) Detection of cry1 genes in Bacillus thuringiensis isolates from south of Brazil and activity against Anticarsia gemmatalis (Lepidoptera: Noctuidae) in Brazil. J Microbiol 32:105–109

    CAS  Google Scholar 

  3. Bohorova N, Maciel AM, Brito RM, Aguilart L, Ibarra JE, Hoisington D (1996) Selection and characterization of Mexican strains of Bacillus thuringiensis active against four major lepidopteran maize pests. Entomophaga 41:153–165

    Google Scholar 

  4. Chang JH, Choi JY, Jin BR, Roh JY, Olszewski JA, Seo SJ, O’Reilly DR, Je YH (2003) Improved baculovírus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin. J Invertebr Pathol 84:30–37

    Article  PubMed  CAS  Google Scholar 

  5. Christov NK, Imaishi H, Ohkawa H (1999) Green-tissue-specific expression of a reconstructed cry1C gene encoding the active fragment of Bacillus thuringiensis delta-endotoxin in haploid tobacco plants conferring resistance to Spodoptera litura. Biosci Biotechnol Biochem 63:1433–1444

    Article  PubMed  CAS  Google Scholar 

  6. Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature of the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813

    PubMed  CAS  Google Scholar 

  7. de Maagd RA, bravo A, Berry C, Crickmore N, Schnepf E (2003) Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Ann Rev Genet 37:409–433

    Article  PubMed  CAS  Google Scholar 

  8. Ferre J, van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Ann Rev Entomol 47:501–533

    Article  CAS  Google Scholar 

  9. Finney DJ (1971) Probit analysis. Cambridge, UK: Cambridge University Press

    Google Scholar 

  10. Ge B, Bideshi D, Moar JW, Federici BA (1998) Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2A inclusions in Bacillus thuringiensis. FEMS Microbiol Lett 165:35–41

    Article  PubMed  CAS  Google Scholar 

  11. Granados RR, Guoxun L, Derksen CG, Mckenna KAA (1994) An insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J Invertebr Pathol 64:260–266

    Article  Google Scholar 

  12. Hofmann C, Vanderbruggen H, Hofte H, Van Rie J, Jansens S, Van Mellaert H (1988) Specificity of Bacillus thuringiensis σ-endotoxin is correlated with the presence of high affinity binding sites in the border membrane of target insect midgets. Proc Natl Acad Sci USA 85:7844–7848

    Article  PubMed  CAS  Google Scholar 

  13. Honée G, Van de Salm T, Visser B, Nucleotide sequence of crystal protein gene isolated from B. thuringiensis subspecies entomocidus 60.5 coding of toxin highly active Spodoptera specie. Nucleic Acids Res 16:6240

  14. Jarvis DL (1997) Baculovirus expression vectors. In: Miller LK (ed) The baculovírus. New York: Plenum Press. pp 389–341

    Google Scholar 

  15. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriphage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  16. Lin CH, Chen YY, Tzeng CC, Tsay HS, Chen LJ (2003) Expression of a Bacillus thuringiensis cry1Ca gene in plastid confers high insecticidal efficacy against tobacco cutworm - a Spodoptera insect. Bot Bull Acad Sin 44:199–210

    CAS  Google Scholar 

  17. Martens JW, Honee G, Zuidema D, Van Lent JWM, Visser B, Vlak JM (1990) Insecticidal activity of a bacterial crystal protein expressed by a recombinant baculovirus in insect cells. Appl Environ Microbiol 56:2764–2770

    PubMed  CAS  Google Scholar 

  18. O’Reilly DR, Miller LK, Luckow VA (1992) Baculovirus expression vectors: A Laboratory Manual. New York: Freeman

    Google Scholar 

  19. Pang Y, Frutos R, Federici BA (1992) Synthesis and toxicity of full length and truncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells a baculovírus vector. J Gen Virol 73:89–101

    Article  PubMed  CAS  Google Scholar 

  20. Ribeiro BM, Crook NE (1998) Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis. Braz J Med Biol Res 31:763–769

    Article  PubMed  CAS  Google Scholar 

  21. Ribeiro BM, Crook NE (1993) Expression of full length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp. Kurstaki in baculovírus and pathogenicity of the recombinant viruses. J Invertebr Pathol 62:121–130

    Article  PubMed  CAS  Google Scholar 

  22. Xue JL, Cai QX, Zheng DS, Yuan ZM (2005) The synergistic between Cry1Aa and Cry1C from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera. Lett Appl Microbiol 40:460–465

    Article  PubMed  CAS  Google Scholar 

  23. Zhao JZ, Cao J, Li Y, Collins HL, Roush RT, Earle ED, Shelton AM (2003) Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnol 21:1493–1497

    Article  CAS  Google Scholar 

  24. Zhao JZ, Cao J, Collins HL, Bates SL, Roush RT, Earle ED, Shelton AM (2005) Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants. Proc Natl Acad Sci USA 102:8426–8430

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Embrapa Recursos Genéticos e Biotecnologia for supplying A. gemmatalis and S. frugiperda larvae. This work was supported by the following Brazilian agencies: FINATEC, CAPES, CNPq, FAPDF, and PRONEX.

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Authors and Affiliations

  1. Cell Biology Department, University of Brasília, 70910-900, Brasília, DF, Brazil

    Raimundo W. S. Aguiar, Érica S. Martins & Bergmann M. Ribeiro

  2. Embrapa-Recursos Genéticos e Biotecnologia, C.P. 02372, 70770-900, Brasília, DF, Brazil

    Érica S. Martins, Andréia C. Batista, Viviane M. Melatti & Rose G. Monnerat

  3. Embrapa-Milho e Sorgo, 35701-970, Sete Lagoas, MG, Brazil

    Fernando H. Valicente & Newton P. Carneiro

Authors
  1. Raimundo W. S. Aguiar
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  2. Érica S. Martins
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  3. Fernando H. Valicente
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  4. Newton P. Carneiro
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  5. Andréia C. Batista
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  6. Viviane M. Melatti
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  7. Rose G. Monnerat
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  8. Bergmann M. Ribeiro
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Correspondence to Bergmann M. Ribeiro.

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Aguiar, R.W., Martins, É., Valicente, F.H. et al. A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells. Curr Microbiol 53, 287–292 (2006). https://doi.org/10.1007/s00284-005-0502-3

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  • DOI: https://doi.org/10.1007/s00284-005-0502-3

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