Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domain II and III in the specificity towards Spodoptera exigua larvae

S. Herrero Sendra, J. González-Cabrera, J. Ferré, P.L. Bakker, R.A. de Maagd

Research output: Contribution to journalArticleAcademicpeer-review

62 Citations (Scopus)

Abstract

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins
Original languageEnglish
Pages (from-to)507-513
JournalBiochemical Journal
Volume384
Issue number3
DOIs
Publication statusPublished - 2004

Keywords

  • brush-border membrane
  • cryia delta-endotoxins
  • manduca-sexta
  • crystal proteins
  • aminopeptidase n
  • irreversible binding
  • heliothis-virescens
  • insecticidal toxin
  • pore formation
  • pink-bollworm

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