Recombinant Bacillus thuringiensis Crystal Proteins with New Properties: Possibilities for Resistance Management

Bosch, Dirk; Schipper, Bert; Kleij, H van der; Maagd, Ruud A. de; Stiekema, Willem J.

doi:10.1038/nbt0994-915

Cited by 101 publications

(96 citation statements)

References 15 publications

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“…Alanine substitution of residues 541-544 of domain III of Cry1Ca in the hybrid G27 toxin abolished the activity of this hybrid against S. exigua, but not against M. sexta [16]. These results suggested an important role for domain III, as well as for domain II, in the mode of action of Cry1Ca, including insect specificity.…”

Section: Introductionmentioning

confidence: 62%

“…Wild-type Cry1Ca and mutant protoxins were expressed in Escherichia coli strain XL-1 Blue, extracted, solubilized and trypsin treated as described previously [16]. Briefly, each 1 g of cell culture expressing the different toxins was resuspended in 3 ml of lysis buffer (50 mM Tris/HCl, pH 8.0, 5 mM EDTA, 100 mM NaCl) by vigorous shaking, and then 800 µg of lysozyme was added per g of pellet.…”

Section: Mutagenesis and Protein Isolationmentioning

confidence: 99%

“…Cry1Ca mutants were constructed by introduction of the mutations Gln 374 Ala and Thr 440 Ala (domain II mutant; pPB19), Ser 541 Ala, Thr 542 Ala, Gly 543 Ala and Val 544 Ala (domain III mutant; pPB20), or all mutations combined (mutant pPB21), in the cry1Ca gene in expression plasmid pBD150 [16], using the QuickChange mutagenesis kit (Stratagene, Amsterdam, The Netherlands).…”

Section: Mutagenesis and Protein Isolationmentioning

confidence: 99%

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

Herrero

González‐Cabrera

Ferré

et al. 2004

Biochemical Journal

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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.

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Section: Introductionmentioning

confidence: 62%

Section: Mutagenesis and Protein Isolationmentioning

confidence: 99%

Section: Mutagenesis and Protein Isolationmentioning

confidence: 99%

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

Herrero

González‐Cabrera

Ferré

et al. 2004

Biochemical Journal

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“…One strategy to delay resistance is to use alternative insecticidal proteins with different modes of action (Bosch et al 1994;Saraswathy and Kumar 2004). Researchers found that the resistance allele of P. xylostella confers cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa and Cry1Ja, but not to Cry1Ba, Cry1Bb, Cry1Ca, Cry1Da, Cry1Ia or Cry2Aa (Tabashnik et al 1997(Tabashnik et al , 2000Heckel et al 1999).…”

Section: Discussionmentioning

confidence: 99%

RecombinantBacillus thuringiensisstrain shows high insecticidal activity againstPlutella xylostellaandLeptinotarsa decemlineatawithout affecting nontarget species in the field

Wang

Zhang

Song

et al. 2008

Journal of Applied Microbiology

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Aims: To construct a recombinant Bacillus thuringiensis (Bt) strain with broad insecticidal spectrum and investigate its impact on nontarget organisms in field. Method and Results: The cry‐type gene of wild Bt strain UV17 was identified and a novel cry1Ba gene was cloned. The cry3Aa7 gene, which was highly toxic to coleopteran pests, was introduced into UV17, and a recombinant strain designated as UV173A was obtained. Bioassay results showed that UV173A was not only highly toxic against Plutella xylostella (50% lethal concentration [LC50] = 18·03 μg ml–1), but also against coleopteran Leptinotarsa decernlineata (LC50 = 0·19 mg ml–1). The recombinant strain was then tested in field trials to monitor its spatial variation of population and to investigate the impact on nontarget invertebrates. Conclusions: A recombinant Bt stain UV173A with broad insecticidal spectrum was obtained, and it did not cause adverse effects on the population of nontarget organisms. Significance and Impact of the Study: The results obtained here indicated that cry1Ba3 gene may be useful for the resistance management of P. xylostella, and the recombinant stain UV173A was potential for field application against some crucifer vegetable pests as well as L. decemlineata.

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“…Cry proteins share a common three domain structure (Li et al 1991). In simple words, hybrid (Synthetic) toxins produced through inclusion of a domain from another toxin results in increased potency of the fused protein by the shift in receptor binding (Bosch et al 1994). In this direction, a chimeric Bt gene cry1AcF with cry1Ac (domain I & II) and cry1F (domain III) was developed to improve the insecticidal property of the toxin (Chakrabarti et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.)

Keshavareddy

Rohini

Ramu

et al. 2013

Physiol Mol Biol Plants

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Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T 2 generation, the cry1AcF gene was effective against S. litura only to some extent.

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Recombinant Bacillus thuringiensis Crystal Proteins with New Properties: Possibilities for Resistance Management

Cited by 101 publications

References 15 publications

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

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

RecombinantBacillus thuringiensisstrain shows high insecticidal activity againstPlutella xylostellaandLeptinotarsa decemlineatawithout affecting nontarget species in the field

Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.)

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