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Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay
L Masson1, A Mazza, L Gringorten
1Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Abstract:
The Bacillus thuringiensis crylA(a) and crylA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CrylA(c) is highly active against cultured Choristoneura fumiferana cells (CF-1) whereas CrylA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis revealed that the CrylA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258-358 and 450-510) were able to confer toxicity, although at lower levels, and one region (358-450) was present where progressive substitutions of crylA(a) with crylA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CrylA(c) with CrylE suggested that the N-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CrylA(b) but shows an 800-fold increase in toxicity to CF-1 cells relative to that shown by CrylA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450-620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.
Insights
Bacillus thuringiensis (Bt) toxin specificity was investigated using hybrid toxins. The CrylA(c) gene region between codons 258-510 determines toxicity, but in vitro and in vivo assays show differing results.
Area of Science:
- Molecular biology
- Insect toxicology
- Biochemistry
Background:
- Bacillus thuringiensis (Bt) produces insecticidal crystal proteins (Cry toxins).
- Understanding Cry toxin specificity is crucial for developing targeted pest control strategies.
- Previous studies have identified broad regions involved in Bt toxin activity.
Purpose of the Study:
- To precisely map the toxicity-determining regions of Bacillus thuringiensis crylA(a) and crylA(c) genes.
- To investigate the role of different toxin regions in insecticidal activity against specific pests.
- To compare in vivo and in vitro assay results for Bt toxin specificity.
Main Methods:
- Creation and in vitro/in vivo testing of hybrid Bacillus thuringiensis crylA(a) and crylA(c) toxins.
- Toxin threshold dose determinations against cultured insect cells (Choristoneura fumiferana) and dissociated midgut epithelial cells.
- Live insect bioassays using Bombyx mori larvae.
- Analysis of hybrid clone MP80 for homology and toxicity.
- Comparative analysis of in vivo and in vitro assay data.
Main Results:
- The CrylA(c) toxicity-determining region is located between codons 258 and 510, with subsections 258-358 and 450-510 conferring toxicity.
- A reversed order of toxicity was observed between in vivo and in vitro assays.
- Hybrid clone MP80 showed significantly increased toxicity to Choristoneura fumiferana cells.
- The N-terminal region does not appear to play a role in specificity.
- Toxicity-determining regions varied between insect species, with codons 283-450 critical for Bombyx mori.
Conclusions:
- Toxin specificity is determined by specific regions within the Bacillus thuringiensis crylA genes, primarily between codons 258-510 for CrylA(c).
- Significant discrepancies exist between in vivo and in vitro insecticidal activity, indicating other factors influence toxicity.
- The study provides detailed insights into Bt toxin structure-activity relationships and highlights the complexity of insecticidal specificity.