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Toxicity and binding properties of the Bacillus thuringiensis delta-endotoxin Cry1C to cultured insect cells
M S Kwa1, R A de Maagd, W J Stiekema
1Department of Molecular Biology, Center for Plant Breeding and Reproduction Research-DLO (CPRO-DLO), Wageningen, 6700 AA, the Netherlands.
Abstract:
A better understanding of the mode of action of Bacillus thuringiensis delta-endotoxins is needed to develop strategies which may prevent or slow down selection for resistance. We studied the effect of Cry1C on several different cultured insect cell lines by means of toxicity assays, ligand blotting, and toxin binding studies. A clear difference in sensitivity toward Cry1C between the insect cell lines was observed. Spodoptera frugiperda cell line Sf9 was most sensitive, whereas Spodoptera exigua cell lines SeUCR and SelZD2109 showed intermediate sensitivity. Mamestra brassicae (Mb0503) and Drosophila melanogaster (Dm1) cells were the least sensitive as compared to Sf9 cells. Ligand blot analysis of SDS-PAGE size-separated proteins showed that Cry1C specifically binds to a 40-kDa protein in Sf9, SeUCR, and SelZD2109 cells. Cry1Ab does not bind to this protein. The Cry1C-binding protein was not observed in Mb0503 and Dm1 cells, suggesting that the presence of the 40-kDa Cry1C-binding protein is correlated with sensitivity toward Cry1C.
Insights
Bacillus thuringiensis Cry1C endotoxin sensitivity varies among insect cell lines. A 40-kDa binding protein correlates with Cry1C toxicity, crucial for managing insect resistance.
Area of Science:
- Insect molecular biology
- Biochemistry of microbial toxins
- Pest resistance mechanisms
Background:
- Bacillus thuringiensis (Bt) delta-endotoxins are vital biopesticides.
- Understanding Bt toxin action is key to overcoming insect resistance.
- Cry1C toxin's specific interactions require further elucidation.
Purpose of the Study:
- To investigate the mode of action of Bacillus thuringiensis Cry1C toxin.
- To identify insect cell factors influencing Cry1C sensitivity.
- To correlate toxin binding with cellular toxicity.
Main Methods:
- Utilized toxicity assays on various cultured insect cell lines.
- Performed ligand blotting to identify toxin-binding proteins.
- Conducted toxin binding studies to confirm interactions.
Main Results:
- Observed differential sensitivity to Cry1C across insect cell lines (Sf9 > SeUCR/SelZD2109 > Mb0503/Dm1).
- Identified a specific 40-kDa Cry1C-binding protein in sensitive cell lines (Sf9, SeUCR, SelZD2109).
- This 40-kDa protein was absent in less sensitive cell lines (Mb0503, Dm1).
Conclusions:
- The presence of a 40-kDa Cry1C-binding protein is strongly correlated with Cry1C sensitivity in insect cells.
- This finding provides insights into the molecular basis of Cry1C action and potential resistance mechanisms.
- Further research can leverage this understanding to develop strategies against Bt resistance.