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Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines
Jizhen Wei1,2, Gemei Liang1, Kongming Wu1
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
Abstract:
While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC50 ) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC50 = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.
Insights
Cry2Ab insecticidal protein receptors differ from Cry1Ac. Cry2Ab binds fat body cells, while Cry1Ac targets midgut proteins, impacting pest control strategies.
Area of Science:
- Molecular biology
- Insect toxicology
- Biochemistry
Background:
- Bacillus thuringiensis (Bt) Cry proteins are widely used biopesticides.
- Cry1Ac targets larval midgut proteins like cadherin and amino peptidase N (APN).
- Receptors for Cry2Ab remain largely uncharacterized.
Purpose of the Study:
- To investigate the cellular targets and binding differences of Cry1Ac and Cry2Ab.
- To identify potential receptors for Cry2Ab in insect cell lines.
Main Methods:
- Cytotoxicity assays using activated Cry1Ac and Cry2Ab against Helicoverpa zea midgut and fat body cell lines, and Spodoptera frugiperda (SF9) ovary cell line.
- Determination of 50% lethal concentration (LC50) values.
- Ligand blot analysis to assess protein binding.
Main Results:
- Cry1Ac exhibited highest cytotoxicity against midgut cells (LC50 = 31.0 μg/mL), followed by fat body (59.0 μg/mL) and SF9 cells (99.6 μg/mL).
- Cry2Ab showed higher cytotoxicity against fat body cells (LC50 = 7.55 μg/mL) compared to midgut cells (16.0 μg/mL) and SF9 cells (27.0 μg/mL).
- Ligand blot analysis revealed distinct binding patterns for Cry1Ac and Cry2Ab across the cell lines.
Conclusions:
- Cry2Ab receptors are primarily enriched in fat body cells, differing significantly from Cry1Ac receptors found in midgut cells.
- These findings suggest distinct molecular interactions for Cry1Ac and Cry2Ab, crucial for understanding their insecticidal mechanisms and developing resistant management strategies.
