Canadian Forest Service Publications
Toxicity and mode of action of insecticidal Cry 1 A proteins from Bacillus thuringiensis in an insect cell line, CF-1. 2014. Portugal, L. Gringorten, J.L., Caputo, G.F., Soberón, M., Muñoz-Garay, C., Bravo, A. 2014. Peptides 53: 292-299
Issued by: Great Lakes Forestry Centre
Catalog ID: 35829
CFS Availability: PDF (request by e-mail)
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Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1.
Plain Language Summary
Bacillus thuringiensis (Bt) has been used to control insect pest for more than 50 years. Many subspecies produce various types of crystal (Cry) proteins that are insecticidal when ingested by susceptible hosts. Bt Cry proteins are also toxic to specific cell lines in vitro. We compared the activity of native and genetically modified variants of Cry1A proteins against a susceptible cell line, CF-1, with the activity against larvae of the tobacco hornworm and spruce budworm and showed that the mechanism of toxicity was the same, demonstrating that toxin mode of action is conserved in vitro and in vivo. This particular cell line, therefore, can serve as a model system for investigating insecticidal activity, particularly in determining the molecular changes underlying the development of insect resistance to Bt.