Abstract
Background Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins.Methodology/Principal FindingsThe DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors.Conclusions/SignificanceWe show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.
Highlights
Bacillus thuringiensis (Bt) bacteria produce insecticidal crystal (Cry) proteins that are used in the control of insect pests important for agricultural crops and against vectors of human diseases [1]
D136N/T143D as dominant negative (DN) inhibitor of other Cry1 toxins, we tested its ability to inhibit the toxicity of other Cry1 toxins that are active against M. sexta such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa
We fed M. sexta larvae with mixtures of Cry1Ab-D136N/T143D mutant with the different wildtype Cry protoxins in a protein ratio 0.5:1
Summary
Bacillus thuringiensis (Bt) bacteria produce insecticidal crystal (Cry) proteins that are used in the control of insect pests important for agricultural crops and against vectors of human diseases [1]. Cry toxins have been characterized as pore-forming toxins Their mechanism of action involves specific interactions with several receptors and insertion of part of the toxin into the apical membrane of insect midgut cells, forming pores that kill the larvae [2]. Cry oligomers bind to aminopeptidase or alkaline phosphatase receptors [3,4], and inserts into the membrane to form toxic pores [1,2]. Helix a-3 of Cry toxin is involved in toxin oligomerization [5] and helix a-4 in membrane insertion and pore formation [6]. Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. It was shown that non-toxic Cry1Ab helix a-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1:1, 0.5:1, mutant:wt) indicating that oligomer formation is a key step in toxicity of Cry toxins
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