Abstract
The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.
Highlights
During sporulation, Bacillus thuringiensis (Bt) produce different kinds of insecticidal toxins such as Cry and Cyt toxins that have been used commercially worldwide to control different insect pests[1,2]
It was previously reported that mutants in loop 3 region from Cry1Ab are affected in toxicity to M. sexta and showed reduced binding to APN1 and CAD receptors as compared with the wild type Cry1Ab toxin[17]
Cyt toxins represent special and interesting proteins since they are toxic to dipteran insects and are able to synergize the insecticidal activity of some 3d-Cry toxins[3]
Summary
Bacillus thuringiensis (Bt) produce different kinds of insecticidal toxins such as Cry and Cyt toxins that have been used commercially worldwide to control different insect pests[1,2]. Domain II loop 3 of Cry1Ab plays an important role in the binding of this toxin to Manduca sexta APN and CAD receptors[17], while loop 2 and loop α-8 participate in CAD binding[18] Mutations in these loops have resulted in proteins with lower or increased toxicity against specific insect pests, correlating with lower or higher binding affinities to brush border membrane vesicles (BBMV) from those insects[19]. In the case of Cyt toxin, construction of hybrid toxins have resulted in improved toxicity against specific targets, such as the Cyt2Aa-hybrid toxins containing the sequence of a 12 amino acid GBP3 peptide that binds to an aphid APN These Cyt2A-hybrid toxins were constructed in three different exposed loops of the toxin, showing enhanced toxicity against the hemipteran insects such as Acyrthosiphon pisum and Myzus persicae[22]. Our data shows that insect specificity of Cyt toxins can be modified by the introduction of loop regions from non-related toxins with different insect specificity
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