Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis (original) (raw)
Related papers
Evolution of Bacillus thuringiensis Cry toxins insecticidal activity
Microbial Biotechnology, 2013
Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.
Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins
Toxins, 2014
Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on OPEN ACCESS Toxins 2014, 6 2394 Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.
FEBS Letters, 2000
We used site-directed mutagenesis to modify the cally cleaved from an inactive protoxin, to an active toxin Bacillus thuringiensis cry3A gene in amino acid residues 350-form within the insect midgut. The activated toxin binds to 354. Two mutant toxins, A1 (Ra4sA,Yss0F, Y3sIF). and A2 receptors in the midgut and is believed to integrate into the _._ _ (R_sA,AYsso,AYssl), showed significantly improved toxicity lipid bilayer of the brush border membrane. Ion channels are O "" against Tenebrio molitor (yellow mealworm). The mutant toxin formed, causing midgut cells to lose their membrane potential. A1 was also more potent against both Leptinotarsa decemlineata If the rate of damage to the midgut exceeds the rate of repair, (Colorado potato beetle) and Cho,somela scripts (cottonwood lesions form. bacteria invade the hemocele, and death results leaf beetle), while A2 displayed enhanced toxicity only in L. from bacterial septicemia. The fi-endotoxins from B.t. comdecemlineata. Competitive binding assays of L. decemlineata prise a group of over 100 related proteins [5}, which were t5 brush border membrane vesicles (BBMV) revealed that binding previously categorized by insecticidal activity [6] but currently affinities for the AI and A2 mutant toxins were ca. 2.5-fold cq" I-higher than for the wild-type Cry3 toxin. S mar bind ng assays by amino acid similarity [7]. The spectrum of toxicity for each O with C. scripta BBMV revealed a ca. 5-fold lower dissociation toxin is relatively narrow, while the collective activity of this rate for the Al mutant as compared to that of Cry3A. group of pesticidal toxins now spans seven orders of insect © 2000 Federation of European Biochemical Societies. and several other invertebrate groups including nematodes. mites and protozoans [8].
2000
We used site-directed mutagenesis to modify the cally cleaved from an inactive protoxin, to an active toxin Bacillus thuringiensis cry3A gene in amino acid residues 350-form within the insect midgut. The activated toxin binds to 354. Two mutant toxins, A1 (Ra4sA,Yss0F, Y3sIF). and A2 receptors in the midgut and is believed to integrate into the _._ _ (R_sA,AYsso,AYssl), showed significantly improved toxicity lipid bilayer of the brush border membrane. Ion channels are O "" against Tenebrio molitor (yellow mealworm). The mutant toxin formed, causing midgut cells to lose their membrane potential. A1 was also more potent against both Leptinotarsa decemlineata If the rate of damage to the midgut exceeds the rate of repair, (Colorado potato beetle) and Cho,somela scripts (cottonwood lesions form. bacteria invade the hemocele, and death results leaf beetle), while A2 displayed enhanced toxicity only in L. from bacterial septicemia. The fi-endotoxins from B.t. comdecemlineata. Competitive binding assays of L. decemlineata prise a group of over 100 related proteins [5}, which were t5 brush border membrane vesicles (BBMV) revealed that binding previously categorized by insecticidal activity [6] but currently affinities for the AI and A2 mutant toxins were ca. 2.5-fold cq" I-higher than for the wild-type Cry3 toxin. S mar bind ng assays by amino acid similarity [7]. The spectrum of toxicity for each O with C. scripta BBMV revealed a ca. 5-fold lower dissociation toxin is relatively narrow, while the collective activity of this rate for the Al mutant as compared to that of Cry3A. group of pesticidal toxins now spans seven orders of insect © 2000 Federation of European Biochemical Societies. and several other invertebrate groups including nematodes. mites and protozoans [8].
Biochemical Journal, 2004
Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.
Peptides, 2014
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 ␣-1 and part of helix ␣-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.
Pest management science, 2016
Bacillus thuringiensis δ-endotoxins are the most widely used biopesticides for controlling economically important crop pests and disease vectors. Improving its efficacy is of great benefit. Here, an improvement of Cry2Aa δ-endotoxins toxicity was conducted via a cry gene over-expression system using P20 from B. thuringiensis israelensis. The co-expression of Cry2Aa with P20 witnessed a 7-fold increase of its production yield in B. thuringiensis. Generated crystals showed to be significantly more toxic (505.207 µg · g(-1) , 1.99 mg · l(-1) and 1.49 mg · l(-1) ) than the P20-lacking control (720.78 µg · g(-1) , 705.69 mg · l(-1) and 508.51 mg · l(-1) ) against Ephestia kuehniella, Aedes aegypti and Culex pipiens larvae, respectively. In vitro, processing experiments revealed a P20-mediated protection of Cry2Aa against degradation under larval gut conditions. Thus, P20 could promote the maintenance of a tightly packaged conformation of Cry2Aa toxins in the larval midgut upon correct ac...
Peptides, 2007
Mode of action a b s t r a c t Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.