Dataset Information

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

ABSTRACT: As a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4-α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4-α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the assembly of the 250 kDa oligomers, suggesting that these mutation residues might be essential for Cry2Ab oligomerization. As expected, all of these variants showed lower insecticidal activity against P. xylostella. Furthermore, we found that the pore-forming activities of these mutants also decreased when compared to wild-type Cry2Ab. Taken together, our data identified key residues for Cry2Ab oligomerization and emphasized that oligomerization was closely related to the insecticidal activity and pore-forming activity of Cry2Ab.

SUBMITTER: Pan ZZ

PROVIDER: S-EPMC8325727 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

Pan Zhi-Zhen ZZ Xu Lian L Liu Bo B Chen Qing-Xi QX Zhu Yu-Jing YJ

AMB Express 20210731 1

As a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4-α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4-α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the ...[more]

PMID: 34331618

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Project description:Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

Project description:The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Dataset Information

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

Publications

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

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