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A cadherin-like protein influences Bacillus thuringiensis Cry1Ab toxicity in the oriental armyworm, Mythimna separata
Ling Wang1, Xingfu Jiang, Lizhi Luo
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection Chinese Academy of Agricultural Sciences, Beijing, China.
Abstract:
Cadherins comprise a family of calcium-dependent cell adhesion proteins that act in cell-cell interactions. Cadherin-like proteins (CADs) in midguts of some insects act as receptors that bind some of the toxins produced by the Bacillus thuringiensis (Bt). We cloned a CAD gene associated with larval midguts prepared from Mythimna separata. The full-length cDNA (MsCAD1, GenBank Accession No. JF951432) is 5642 bp, with an open reading frame encoding a 1757 amino acid and characteristics typical of insect CADs. Expression of MsCAD1 is predominantly in midgut tissue, with highest expression in the 3rd- to 6th-instars and lowest in newly hatched larvae. Knocking-down MsCAD1 decreased Cry1Ab susceptibility, indicated by reduced developmental time, increased larval weight and reduced larval mortality. We expressed MsCAD1 in E. coli and recovered the recombinant protein, rMsCAD1, which binds Cry1Ab toxin. Truncation analysis and binding experiments revealed that a contiguous 209-aa, located in CR11 and CR12, is the minimal Cry1Ab binding region. These results demonstrate that MsCAD1 is associated with Cry1Ab toxicity and is one of the Cry1Ab receptors in this insect. The significance of this work lies in identifying MsCAD1 as a Cry1Ab receptor, which helps understand the mechanism of Cry1Ab toxicity and of potential resistance to Bt in M. separata.
Insights
Cadherin-like protein 1 (MsCAD1) in Mythimna separata acts as a receptor for Bacillus thuringiensis (Bt) Cry1Ab toxin. Identifying this Bt receptor aids understanding of toxin mechanisms and insect resistance.
Area of Science:
- Molecular Biology
- Insect Toxicology
- Biochemistry
Background:
- Cadherins are calcium-dependent cell adhesion proteins involved in cell interactions.
- Cadherin-like proteins (CADs) in insect midguts function as receptors for Bacillus thuringiensis (Bt) toxins.
- Understanding these interactions is crucial for pest control and Bt resistance management.
Purpose of the Study:
- To clone and characterize a cadherin-like gene from Mythimna separata midgut.
- To investigate the role of this gene in susceptibility to Bt Cry1Ab toxin.
- To identify the specific binding region of the protein for Cry1Ab toxin.
Main Methods:
- Cloning of the MsCAD1 gene and sequencing of its full-length cDNA.
- Quantitative analysis of MsCAD1 gene expression in different larval stages.
- RNA interference (RNAi) to knock down MsCAD1 expression and assess its effect on Cry1Ab susceptibility.
- Recombinant expression of MsCAD1 in E. coli and binding assays with Cry1Ab toxin.
- Truncation analysis to determine the minimal toxin-binding domain.
Main Results:
- A full-length cDNA, MsCAD1, encoding a 1757 amino acid protein typical of insect CADs was identified.
- MsCAD1 expression was highest in later larval instars (3rd-6th) and predominantly in midgut tissue.
- Knockdown of MsCAD1 reduced susceptibility to Cry1Ab, leading to decreased larval mortality and increased weight.
- Recombinant MsCAD1 (rMsCAD1) directly bound to Cry1Ab toxin.
- A 209-amino acid region within CR11 and CR12 of MsCAD1 was identified as the minimal Cry1Ab binding site.
Conclusions:
- MsCAD1 is a functional receptor for the Bacillus thuringiensis Cry1Ab toxin in Mythimna separata.
- This finding elucidates a key mechanism underlying Cry1Ab toxicity in this insect.
- Identification of MsCAD1 provides insights into potential mechanisms of Bt resistance in M. separata.
