SfABCC2 transporter extracellular loops 2 and 4 are responsible for the Cry1Fa insecticidal specificity against Spodoptera frugiperda
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Published on: August 23, 2018
Yuanyuan Liu1, Minghui Jin2, Ling Wang3
1Institute of Entomology, School of Life Sciences, Central China Normal University, Wuhan, 430079, PR China.
Abstract:
Bacillus thuringiensis (Bt) bacteria produce Cry toxins that kill insect pests. Insect specificity of Cry toxins relies on their binding to larval gut membrane proteins such as cadherin and ATP-binding cassette (ABC) transporter proteins. Mutations in ABC transporters have been implicated in high levels of resistance to Cry toxins in multiple pests. Spodoptera frugiperda is an insect pest susceptible to Cry1Fa and Cry1Ab toxins while Mythimna separata is tolerant to Cry1Fa and less susceptible to Cry1Ab. Here, we analyzed the potential role of ABCC2 in determining the susceptibility of S. frugiperda to Cry1Fa and Cry1Ab, by expressing SfABCC2 or MsABCC2 in Hi5 insect cell line and by the systematic replacements of extracellular loops (ECLs) between these two proteins. Expression of SfABCC2 in Hi5 conferred susceptibility to both Cry1Fa and Cry1Ab, in contrast to the expression of MsABCC2 that mediated low toxicity to Cry1Ab and no toxicity to Cry1Fa in agreement with their larvicidal toxicities. The SfABCC2 and MsABCC2 amino acid sequences showed differential residues among ECL1, ECL2, ECL4 and ECL6 loops, while ECL3 and ECL5 share the same primary sequence. The exchange of ECLs between SfABCC2 and MsABCC2 demonstrated that ECL4 and ECL2 contribute to Cry1Fa toxicity, where ECL4 plays a major role. The medium region (named M2) of ECL4 was identified as the most important region of SfABCC2 involved in Cry1Fa toxicity as shown by point mutations in this region. These findings will be helpful to understand the mechanisms of action of Bt toxins in S. frugiperda.
Insights
Bacillus thuringiensis (Bt) Cry toxins target insect pests by binding to gut proteins. This study reveals that the ABCC2 protein
Area of Science:
- Insect toxicology
- Molecular biology
- Biochemistry
Background:
- Bacillus thuringiensis (Bt) produces Cry toxins lethal to insect pests.
- Cry toxin specificity depends on binding to insect gut membrane proteins like cadherin and ATP-binding cassette (ABC) transporters.
- Mutations in ABC transporters are linked to pest resistance against Bt toxins.
Purpose of the Study:
- To investigate the role of the ABCC2 protein in determining the susceptibility of Spodoptera frugiperda (S. frugiperda) to Cry1Fa and Cry1Ab toxins.
- To compare the function of S. frugiperda ABCC2 (SfABCC2) and Mythimna separata ABCC2 (MsABCC2) in mediating Cry toxin toxicity.
- To identify specific regions within ABCC2 responsible for differential Cry toxin susceptibility.
Main Methods:
- Expression of SfABCC2 and MsABCC2 in the Hi5 insect cell line.
- Systematic exchange of extracellular loops (ECLs) between SfABCC2 and MsABCC2.
- Analysis of Cry1Fa and Cry1Ab toxicity in cell lines expressing wild-type and chimeric ABCC2 proteins.
- Site-directed mutagenesis of specific regions within the ECL4 loop of SfABCC2.
Main Results:
- SfABCC2 expression conferred susceptibility to both Cry1Fa and Cry1Ab in Hi5 cells.
- MsABCC2 expression resulted in low toxicity to Cry1Ab and no toxicity to Cry1Fa.
- Exchange experiments indicated that ECL4 and ECL2 of ABCC2 are crucial for Cry1Fa toxicity, with ECL4 being the primary determinant.
- A specific region (M2) within the ECL4 loop of SfABCC2 was identified as critical for Cry1Fa toxicity.
Conclusions:
- The ABCC2 protein plays a significant role in mediating the toxicity of Bt Cry1Fa and Cry1Ab toxins.
- Differential residues within extracellular loops, particularly ECL4, of ABCC2 contribute to the varying susceptibility of different insect species to Bt toxins.
- Understanding the structural basis of ABCC2 function in toxin binding can inform strategies for managing Bt resistance in insect pests.
