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Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer.

ABSTRACT: The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin.

SUBMITTER: Feng D

PROVIDER: S-EPMC4546665 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer.

Feng Dongmei D Chen Zhen Z Wang Zhiwen Z Zhang Chunlu C He Kanglai K Guo Shuyuan S

PloS one 20150821 8

The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported ...[more]

PMID: 26295704

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Project description:Maize with enhanced β-carotene production was engineered to counteract pervasive vitamin A deficiency in developing countries. Second-generation biofortified crops are being developed with additional traits that confer pest resistance. These include crops that can produce Bacillus thuringiensis Berliner (Bt) insecticidal proteins. Currently, it is unknown whether β-carotene can confer fitness benefits through to insect pests, specifically through altering Ostrinia nubilalis foraging behaviour or development in the presence of Bt insecticidal toxin. Therefore the effects of dietary β-carotene plus Bt insecticidal protein on feeding behaviour, mortality, and physiology in early and late instars of O. nubilalis larvae were investigated. The results of two-choice experiments showed that irrespective of β-carotene presence, at day five 68%-90% of neonates and 69%-77% of fifth-instar larvae avoided diets with Cry1A protein. Over 65% of neonate larvae preferred to feed on diets with β-carotene alone compared to 39% of fifth-instar larvae. Higher mortality (65%-97%) in neonates fed diets supplemented with β-carotene alone and in combination with Bt protein was found, whereas <36% mortality was observed when fed diets without supplemented β-carotene or Bt protein. Diets with both β-carotene and Bt protein extended 25 days the larval developmental duration from neonate to fifth instar (compared to Bt diets) but did not impair larval or pupal weight. Juvenile hormone and 20-hydroxyecdysone regulate insect development and their levels were at least 3-fold higher in larvae fed diets with β-carotene for 3 days. Overall, these results suggest that the effects of β-carotene and Bt protein on O. nubilalis is dependent on larval developmental stage. This study is one of the first that provides insight on how the interaction of novel traits may modulate crop susceptibility to insect pests. This understanding will in turn inform the development of crop protection strategies with greater efficacy.

Project description:BACKGROUND:Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is the major insect pest of maize in China and countries of East and Southeast Asia, the Pacific and Australasia. ACB can develop strong resistance to the transgenic Bt maize expressing Cry1Ab, the most widely commercialized Bt maize worldwide. However, the molecular basis for the resistance mechanisms of ACB to Cry1Ab remained unclear. Two biological replicates of the transcriptome of Bt susceptible (ACB-BtS) and Cry1Ab resistant (ACB-AbR) strains of ACB were sequenced using Solexa/Illumina RNA-Seq technology to identify Cry1Ab resistance-relevant genes. RESULTS:The numbers of unigenes for two biological replications were 63,032 and 53,710 for ACB-BtS and 57,770 and 54,468 for ACB-AbR. There were 35,723 annotated unigenes from ACB reads found by BLAST searching NCBI non-redundant, NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology databases. Based on the NOISeq method, 3,793 unigenes were judged to be differentially expressed between ACB-BtS and ACB-AbR. Cry1Ab resistance appeared to be associated with change in the transcription level of enzymes involved in growth regulation, detoxification and metabolic/catabolic process. Among previously described Bt toxin receptors, the differentially expressed unigenes associated with aminopeptidase N and chymotrypsin/trypsin were up-regulated in ACB-AbR. Whereas, other putative Cry receptors, cadherin-like protein, alkaline phosphatase, glycolipid, actin, V-type proton ATPase vatalytic, heat shock protein, were under-transcripted. Finally, GPI-anchor biosynthesis was found to be involved in the significantly enriched pathway, and all genes mapped to the pathway were substantially down-regulated in ACB-AbR. CONCLUSION:To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in ACB Bt resistance. This study identified differentially expressed unigenes related to general Bt resistance in ACB. The assembled, annotated transcriptomes provides a valuable genomic resource for further understanding of the molecular basis of ACB Bt resistance mechanisms.

Dataset Information

Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer.

Publications

Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer.

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