Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from<i>Helicoverpa armigera</i>by an improved pull‐down assay

Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A 548 to S 798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera. IMPORTANCE Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

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“…The Cry1Ah-binding proteins on BBMV from H. armigera were identified by pulldown assays as previously described (26). As Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Finally, CNBr-Cry1Ah was stored in 20% ethanol at 4°C. The pulldown assays were done as previously described (26). After 2 h of incubation at 4°C of 50 l CNBr-Cry1Ah with 100 g solubilized BBMV proteins (1% CHAPS), the unbound BBMV proteins were removed by centrifugation at 400 ϫ g for 30 s at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

Zhou

Liu

Liang

et al. 2017

Appl Environ Microbiol

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“…In a previous work, we identified 10 Cry8Ea-binding proteins from H. parallela using a similar strategy (18). In addition, Zhou et al identified ABCC2 (ATP-binding cassette transporter subfamily C protein) as a Cry1Ac-binding protein in Helicoverpa armigera using the same methodology (21). These Cry8-like-binding proteins in H. oblita, as well as the Cry8Ea-binding H. parallela proteins (18), were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase (22), alkaline phosphatase (23), and cadherin (24).…”

Section: Discussionmentioning

confidence: 99%

“…After silver staining, protein bands on the SDS-PAGE gel were cut out for subsequent identification by LC-MS/MS, as described elsewhere (21). The LC-MS/MS data were searched with Mascot MS/MS Ion Search, with the H. oblita midgut unigenes as the database.…”

Section: Methodsmentioning

confidence: 99%

Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

Jian

Huang

Shu

et al. 2017

Appl Environ Microbiol

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The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), containing two cry genes (cry8-like and cry8Ga), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from H. oblita, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from H. oblita larvae were identified by pulldown assays and liquid chromatographytandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the H. oblita transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of Tribolium castaneum, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin.IMPORTANCE Holotrichia oblita is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. H. oblita is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of H. oblita and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against H. oblita larvae.

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“…V‐ATPases are located in the goblet cell apical membrane, which is involved in energy production and conversion (Nishi and Forgac, ; Beyenbach and Wieczorek, ). Evidence from a number of studies appears to indicate a role for V‐ATPase in the toxicity of Cry proteins in susceptible insects (Krishnamoorthy et al ., ; Chen et al ., ; Nakasu et al ., ; Contreras et al ., ; Xu et al ., ; Zhou et al ., ). The potential role of the V‐ATPase subunit A in mediating the toxicity of Bt Cry toxins in C. suppressalis has not previously been examined.…”

Section: Introductionmentioning

confidence: 97%

The midgut V‐ATPase subunit A gene is associated with toxicity to crystal 2Aa and crystal 1Ca‐expressing transgenic rice in Chilo suppressalis

Qiu

Sun

Jiang

et al. 2019

Insect Molecular Biology

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Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) are toxic to a diverse range of insects. Transgenic rice expressing Cry1A, Cry2A and Cry1C toxins have been developed that are lethal to Chilo suppressalis, a devastating insect pest of rice in China. Identifying the mechanisms underlying the interactions of Cry toxins with susceptible hosts will improve both our understanding of Cry protein toxicology and long‐term efficacy of Bt crops. In this study, we tested the hypothesis that V‐ATPase subunit A contributes to the action of Cry1Ab/1Ac, Cry2Aa and Cry1Ca toxins in C. suppressalis. The full‐length V‐ATPase subunit A transcript was initially cloned from the C. suppressalis larval midgut and then used to generate double‐stranded RNA (dsRNA)‐producing bacteria. Toxicity assays using transgenic rice lines TT51 (Cry1Ab and Cry1Ac fusion genes), T2A‐1 (Cry2Aa), and T1C‐19 (Cry1Ca) in conjunction with V‐ATPase subunit A dsRNA‐treated C. suppressalis larvae revealed significantly reduced larval susceptibility to T2A‐1 and T1C‐19 transgenic rice, but not to TT51 rice. These results suggest that the V‐ATPase subunit A plays a crucial role in mediating Cry2Aa and Cry1Ca toxicity in C. suppressalis. These findings will have significant implications on the development of future resistance management tools.

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Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles fromHelicoverpa armigeraby an improved pull‐down assay

Cited by 35 publications

References 56 publications

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

The midgut V‐ATPase subunit A gene is associated with toxicity to crystal 2Aa and crystal 1Ca‐expressing transgenic rice in Chilo suppressalis

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