Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters

doi:10.1016/j.peptides.2017.04.003

Peptides

Volume 98, December 2017, Pages 86-92

https://doi.org/10.1016/j.peptides.2017.04.003 Get rights and content

Highlights

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Moth ABCC2 and ABCC3 function as Cry1A toxin receptors.
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A beetle ABCC transporter is a receptor of a beetle-specific Cry8C toxin.
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Mosquito and human ABCC proteins do not respond to any of the tested Cry toxins.

Abstract

In this study, we examined insect and human ABCC transporters closely related to the lepidopteran ABC transporter C2 (ABCC2), a powerful receptor for the Bacillus thuringiensis Cry toxin, for their responses to various Cry toxins. ABCC2 and the lepidopteran ABC transporter C3 (ABCC3) conferred cultured cells with susceptibility to a lepidopteran-specific Cry1Aa toxin but not to lepidopteran-specific Cry1Ca and Cry1Da. One coleopteran ABCC transporter specifically responded to a coleopteran-specific Cry8Ca toxin. ABCC transporters from a dipteran insect and humans did not respond to any of the tested Cry toxins that are active to lepidopteran and coleopteran insects. These results yield important information for our understanding of insect specificity of Cry toxins and provide the first demonstration of a coleopteran ABCC transporter that serves as a Cry toxin receptor.

Introduction

The ATP-binding cassette (ABC) transporter family of proteins are membrane proteins that transport a broad range of substrates such as lipids, sugars, vitamins, nucleotides, and xenobiotics [1]. With regard to insect ABC transporters, many studies thus far have focused on physiological roles involved in specialized transport activity, such as pigment, as represented by a Drosophila white mutant [2], and chemical insecticide resistance [3], [4]. The insect ABC transporter family has been paid much recent attention in another aspect: some of these family members serve as receptors of an insecticidal protein produced by Bacillus thuringiensis, a bacterial insect pathogen.

B. thuringiensis produces various insecticidal proteins, including Cry toxins, which are widely used in pest control throughout the world. Cry toxins contained in crystalline inclusions are solubilized and partially degraded into an activated toxin core by proteases in the digestive fluids of insects after ingestion. The activated toxin forms a pore on the cell membrane of midgut epithelial cells following specific interactions with a receptor(s), resulting in osmotic cell lysis that leads to disintegration of midgut epithelial tissue and death of the insect [5], [6]. Many previous reports have suggested that specific toxin-receptor interactions were one of the main factors involved in target insect specificity [7]; however, the functional receptors of most Cry toxins that strongly mediate toxicity have not yet been identified.

The ABC transporter subfamily C2 (ABCC2) gene in lepidopteran insects was identified as responsible for insect strains with high-resistance against Cry1A toxins [8]. Since then, the involvement of ABCC2 in Cry toxin intoxication has become widely studied. We previously showed that silkworm Bombyx mori ABCC2 (BmABCC2) conferred Sf9 cells with high susceptibility to Cry1A and Cry1F toxins [9]. A voltage clamp experiment using Xenopus laevis oocytes demonstrated that BmABCC2 functions as the most powerful mediator among the Cry1A toxin receptors to generate toxin pores in the membrane [10]. Therefore, to date, ABCC2 is regarded as one of the most important receptors, especially in mediating pore formation of Cry1A toxins, at least in lepidopteran insect cells. Recently, ABCC3, a paralog of ABCC2, was also reported to play a role in determining susceptibility to Cry1Ac and Cry1Ca in Spodoptera exigua larvae [11] and Cry1Ac in Plutella xylostella larvae [12] and cultured Sl-HP insect cells from Spodoptera litura [13]. Chen et al. also showed that S. litura ABCC3 confers Cry1Ac susceptibility to Hi5 insect cells [13], suggesting that ABCC3 is also a functional receptor of Cry1A toxins. These two ABCC transporters in lepidopteran insects are likely to mediate susceptibility to various Cry1 toxins mainly targeting lepidopteran insects; however, their precise Cry toxin specificities have not been fully investigated. Moreover, we also reported that Sf9 cells expressing BmABCC2 showed low susceptibility to Cry8Ca that is mainly active to coleopteran insects [9]. Cry8Ca is phylogenetically distant from Cry1 toxins, but both have similar three-dimensional structures with three conserved domains [14]. This suggests that Cry8Ca exhibits toxicity mediated by ABCC2 or other ABCC transporters in coleopteran insects, although no case of ABCC transporters involved in this mode of action of coleopteran-specific Cry toxins has yet to be reported. Conversely, human MRP4/ABCC4 (hABCC4) is the most closely related human ABC transporter to lepidopteran ABCC2 [15]. Therefore, it is important to determine whether hABCC4 functions as a Cry toxin receptor for a better understanding of Cry toxin safety for humans.

In this study, we assess the Cry toxin specificities of two S. exigua ABCC2 and ABCC3 proteins against five Cry toxins. Based on the hypothesis that non-lepidopteran insect ABCC transporters may be involved in Cry toxin intoxication, we also investigate the Cry toxin specificities of non-lepidopteran insect ABCC transporters that are closely related to lepidopteran ABCC2.

Section snippets

Cry toxin preparation

Cry1Aa, Cry1Da, Cry3Bb, and Cry8Ca toxins were produced as recombinant proteins from Escherichia coli as described before [9]. The DNA fragment coding for Cry1Da toxin from the Bacillus Genetic Stock Center (BGSC, Ohio, USA) was amplified using primers shown in Table S1 to insert into pGEX4t-3 vector (GE Healthcare, Calfont, UK). Cry1Ca toxin was produced by B. thuringiensis recombinant stain [16]. The inclusion bodies were solubilized and activated as described elsewhere [17]. The toxin

Phylogenetic analysis of ABCC transporters

To determine whether the ABCC2 transporter is conserved among insects and to identify ABCC2 transporter orthologs in dipteran and coleopteran insects, we performed a phylogenetic analysis among the ABCC transporters from insects and humans using amino acid sequences of insect ABC transporters deduced from the genomic information of the silkworm B. mori, the fruit fly Drosophila melanogaster, the red floor beetle T. castaneum, the mosquito Anopheles gambiae, and the honey bee Apis mellifera [18]

Discussion

SeABCC3 conferred HEK293T cells with lower Cry1Aa susceptibility relative to SeABCC2 (Fig. 4A). Many Cry1A-resistant strains have mutations in the ABCC2 gene [29] whereas no Cry1A-resistant strains with ABCC3 mutations have been reported, indicating that, in many cases, the contribution of ABCC2 in determining larval Cry1A susceptibility is much higher than that of ABCC3. However, the relative responses of ABCC2 and ABCC3 may vary among different Cry1A toxins and lepidopteran insect species.

Conflict of interest

None declared.

Author contributions

HE and RS conceived, designed experiments, and wrote the manuscript. HE, ST, KI, and SA performed experiments. HE analyzed data. SK contributed regents/material/analysis tools for HEK293T cells.

Acknowledgements

Authors acknowledge Dr. Salvador Herrero and Dr. Baltasar Escriche (University of Valencia, Spain) for SeABCC2 cDNA and S. exgua larvae, Dr. Toshinori Sasaki (National Institute of Infectious Diseases, Japan) for A. albopictus, and Drs. Sumio Ohtsuki and Tetsuya Terasaki for Human MRP4/ABCC4 cDNA (Tohoku University, Japan). HEK293T cells were provided by RIKEN Cell Bank (Tsukuba, Japan). This research was financially supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant

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Mutations induced by TALENs were identified according to the sequencing results. Cry1Ab (AAA22551), Cry1Ac (AAA22331) and Cry1Fa (AAA22348) protoxins were produced as recombinant proteins by Escherichia coli BL21 (for production of Cry1Ab and Cry1Ac) or Rosetta (DE3) pLysS (for production of Cry1Fa) as described previously (Tanaka et al., 2013; Endo et al., 2017). The inclusion body of these protoxins were suspended in 5 mL solution containing 100 μL of 2 M Tris-HCl and 10 μL of 1M dithiothreitol (DTT).
Field-evolved resistance of insect pests to Bacillus thuringiensis (Bt) toxins (Cry toxins) is a threat to the efficacy of Bt-based bio-insecticides and transgenic crops. Recent reports have suggested that ATP-binding cassette transporter subfamily C2 (ABCC2) and cadherin-like receptor play important roles in conferring susceptibility to Cry1 toxins. However, the receptors involved in Bt susceptibility in each insect remain unclear. To determine the receptors that are involved in the susceptibility of Bombyx mori to Cry1 toxins (1Ab, 1Ac and 1Fa), we conducted diet overlay bioassay using B. mori strains disrupted with one or two receptor (s) among BmABCC2, BmABCC3, and cadherin-like receptor (BtR175) generated by transcription activator-like effector nuclease (TALEN)-mediated gene editing. The single-knockout strains for BmABCC2 showed resistance to Cry1Ab and Cry1Ac, whereas only strains with double knockout of BmABCC2 and BmABCC3 exhibited high resistance to Cry1Fa. Progeny populations generated from the crossing of heterozygotes for BtR175 knockout allele included 25% theoretical homozygotes for the BtR175 knockout allele and they showed resistance to Cry1Ab and Cry1Ac. Then, through a cell swelling assay using Sf9 cells ectopically expressing the receptor, we analyzed the mechanisms underlying the different contributions of BmABCC2, BmABCC3, and BtR175 to larval susceptibility. The receptor activity of BmABCC2 for Cry1Ab and Cry1Ac was far higher than that of BmABCC3, and BtR175 synergistically enhanced the receptor activity of BmABCC2. This result well explained the important involvement of BmABCC2 and BtR175 in the larval susceptibility to Cry1A toxins. By contrast, the receptor activities of BmABCC2 and BmABCC3 for Cry1Fa were observed at a similar level and synergistic effect of BtR175 was small. This finding explains the equal importance of BmABCC2 and BmABCC3 and very small contribution of BtR175 on larval susceptibility to Cry1Fa. Thus, we demonstrated the different importance of BmABCC2, BmABCC3, and BtR175 to various Cry1 toxins as susceptibility-determining factors in B. mori larvae and the underlying basis for the observed differences. Furthermore, a weak correlation was indicated between the binding affinity and receptor activities of BmABCC2 and BmABCC3 to Cry1 toxins.
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Thus, it was proposed that using a combination of different Bt toxins that do not share insect gut receptors could be an important strategy to delay the evolution of insect resistance (Li et al., 2021；Zhao et al., 2003). Lepidopteran midgut ATP-binding cassette (ABC) transporters proteins have been shown to serve as receptors of different Cry toxins such as Cry1A and Cry1Fa (Gahan et al., 2010; Tanaka et al., 2013; Endo et al., 2017; Chen et al., 2015; Ocelotl et al., 2017; Sato et al., 2019; Wang et al., 2020a). It was shown that the knock-out mutations induced by CRISPR/CAS9 of both ABCC2 and ABCC3 were associated with high levels of resistance to Cry1Ac in Helicoverpa armigera and Plutella xylostella (Wang et al., 2020b; Liu et al., 2020).
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Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) can provide safe and effective control of some major pests, but evolution of resistance by pests diminishes these benefits. Better understanding of the genetics and mechanisms of resistance is urgently needed to improve methods for monitoring, managing, and countering pest resistance to Bt toxins. Here we used CRISPR-mediated knockouts to evaluate the role of five genes encoding candidate Bt toxin receptors in Spodoptera exigua (beet armyworm), a devastating pest of vegetable, field and flower crops. We compared susceptibility to Bt toxins Cry1Ac, Cry1Fa, and Cry1Ca between the parent susceptible strain and each of five strains homozygous for the knockout of one of the candidate genes (SeAPN1, SeCad1, SeABCC1, SeABCC2 or SeABCC3). The results from the 15 pairwise comparisons reveal that SeABCC2 has a major role and SeCad1 a minor role in mediating toxicity of Cry1Ac and Cry1Fa. SeABCC2 also has a minor role in toxicity of Cry1Ca. In addition, the results imply little or no role for the other three candidate receptors in toxicity of Cry1Ac or Cry1Fa; or for the four candidate receptors other than SeABCC2 in toxicity of Cry1Ca.

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Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters

Highlights

Abstract

Introduction

Section snippets

Cry toxin preparation

Phylogenetic analysis of ABCC transporters

Discussion

Conflict of interest

Author contributions

Acknowledgements

Biochim. Biophys. Acta

Insect Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

Trends Genet.

Biochim. Biophys. Acta

Mol. Cells

Insect Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

Biochim. Biophys. Acta

Adv. Genet.

Pestic. Biochem. Physiol.

Insect Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

ABC transporters: from microorganisms to man

Annu. Rev. Cell Biol.

Genetic analysis of the xenobiotic resistance-associated ABC gene subfamilies of the Lepidoptera

Insect Mol. Biol.

Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different insect specificity

Biochim. Biophys. Acta

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

Microb. Biotechnol.