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Chen R.-R.,Nanjing Agricultural University | Ren X.-L.,Nanjing Agricultural University | Han Z.-J.,Nanjing Agricultural University | Mu L.-L.,Nanjing Agricultural University | And 3 more authors.
Archives of Insect Biochemistry and Physiology | Year: 2014

In S. exigua, ingestion of Cry1Ac reduces larval growth, shortens lifespan, and decreases copulation and oviposition of the adults. Cadherin-like protein SeCad1b in S. exigua has recently been published. Here, we tested whether SeCad1b mediates the negative effects of Cry1Ac. We identified three potential Cry toxin binding regions in SeCad1b, i.e., 879EIAIQITDTNN889, 1357SLLTVTI1363, and 1436GVISLNFQ1443. We expressed and purified a truncated cadherin, rSeCad1bp, and its interspecific homologue, rHaBtRp, from H. armigera that contain the putative toxin binding regions. Using a toxin overlay assay, we found that rSeCad1bp specifically binds to biotinylated Cry1Ac in a dose-dependent manner. We also discovered that an addition of rSeCad1bp and rHaBtRp enhances the suppression of larval growth by Cry1Ac, although rSeCad1bp is less suppressive than rHaBtRp. Finally, RNA interference-mediated knockdown of SeCad1b reduced approximately 80% of the target gene and significantly alleviated the negative effect of CrylAc on larval growth. We infer that the S. exigua SeCad1b is a functional receptor of Cry1Ac. © 2014 Wiley Periodicals, Inc.


Ren X.-L.,Nanjing Agricultural University | Chen R.-R.,Nanjing Agricultural University | Zhang Y.,Nanjing Agricultural University | Ma Y.,Institute of Cotton Research of CAAS State Key Laboratory of Cotton Biology | And 4 more authors.
Applied and Environmental Microbiology | Year: 2013

Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca. ©2013, American Society for Microbiology.


PubMed | Institute of Cotton Research of CAAS State Key Laboratory of Cotton Biology and Nanjing Agricultural University
Type: Journal Article | Journal: Journal of economic entomology | Year: 2016

In insects, the mode of Cry1A toxins action has been studied in detail and many receptors that participate in the process are known. Recent evidence has revealed that an ABC transporter (ABCC2) is involved in conferring resistance to Cry1A toxins and that ABCC2 could be a receptor of Cry1A. However, it is not known whether Cry1Ca interacts with the same receptor proteins as Cry1A. In this study, we report the cloning of an ABC transporter gene, SeABCC2b, from the midgut of Spodoptera exigua (Hbner) larvae, and its expression in Sf9 cells for a functional analysis. The addition of Cry1Ca and Cry1Ac to Sf9 cell culture caused swelling in 28.5% and 93.9% of the SeABCC2-expressing cells, respectively. In contrast, only 7.4% and 1.3% of the controls cells swelled in the presence of Cry1Ca and Cry1Ac. Thus, SeABCC2b-expressing Sf9 cells had increased susceptibility to Cry1Ca and Cry1Ac. Similarly, S. exigua cadherin (SeCad1b) expressed in Sf9 cells caused 47.1% and 1.8% of the SeCad1b-expressing cells to swell to Cry1Ca and Cry1Ac exposure. Therefore, Sf9 cells expressing SeCad1b were more sensitive to Cry1Ca than Cry1Ac. Together, our data suggest that SeABCC2b from S. exigua mediates Cry1Ac cytotoxicity and, in conjunction with SeCad1b, contributes to enhance Cry1Ca toxicity in Sf9 cells.

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