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Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells

1
Departamento de Genética/ERI BioTecMed, Universitat de València, Burjassot, 46100 València, Spain
2
Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj 31578-77871, Alborz, Iran
3
Departamento de Agronomía, Biotecnología y Alimentación, Universidad Pública de Navarra, Pamplona, 31006 Navarra, Spain
*
Author to whom correspondence should be addressed.
Toxins 2020, 12(2), 133; https://doi.org/10.3390/toxins12020133
Received: 16 December 2019 / Revised: 31 January 2020 / Accepted: 19 February 2020 / Published: 21 February 2020
Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins. View Full-Text
Keywords: Cry1Ab; oligomer formation; Sf21 cell line; Ostrinia nubilalis; Lobesia botrana; Leptinotarsa decemlineata; bioassay Cry1Ab; oligomer formation; Sf21 cell line; Ostrinia nubilalis; Lobesia botrana; Leptinotarsa decemlineata; bioassay
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    Doi: 10.5281/zenodo.3578309
    Link: https://zenodo.org/record/3578309#.XfeFBehKiM8
    Description: Figure S1. Biotin labelled Cry1Ab oligomer formation promoted by O. nubilalis BBMV. Lanes 1 and 3- Proteins associated with the BBMV (pellet) after incubation with Cry1Ab protein. Lanes 2 and 4- Supernatant after incubation of BBMV-proteins. Lane 5- Control of Cry1Ab protein incubated without BBMV. Lane 6- O. nubilalis BBMV. Lane M- molecular weight marker. The arrowhead points to the Cry1Ab oligomer (about 250 kDa).
MDPI and ACS Style

Khorramnejad, A.; Domínguez-Arrizabalaga, M.; Caballero, P.; Escriche, B.; Bel, Y. Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells. Toxins 2020, 12, 133.

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