ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth

Josue Ocelotl,Jorge Sánchez,Alejandra Bravo,Mario Soberón,Isabel Gómez,Bruce E Tabashnik

doi:10.1038/s41598-017-02545-y

Josue Ocelotl, Jorge Sánchez + Show 4 more

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https://doi.org/10.1038/s41598-017-02545-y

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Abstract

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

Highlights

ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth 2017, 7 (1) Scientific Reports
Characterization of the binding of Cry1Ab or Cry1Ac in resistant colonies from different lepidopteran species suggested that ABCC2 might function as receptor molecule since in most cases at least one Cry1A toxin is affected in binding to brush border membrane vesicles (BBMV) from the resistant colony[22, 29]
To gain insight into the possible role of ABCC2 in the mode of action of Cry1Ac toxin we compared the binding and oligomerization of Cry1Ac and Cry1AcMod toxins to BBMV from a strain of P. xylostella susceptible to Cry1Ac and a strain in which resistance to Cry1Ac is linked to an ABCC2 mutation[23]

Summary

Introduction

ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth 2017, 7 (1) Scientific Reports. Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. The protease resistant fragment or activated toxin, composed of the three-domain structure, undergoes a complex sequential binding events with different insect proteins leading to oligomerization, membrane insertion and pore formation, resulting in colloidal osmotic lysis of the midgut cells[13,14,15]. In lepidopteran larvae the first binding event of activated Cry1A toxins is proposed to be a low affinity interaction with the highly abundant GPI-anchored-receptors, alkaline phosphatase (ALP) or aminopeptidase-N (APN)[15,16,17,18] This interaction is proposed to concentrate the toxin in proximity to the brush border microvilli membrane of the midgut cells where the toxin binds, in a high affinity interaction, to the cadherin receptor[15]. Incubation of Cry1Ab activated toxin with a www.nature.com/scientificreports/

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