Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera

Abstract

Toxins from the bacterium Bacillus thuringiensis (Bt) are used widely for insect control in sprays and transgenic plants, but their efficacy is reduced when pests evolve resistance. Previous work showed that mutations in a gene encoding the transporter protein ABCC2 are linked with resistance to Bt toxins Cry1Ab, Cry1Ac or both in four species of Lepidoptera. Here we compared the ABCC2 gene of Helicoverpa armigera (HaABCC2) between susceptible strains and a laboratory-selected strain with >1,000-fold resistance to Cry1Ac relative its susceptible parent strain. We discovered a 73-base pair (bp) insertion in the cDNA of the resistant strain that generates a premature stop codon expected to yield a truncated ABCC2 protein. Sequencing of genomic DNA revealed that this insertion is an intron that is not spliced out because of a 6-bp deletion at its splicing site. Analysis of progeny from crosses revealed tight genetic linkage between HaABCC2 and resistance to Cry1Ac. These results provide the first evidence that mis-splicing of a gene encoding an ABCC2 protein confers resistance to a Bt toxin.

Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

“…Only Group C produced enough progeny to maintain the population. 19 The second instar larvae of G 2 of Group C were screened with a diagnostic concentration of There are no double peaks observed in the sequence chromatograms for the two genes (Fig. 5), 10 suggesting that the sgRNA used in our study is relatively specific.…”

“…The CRISPR/Cas9 system will 18 be an ideal tool to address these questions. We believe reverse genetic manipulation of 19 candidate genes in target insects, mediated by CRISPR/Cas9 system, will be a promising tool 20 for revealing the complex modes of action of and diverse resistance mechanisms to Bt toxins 21 in future. 22 Resistance is an evolutionary phenomenon and there are several ways for insect 23 populations to become resistant to chemical insecticides or toxins from entomopathogens.…”

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

“…There has been much interest recently in the interaction of Cry toxins with members of the ATP binding cassette (ABC) protein family, particularly subfamilies C2 and A. Alterations in these ABC proteins are linked with resistance against Cry1 toxins (Heckel, 2012;Park et al, 2014;Xiao et al, 2014) and Cry2Ab (Tay et al, 2015). Although the exact role of these proteins remains unclear it has been proposed that they may act as receptors but only when in a particular conformational state (Heckel, 2012) …”

Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.)

“…However, the structure and function of white protein in the insect midgut tissue remain elusive, and how the white protein acts as both transporter and Cry1Ac receptor in the midgut warrants further investigation. Recently, mutations of an ABCC gene (ABCC2) have been found to be linked with Bt Cry resistance in several lepidopteran insects (Gahan et al, 2010;Baxter et al, 2011;Atsumi et al, 2012;Park et al, 2014;Xiao et al, 2014), and it has been speculated that ABCC2 might play a role in facilitating the insertion of oligomeric pre-pore structure into the insect midgut membrane in the mode of action of Bt Cry toxins (Heckel, 2012). ABCC2 has recently been proved to be a functional Cry toxin receptor in B. mori (Tanaka et al, 2013), and we suspect that the role of the possibly formed white heterodimer in P. xylostella is similar to that of ABCC2 in B. mori.…”