ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

Abstract

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.

Figure 1

Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. (a) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. (b) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio^® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio^® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

Figure 2

The r_A1 mutation at the junction of exon and intron 20 in PgABCA2. (a) gDNA sequence for exon and intron 20 of the wild-type PgABCA2 allele (s_A1) in the susceptible APHIS-S strain and the mutant r_A1 allele from the Cry2Ab-resistant Bt4-R2 strain. The r_A1 indel mutation has a 7-bp insertion (red letters) and 44-bp deletion (red dashes) in exon 20 (blue) and spanning the 5′ splice junction with intron 20 (not highlighted). The allele-specific primers r_A1-F and r_A1-R match the sequences highlighted in orange. (b) Allele-specific PCR using the primers in (a) yielded a 343-bp fragment from APHIS-S (s_A1s_A1), a 305-bp fragment from Bt4-R2 (r_A1r_A1), and both fragments in the offspring from crosses between the two strains (r_A1s_A1). Unprocessed image of agarose gel is shown in Supplementary Fig. S13.

Figure 3

Linkage analysis. Biphasic genetic linkage analysis was initiated with single-pair reciprocal crosses between F₀ adults, with either a resistant Bt4-R2 female and a susceptible APHIS-S male (shown here) or a susceptible APHIS-S female and a resistant Bt4-R2 male. The resulting F₁ progeny were backcrossed in single pairs with the resistant Bt4-R2 strain. Backcross (BC) progeny were tested either on diet containing 1 microgram Cry2Ab per mL diet or control diet. Survivors from 10 reciprocal backcross families (five F₁ ♀ X Bt4-R2 ♂ and five Bt4-R2 ♀ X F₁ ♂) were weighed and genotyped with allele-specific PCR.

Figure 4

Pink bollworm sampling sites in India. After widespread resistance to Cry2Ab was reported in India, Cry2Ab-resistant larvae were collected in 2015 and 2016 as fourth instars from dual-toxin cotton producing Cry2Ab and Cry1Ac from seven districts of four states: Ahmednagar and Jalna, Maharashtra (AM and JM), Karimnagar, Telangana (KT), Guntur, Andhra Pradesh (GAP), and Chitradurga, Raichur and Yadgir, Karnataka (CK, RK and YK) (red). Several years before resistance to Cry2Ab was detected in India, Cry2Ab-susceptible larvae were collected in 2010 from non-Bt cotton in Akola, Maharashtra (AMH) (green).