Parallel evolution of Bacillus thuringiensis toxin resistance in lepidoptera

Abstract

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function.

Figure 1

Bt resistance loci in Lepidoptera. Linkage maps for (A) P. xylostella and (C) T. ni in comparison with (B) B. mori’s sequenced chromosome 15 (partial). P. xylostella and B. mori show multiple chromosomal rearrangements while maintaining genetic synteny. Blocks of common genes are shaded and arrows depict inverted orientation in P. xylostella. T. ni and B. mori show a conserved gene order in this data set. Bt Cry1Ac resistance loci are in complete linkage with the ABCC2 gene. P. xylostella showed low levels of recombination across this region, and five mapped genes are in complete linkage with BtR-1. RpL8 also maps to BtR-1 (see Table 1), but was not polymorphic in these mapping crosses. Both linkage maps were constructed using JoinMap 3.0 (grouping = LOD 10, Kosambi’s mapping function). P. xylostella were fed on transgenic Bt Cry1Ac-expressing canola, and T. ni had purified Cry1Ac toxin incorporated into their artificial diet. Centimorgan distances may be affected by linkage disequilibrium caused by selection for resistance. B. mori gene identifiers refer to the final four numbers (note underlining) of the gene ID (e.g., BGIBMGA003337-TA). Accession numbers are provided in Table S1.

Figure 2

P. xylostella ABCC2 schematic. A P. xylostella BAC library was screened with partial gene sequence from ABCC2, which was identified from an EST library, and two clones were sequenced (25L19 and 11G15). P. xylostella ABCC2 genomic sequence was predicted on the basis of similarity with the H. virescens homolog and then confirmed through amplification from midgut cDNA, using backcross progeny that survived a Cry1Ac bioassay (Bt-resistant) or from the control strain Geneva88 (Bt-susceptible). (A) Partial amino acid alignment of ABCC2 of Bt-susceptible (JN030490) and Bt-resistant (JN030491) P. xylostella. A 30-bp, 10-amino-acid deletion is predicted within transmembrane (TM) domain 12. (B) Schematic of ABCC2 displaying 12 transmembrane domains predicted using Phobius (http://phobius.sbc.su.se/) and two nucleotide-binding domains (NBD). The 10-amino-acid deletion in Bt-resistant individuals is predicted to remove TM12, leaving the carboxyl terminus in a noncytoplasmic region (dashed line).