Transposon insertion causes cadherin mis-splicing and confers resistance to Bt cotton in pink bollworm from China

Abstract

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively, but rapid evolution of resistance by pests reduces their efficacy. We report a 3,370-bp insertion in a cadherin gene associated with resistance to Bt toxin Cry1Ac in the pink bollworm (Pectinophora gossypiella), a devastating global cotton pest. We found the allele (r15) harboring this insertion in a field population from China. The insertion is a miniature inverted repeat transposable element (MITE) that contains two additional transposons and produces two mis-spliced transcript variants (r15A and r15B). A strain homozygous for r15 had 290-fold resistance to Cry1Ac, little or no cross-resistance to Cry2Ab, and completed its life cycle on Bt cotton producing Cry1Ac. Inheritance of resistance was recessive and tightly linked with r15. For transformed insect cells, susceptibility to Cry1Ac was greater for cells producing the wild-type cadherin than for cells producing the r15 mutant proteins. Recombinant cadherin protein occurred on the cell surface in cells transformed with the wild-type or r15A sequences, but not in cells transformed with the r15B sequence. The similar resistance of pink bollworm to Cry1Ac in laboratory- and field-selected insects from China, India and the U.S. provides a basis for developing international resistance management practices.

Figure 1

Predicted cadherin protein in pink bollworm strain JL46. The amino-terminal membrane signal sequence (S), cadherin repeats (1–12), membrane proximal region (MPR), transmembrane region (T), and cytoplasmic domain (C) are shown. Red numbers indicate deletions in the cDNA from r15A (303 bp) and r15B (193 bp). The red triangle indicates the truncation of the protein predicted from r15B because of the premature stop codon (red letters TGA).

Figure 2

Map of the PgCad1 r15 mutation. (A) r15 allele of PgCad1. The 3,370-bp insertion in exon 28 contains remnants of three nested transposons, each shown as a colored triangle. (B) Details of the three nested transposons. The primary structure of each transposon is drawn to scale. Gray arrows show target site duplications (TSDs) (sequences below the arrows). Blue arrowheads represent the 31-bp terminal inverted repeats (TIRs). (AYG)₄(ACAT)₄ in RTE-5_PGo and (TTAY)₄ in SINE-1_PGo are microsatellite repeats.

Figure 3

Cellular localization of PgCad1 proteins within Hi5 cells. Hi5 cells transfected with pIE2-sPgCad1-GFP (a–d), pIE2-r15APgCad1-GFP (e–h), or pIE2-r15BPgCad1-GFP (i–l). Nuclei stained with Hoechst 3342 are shown in blue, dsRED-labeled endoplasmic reticulum shown in red, and GFP-labeled PgCad1 fusion proteins are shown in green. Superimposed images from (a–c) are shown in (d), from (e–g) in (h), and from (i–k) in (l). The arrow in (d) indicates cell membrane. Bar = 20 μm.

Figure 4

Susceptibility to Cry1Ac of Hi5 cells producing PgCad1 proteins. Hi5 cells transfected with pIE2-sPgCad1-GFP (a–d), pIE2-r15APgCad1-GFP (e–h), pIE2-r15BPgCad1-GFP (i–l) or the empty vector pIE2-GFP (m–p) were treated with Cry1Ac (10 μg Cry1Ac per ml for cells producing sPgCad1-GFP and 40 μg Cry1Ac per ml for r15A- and r15BPgCad1-GFP and GFP cells) and observed for swelling using fluorescence microscopy. Nuclei stained with Hoechst 3342 are shown in blue and PgCad1-GFP fusion proteins are shown in green. Superimposed images from (a,b) are shown in (c), from (e,f) in (g), from (i,j) in (k) and from (m,n) in (o). Arrows in (d) indicate representative swollen cells. Bars shown in (d,h,l,p) = 200 μm.