Shared and Independent Genetic Basis of Resistance to Bt Toxin Cry2Ab in Two Strains of Pink Bollworm

Abstract

Evolution of pest resistance threatens the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Field populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton, have evolved practical resistance to transgenic cotton producing Bt toxin Cry2Ab in India, but not in the United States. Previous results show that recessive mutations disrupting an autosomal ATP-binding cassette gene (PgABCA2) are associated with pink bollworm resistance to Cry2Ab in field-selected populations from India and in one lab-selected strain from the United States (Bt4-R2). Here we discovered that an independently derived, lab-selected Cry2Ab-resistant pink bollworm strain from the United States (BX-R) also harbors mutations that disrupt PgABCA2. Premature stop codons introduced by mis-splicing of PgABCA2 pre-mRNA were prevalent in field-selected larvae from India and in both lab-selected strains. The most common mutation in field-selected larvae from India was also detected in both lab-selected strains. Results from interstrain crosses indicate BX-R has at least one additional mechanism of resistance to Cry2Ab that does not involve PgABCA2 and is not completely recessive or autosomal. We conclude that recessive mutations disrupting PgABCA2 are the primary, but not the only, mechanism of resistance to Cry2Ab in pink bollworm.

Figure 1

Survival of pink bollworm larvae from two resistant strains (Bt4-R2 and BX-R), a susceptible strain (APHIS-S), and their F₁ progeny from mass crosses. Asterisks indicate 0% survival for APHIS-S and the F₁ progeny from the two reciprocal mass crosses between Bt4-R2 and APHIS-S. Light blue: 3 μg Cry2Ab per mL diet (n = 32 larvae per bar or asterisk). Dark blue: 10 μg Cry2Ab per mL diet (n = 48 per bar or asterisk).

Figure 2

Survival of pink bollworm larvae from single-pair crosses between resistant strains Bt4-R2 and BX-R tested at 10 μg Cry2Ab per mL diet. Families A-J (blue) from female Bt4-R2 X male BX-R and K-T (pink) from female BX-R X male Bt4-R2.

Figure 3

Mutations in 28 PgABCA2 cDNA sequences from Cry2Ab-resistant strain BX-R and Cry2Ab-resistant F₁ progeny from BX-R X Bt4-R2. (a) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (purple) and two transmembrane domains (TMD1 and TMD2). Each TMD contains six transmembrane-spanning regions (TM, orange), three extracellular loops (ECL, green), and two intracellular loops (ICL, blue). The two TMDs are connected by a single intracellular loop (ICL3). ICL3 and the C-terminal domain each contain a nucleotide-binding domain (NBD, yellow). (b) Mutations in PgABCA2 cDNAs from BX-R (three clones from each of three individuals: 1, 2 and 5) and resistant F₁ progeny from BX-R X Bt4-R2 (4-5 clones from each of four individuals: A1 and A3 from family A, N1 and N3 from family N, see Fig. 2) relative to the susceptible strain APHIS-S (MG637361). Numbers to the right of the decimal point for each individual indicate the clone sequenced. Exon numbers are shown for APHIS-S. Red triangles above bars indicate premature stop codons, which occur in all sequences except 2.12 from BX-R. Colors within bars show mutations: red for deletions, blue for substitutions, green for insertions and deletions, and orange for the single-bp insertion in exon 2 of N1.8 from the resistant F₁ progeny.