Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

Abstract

The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Fig 1. Tests of the association of AFLP linkage groups with Cry2Ab1 resistance in a bioassay of backcross progeny.

The signed interaction χ² value shows the positive or negative association of the AFLP linkage group from the resistant grandparent with resistance in the progeny. Horizontal lines depict the Bonferroni-corrected probability values of P = 0.05, P = 0.005, and P = 0.001. Only AFLP linkage group 8 shows a significant positive association with resistance.

Fig 2. Linkage map of B. mori Chromosome 17 (BmChr17), showing syntenic genes used in mapping of H. armigera.

Approximate recombination rates between genes are provided in relation to the putative Cry2Ab resistance gene, indicated by ‘R’. The B. mori scaffolds on BmChr17 are also provided as obtained from KAIKObase <http://sgp.dna.affrc.go.jp/KAIKObase/>.

Fig 3. Predicted protein sequence of HaABC2.

Feature predictions include 12 transmembrane helices (TM I to XII), two ATP-binding domains ATP1 and ATP2, two transporter motifs TpM1 and TpM2, and predicted glycosylation sites (highlighted in blue, and indicated by ‘Y’). Amino acid differences between H. armigera and H. punctigera are indicated by ‘.’ mutation sites are indicated by red and blue arrows for H. armigera and H. punctigera respectively—Ha2Ab-R01 (amino acid position 964); Ha2Ab-R02 (amino acid position 1,043); Ha2Ab-R03 (amino acid position 1,368); and Hp2Ab-R04 (amino acid position 1,504).

Fig 4. A summary of the three Cry2Ab resistance alleles (Ha2Ab-R01, R02, R03) in the ABCA2 gene in Helicoverpa armigera, and one H. punctigera Cry2Ab resistance allele (Hp2Ab-R04).

The ABCA2 gene consists of 31 exons (Fig 4a), with mutations at exons 16, 18, 24 and 27 indicated by red (for H. armigera) and blue (for H. punctigera) boxes. Fig 4b: The Ha2Ab-R01 allele was the result of a 73 base pair (bp) deletion at the c-terminus of exon 16, and the insertion of a 8bp ‘CGGTTAAG’ sequence, and resulted in a glycine (G) replacing the leucine (L) amino acid followed by a premature stop codon (*) (in red). The Ha2Ab-R02 allele was the result of a 5bp ‘ACAAG’ deletion at the start of exon 18 that resulted in a premature stop codon. The Ha2Ab-R03 allele was the result of a 5bp ‘GAATA’ target site duplication signature reminiscent of past insertions by transposable elements. This duplication mutation also resulted in a premature stop codon in exon 24. The H. punctigera Hp2Ab-R04 allele was the result of a 14bp deletion, and resulted in missense mutations.

Fig 5. Diagram of the ABCA2 protein structure and location of mutations in H. armigera and H. punctigera.

Glycosylation sites on the two large extracellular loops are represented by ‘Y’. Two highly conserved ATP Nucleotide Binding Folds (NBF1, NBF2) that included the Transporter signature motifs 1 and 2 (TpM1, TpM2) are present in the intracellular environment. The Helicoverpa ABCA2 protein structure consists of two transmembrane domains (TMD 1, TMD 2), each with six transmembrane helices (TM I-VI in TMD 1; TM VII-XII in TMD 2). The approximate positions of the mutations in H. armigera and H. punctigera are indicated by red and blue arrows, respectively.

Fig 6. Phylogenetic tree showing clustering of ABCA1 and ABCA2 proteins of various Lepidoptera, with Drosophila homolog.

Species abbreviations are: Bmor, Bombyx mori; Harm, Helicoverpa armigera; Hvir, Heliothis virescens; Pxyl, Plutella xylostella; Dple, Danaus plexippus; Hmel, Heliconius melpomene; Dmel, Drosophila melanogaster. The tree is based on the alignment shown in S1 Fig