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, labselected 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. Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have revolutionized management of some of the world's most devastating pests 1. In 2018, millions of farmers in 15 countries grew more than 104 million hectares of transgenic Bt crops 2. Bt crops can suppress pests, increase yield and farmer profits, and decrease conventional insecticide use, which translates to health and environmental benefits 1,3-7. However, evolution of resistance to Bt crops by pests reduces such benefits 8-11. Practical resistance to Bt crops, which is field-evolved resistance that has practical consequences for pest management, has been documented in at least nine species of major pests targeted by Bt crops 8-11. Better understanding of the mechanisms and genetic basis of resistance to Bt toxins is needed to monitor, manage, and counter pest resistance. For Cry1 toxins, the first family of lepidopteran-active crystalline (Cry) Bt toxins deployed widely in transgenic crops, the molecular genetic basis of resistance has been studied extensively 12-16. However, much less is known about this issue for Cry2 toxins, the second such family. While each of the first Bt crops produced a single toxin from the Cry1 family (e.g., Cry1Ac), most Bt crops grown now produce Cry2Ab in combination with one or more Cry1 toxins 17. For lab-selected strains of the lepidopteran pests Helicoverpa armigera and Helicoverpa punctigera, resistance to Cry2Ab is associated with reduced binding to larval midgut membranes and linked with autosomal recessive mutations that disrupt the ATP-binding cassette protein ABCA2 18,19. Deleting portions o...

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab

“…Larva 18 had only one mutation in all three clones sequenced: an indel in exon 20 (c.3556_3588delins) that matches the r A1 mutation previously reported in the lab-selected Cry2Ab-resistant strain Bt4-R2 29 . Collectively, three of the five larvae (5, 10 and 18) from CRISPR-R2 that lacked gDNA mutations in sgRNA sites 1–5 had five cDNA mutations that do not correspond to sgRNA sites and were reported previously in Cry2Ab-resistant laboratory-selected strains from Arizona (Bt4-R2 and BX-R), field-selected populations from India, or both (c.1090_1234del, c.3097_3100del, c.3313_3589del, c.3418_3589del, and c.3556_3588delins; Table 2 ) 29 , 30 .…”

“…We discovered a novel mutation (c.4598_4599ins) in clone 6 from larva C.3 (clone C.3.6) that does not correspond to sgRNA target sites 1–5 (Fig. 2 ) and was not previously found in Cry2Ab-resistant pink bollworm 29 , 30 . Collectively, four F 2 survivors (A.1, A.2, C.3 and C.4) had the same five cDNA mutations seen in CRISPR-R2 that were previously found in Cry2Ab-resistant lab-selected strains from Arizona, field-selected populations from India, or both (Table 2 ).…”

“…h Source of larvae in the current study: CRISPR-R2, F 2 progeny of single-pair families from CRISPR-R2 X APHIS-S, or both. i No indicates not found in previous studies; other entries indicate the same cDNA mutation was previously found in one or more of the following sets of Cry2Ab-resistant insects: lab-selected Bt4-R2 and BX-R strains from Arizona and field-selected populations from India 29 , 30 .…”

“…However, this lepidopteran remains an important pest in many other countries and has evolved practical resistance to Cry1Ac and Cry2Ab in India 26 , 28 . Previous work shows that mutations disrupting PgABCA2 , the pink bollworm gene encoding ABCA2 protein, are associated with resistance to Cry2Ab in field-selected populations from India and in two lab-selected strains from the United States 29 , 30 . However, gene editing has not been used previously to determine if mutations in any pink bollworm gene can cause resistance to a Bt toxin.…”

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture

“…Mis-splicing mutants in ABCA2 were associated with Cry2Ab resistance in P. gossypiella in a laboratory-selected strain from Arizona and field populations from India [ 70 ]. Additional crosses confirmed these mutations but suggested that an additional, uncharacterized mechanism was also involved in Cry2Ab resistance in this species [ 71 ].…”