Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Authors: Fabrick, Jeffrey; HEU, CHAN - Oak Ridge Institute For Science And Education (ORISE); Leroy, Dannialle; DEGAIN, BEN - University Of Arizona; YELICH, ALEX - University Of Arizona; UNNITHAN, GOPALAN - University Of Arizona; WU, YIDONG - Nanjing Agricultural University; LI, XIANCHUN - University Of Arizona; CARRIERE, YVES - University Of Arizona; TABASHNIK, BRUCE - University Of Arizona

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/21/2022
Publication Date: 10/6/2022
Citation: Fabrick, J.A., Heu, C.C., LeRoy, D.M., DeGain, B.A., Yelich, A.J., Unnithan, G.C., Wu, Y., Li, X., Carriere, Y., Tabashnik, B.E. 2022. Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea. Scientific Reports. 12. Article 16706. https://doi.org/10.1038/s41598-022-21061-2.
DOI: https://doi.org/10.1038/s41598-022-21061-2

Interpretive Summary: Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage many economically important insect pests. However, the evolution of pest resistance to Bt crops decreases their effectiveness and lessens their many benefits. Better understanding of the genetic basis of resistance to Bt crops is needed to monitor, manage, and counter pest resistance to Bt crops. Helicoverpa zea is a major New World pest of many crops and has evolved practical resistance to Bt corn and cotton producing Cry1 and Cry2A toxins throughout much of the southern United States. Previous work shows that in several moth species, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. Here, an ARS scientist at Maricopa, AZ and collaborators show that mutations introduced by CRISPR/Cas9 gene editing in the H. zea gene encoding ABCA2 (HzABCA2) causes resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant H. zea strains with one having more than 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2 with the most common mutation consisting of 4-bp deletion in the expected Cas9 guide RNA target site. The results demonstrate that HzABCA2 mutations can cause high levels of resistance to Cry2Ab, and that this gene is a leading candidate for monitoring H. zea for Cry2Ab resistance in the field.

Technical Abstract: Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had >200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in the field.