Mechanisms of Pest Resistance to Second Generation Bt Crops - AGRICULTURAL RESEARCH SERVICE

Goals / Objectives
Evolution of pest resistance is the most serious risk threatening the continued efficacy of transgenic Bt crops. Understanding the underlying genetics and mechanisms of resistance to Bacillus thuringiensis Cry toxins is therefore critical for designing resistance management strategies and by identification of the gene(s) linked with resistance to Cry1Ac and Cry2Ab we will enhance fundamental understanding of resistance and toxin mode of action. Previous research on Bt toxins has primarily focused on Cry1 toxins used in first generation Bt crops and understanding of resistance to the Cry2Ab toxin now deployed widely in second generation Bt cotton and Bt corn is limited. The long-term goal of this project is to provide the fundamental knowledge needed to enhance the ability to design sound strategies for managing resistance to Bt crops. To address this goal, we will achieve the following specific objectives: Objective 1: Determine if resistance to Bt toxin Cry2Ab is genetically linked with ABCA2 in Pectinophora gossypiella; Objective 2: Compare the genetic basis of resistance to Cry2Ab among field- and lab-selected strains of pink bollworm; Objective 3: Determine if fitness costs are associated with resistance to Cry2Ab; Objective 4: Determine if CRISPR/Cas9 knockout of ABCA2 causes resistance to Cry2Ab in Pectinophora gossypiella and Helicoverpa zea. The understanding of resistance to second-generation Bt corn and Bt cotton pyramids in Helicoverpa zea (corn earworm) and Pectinophora gossypiella (pink bollworm) has immediate practical implications. Namely, by elucidating the molecular mechanisms of resistance to Bt toxins and associated fitness costs, this project will strengthen the scientific framework for sustainable, environmentally sound pest management.

Project Methods
Pest resistance is the major threat to the continued success of Bt crops. Knowledge of the genetics and mechanism of pest resistance to Bt toxins is essential for developing and evaluating new products that will be effective against resistant pests. Here we propose to characterize resistance to Cry2Ab in a key lepidopteran pest targeted by Bt crops, Pectinophora gossypiella (pink bollworm, PBW). The long-term goal of this project is to provide the fundamental knowledge needed to enhance the ability to design sound strategies for managing resistance to Bt crops. To address this goal, we will achieve the following specific objectives: We have recently generated strains of PBW extremely resistant to both Cry1Ac and Cry2Ab. Unlike all other resistant strains of PBW studied previously, these new resistant strains survive on pyramided Bt plants that produce Cry1Ac and Cry2Ab. With Cry2Ab increasingly important in corn, PBW resistance to Cry2Ab becomes especially relevant as a model for corn, particularly with the lack of basic knowledge about Cry2Ab receptors and resistance. By elucidating the genetics and mechanism of PBW resistance to Cry2Ab and extending this fundamental knowledge into Cry2Ab resistance in other important pests (namely the corn earworm, Helicoverpa zea), this project will provide information critical for understanding and countering pest resistance to currently deployed Bt crops. For objective 1, F1 progeny from single-pair reciprocal crosses between our Cry2Ab-resistant and susceptible strain will be backcrossed in single-pair matings to the resistant strain (as we know that inheritance of resistance is recessive). The resulting single-pair F2 families will be tested at a diagnostic concentration of Cry2Ab. Parents (F0), F1 progeny used as parents to generate F2 families, and their F2 progeny will be used to test the candidate gene ABCA2 for genetic linkage with Cry2Ab resistance. For objective 2, we will determine if field- and lab-selected strains of pink bollworm with resistance to Cry2Ab share common genetic basis. Namely, we will test if ABCA2 contains mutations from Cry2Ab-resistant individuals from U.S. lab-selected strains and those collected from India second-generation Bt cotton fields. For objective 3, we will evaluate fitness costs associated with two Cry2Ab resistant strains of PBW. Namely, artificial diet bioassays will be used to test survival and overall fitness of susceptible and Cry2Ab-resistant strains of PBW. Survival, development time, weight, pupation rates, eclosion rates, adult fecundity, etc. will serve as proxies for lifetime fitness of PBW. Comparisons will be made between strains on both untreated (control) and Cry2Ab-treated diet (at several concentrations). For objective 4, CRISPR/Cas9 will be used to knockout ABCA2 in PBW to establish that mutations to this gene can cause resistance to Cry2Ab in this important pest species. Our studies will provide valuable knowledge about the underlying mechanisms of resistance to Gt crops in an important pest of U.S. cotton. Such Information is critical for resistance monitoring and for the delay of the spread of resistance to this technology.