Entomology & Plant Path - RES
Non Technical Summary
Evolution of resistance is the most serious threat to sustainability of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt), which represent >80% of the corn and cotton grown in the USA. The fall armyworm is a devastating pest that has rapidly developed resistance to the Cry1Fa Bt toxin produced by transgenic corn in several locations in the USA, Brazil and Argentina. We have identified an ATP Binding Cassette subfamily C2 (ABCC2) gene in fall armyworm (SfABCC2) as a Cry1Fa receptor, and mutations in this gene linked to resistance to Cry1Fa corn. In this project, we will elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility (Objective 1) and use targeted next generation sequencing to screen up to 6,000 fall armyworm from locations in the USA, Brazil, Colombia and Africa for mutations in the SfABCC2 gene. Expected outcomes are the identification of regions in the SfABCC2 protein that are critical for Cry1 toxicity, and the deployment of an innovative and highly sensitive system for monitoring resistance to transgenic Bt crops.
Animal Health Component
Research Effort Categories
Goals / Objectives
The goal of this project is to develop sensitive high throuhput DNA-based methods to screen for resistance to Cry insecticidal proteins from the bacterium Bacillus thuringiensis, which are presently the most used biological insecticides and are also produced by widely used transgenic crops. The project will test the role of the ABCC2 protein as a functional receptor for Cry toxins, and determine the frequency of candidate resistance alleles against Cry toxins in field populations of the fall armyworm (Spodoptera frugiperda) in the American and African continents. The project includes the following objectives:Objective 1- Elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility. The proposed work includes in vitro tests to determine Cry toxin binding sites in the ABCC2 protein from fall armyworm (SfABCC2), and testing the identified regions for their receptor functionality using insect cell cultures and editing of the SfABCC2 gene using the CRISPR/Cas9 system.Objective 2- Develop a highly sensitive, high throuhgput, multiplexed targeted sequencing method for screening of resistance to Cry toxins. This method will then be used to screen and identify candidate resistance alleles to Cry toxins in the SfABCC2 gene of fall armyworm collected in the USA, Brazil, Colombia, and African countries where the fall armyworm has been reported as a devastating invasive species.
The project will build on preliminary data findings to identify functional interactions between Cry1 toxins and the SfABCC2 receptor (Objective 1) and to deploy a high throughput DNA sequence-based technology to screen fall armyworm populations, on a large scale, for mutations in the SfABCC2 gene as candidate for resistance alleles (Objective 2). In the fist Objective we will express truncated forms of SfABCC2 and test their Cry1 binding to locate the toxin binding site/s. Relevance of the identified binding regions for Cry1 toxicity will be evaluated using expression of wild type and modified SfABCC2 proteins in insect cell cultures and determining their susceptibility to Cry1 toxins. Relevance to in vivo toxicity will be evaluated through genome editing of SfABCC2 in fall armyworm using the CRISPR/Cas9 system. These efforts will result in identification of critical SfABCC2-Cry1 toxin interactions, leading to a change in knowledge and identifying targets in the development of improved insecticidal proteins.In Objective 2 we will adapt a next generation sequencing technology (Hi-Plex) to sequence the SfABCC2 gene in thousands of field-collected fall armyworm from populations in the American and African continents. Gene sequences will be used in predictive software to identify and evaluate disruptive mutations and mutations affecting regions critical to interactions with Cry1 toxins as identified in Objective 1. These efforts will allow determination of the relative frequency of candidate resistance alleles in fall armyworm populations of diverse geographic origin, leading to a change in knowledge by detecting field-evolved resistance alleles in a pest that is emerging as a threat to the use of transgenic corn technology in the Western hemisphere. More importantly, efforts to develop high throughput technologies to detect and quantify the risk of evolution of resistance tackle the current lack of sensitive DNA-based techniques for resistance monitoring.