CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins |
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| Institution: | 1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;2. CSIRO, Black Mountain Laboratories, Canberra, Australian Capital Territory, Australia;1. USDA-ARS, Corn Insects & Crop Genetics Research Unit, Genetics Laboratory, Iowa State University, Ames, IA 50011, USA;2. Department of Entomology, Iowa State University, Ames, IA 50011, USA;3. Department of Entomology, University of Nebraska, Lincoln, NE, USA;1. School of Life Sciences, Central China Normal University, Wuhan 430079, PR China;2. State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China;1. Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;2. Sericultural Research Institute, Jiangsu University of Science and Technology, Zhengjiang 212018, Jiangsu, China;3. Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhengjiang 212018, Jiangsu, China;1. Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan;2. Japan Society for the Promotion of Science, Japan;1. Justus-Liebig-University Gießen, Institute for Insect Biotechnology, Department of Insect Biotechnology in Plant Protection, Winchesterstr. 2, 35394 Gießen, Germany;2. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Division of Bioresources, Department of Insect Pest and Vector Control, 35394 Gießen, Germany |
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| Abstract: | High levels of resistance to Bt toxin Cry2Ab have been identified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two lepidopteran insects, Helicoverpa armigera and Helicoverpa punctigera. To further confirm the causal relationship between the ABCA2 gene (HaABCA2) and Cry2Ab resistance in H. armigera, two HaABCA2 knockout strains were created from the susceptible SCD strain with the CRISPR/Cas9 genome editing system. One strain (SCD-A2KO1) is homozygous for a 2-bp deletion in exon 2 of HaABCA2 created by non-homologous end joining (NHEJ). The other strain (SCD-A2KO2) is homozygous for a 5-bp deletion in exon 18 of HaABCA2 made by homology-directed repair (HDR), which was produced to mimic the r2 resistance allele of a field-derived Cry2Ab-resistant strain from Australia. Both knockout strains obtained high levels of resistance to both Cry2Aa (>120-fold) and Cry2Ab (>100-fold) compared with the original SCD strain, but no or very limited resistance to Cry1Ac (<4-fold). Resistance to Cry2Ab in both knockouts is recessive, and genetic complementary tests confirmed Cry2Ab resistance alleles are at the same locus (i.e. HaABCA2) for the two strains. Brush border membrane vesicles (BBMVs) of midguts from both knockout strains lost binding with Cry2Ab, but maintained the same binding with Cry1Ac as the SCD strain. In vivo functional evidence from this study demonstrates knockout of HaABCA2 confers high levels of resistance to both Cry2Aa and Cry2Ab, confirming that HaABCA2 plays a key role in mediating toxicity of both Cry2Aa and Cry2Ab against H. armigera. |
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| Keywords: | Cry2Aa Cry2Ab Bt resistance ABC transporter CRISPR/Cas9 |
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