Knockout of SfVipR2 confers Vip3Aa resistance in Spodoptera frugiperda with evidence of genetic complementation among non-allelic mutations of SfVipR1, SfVipR2, and SfCHS2.

The Vip3Aa protein from Bacillus thuringiensis is widely deployed in transgenic crops to control lepidopteran pests, including the fall armyworm Spodoptera frugiperda, a highly invasive, polyphagous pest of global importance. Loss-of-function mutations in SfVipR1 (a thyroglobulin-like gene) and SfCHS2 (chitin synthase 2) have previously been confirmed to confer high-level, recessive resistance to Vip3Aa in S. frugiperda. A second thyroglobulin-like gene (HaVipR2) has recently been recognized as an important determinant of Vip3Aa resistance in Helicoverpa armigera. In this study, we first confirmed that knockout of SfVipR2 using CRISPR/Cas9 gene editing also confers high-level, recessive resistance to Vip3Aa. Then, three isogenic knockout strains (SfVipR1-KO established previously; and SfVipR2-KO and SfCHS2-KO, newly generated; all in the YJ-19 genetic background) were used to investigate functional relationships among SfVipR1, SfVipR2, and SfCHS2. Reciprocal crosses between any pair of these resistant strains yielded fully susceptible hybrid progeny, demonstrating complete genetic complementation and no epistasis among the non-allelic mutations. Disruption of any of these genes can result in high-level resistance to Vip3Aa, indicating a substantial resistance risk in S. frugiperda and underscoring the need for resistance management tactics such as establishing an adequate proportion of non-Bt host refuges and deploying pyramided crops expressing Vip proteins and Bt proteins with a different mode of action than Vip3Aa (e.g., Cry proteins).