Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

Abstract

BACKGROUND The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. RESULTS Here, we successfully constructed a homozygous strain (Pxα6‐KO) with a 2‐nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non‐homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3–TM4 loop region. When compared to the background strain IPP‐S, the knockout strain Pxα6‐KO exhibited 229‐ and 1462‐fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3‐fold) effects on the toxicities of imidacloprid, abamectin, β‐cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2‐nt deletion mutation of nAChRα6 in the Pxα6‐KO strain. CONCLUSION In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6‐KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry

Functional validation of nicotinic acetylcholine receptor (nAChR) α6 as a target of spinosyns in Spodoptera exigua utilizing the CRISPR/Cas9 system

“…The genetically modified Seα6‐KO strain exhibits high levels of resistance to both spinosad and spinetoram compared with the WH‐S strain. Similar levels of resistance to spinosyns were observed in D. melanogaster and P. xylostella by using the CRISPR/Cas9 system.…”

CRISPR-Mediated Knockout of the ABCC2 Gene in Ostrinia furnacalis Confers High-Level Resistance to the Bacillus thuringiensis Cry1Fa Toxin

“…In the present work, the obtained Cry1Fa resistance was confirmed to be genetically associated with the 8-bp deletion of OfABCC2, which excludes the CRISPR-mediated off-target effects on resistance phenotype. We analyzed 18 research cases that employed the CRISPR/Cas9 system to manipulate the resistance genes to Bt toxins or insecticides, and found that only five of them performed linkage analysis between acquired resistance and the introduced mutation, including the knockout of the cadherin gene in H. armigera [40], nicotinic acetylcholine receptor α6 subunit in P. xylostella and S. exigua [41,42], the ryanodine receptor G4946E mutation in Drosophila melanogaster [43], and a CYP9M10 gene in Culex quinquefasciatus [44]. We therefore recommend that when CRISPR-based gene editing is conducted to verify the function of a candidate gene, it is necessary to perform a genetic linkage analysis in order to clarify whether there are off-target effects.…”

CRISPR‐mediated gene knockout reveals nicotinic acetylcholine receptor (nAChR) subunit α6 as a target of spinosyns in Helicoverpa armigera

“…The genetically modified Haα6‐KO strain exhibits high levels of resistance to both spinosad and spinetoram compared with the parent SCD strain. Comparable levels of resistance to spinosyns are also observed in the α6 knockouts of D. melanogaster , P. xylostella and S. exigua established by using the CRISPR/Cas9 technology. Further, we investigated the role of Haα7 of H. armigera in spinosyn toxicity, but found knockout of Haα7 had no effect on susceptibility to spinosyns and the other four insecticides.…”