CYP6AE gene cluster knockout in Helicoverpa armigera reveals role in detoxification of phytochemicals and insecticides

Abstract

The cotton bollworm Helicoverpa armigera, is one of the world's major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.

Fig. 1

CRIPSPR-Cas9-based knock out of the CYP6AE cluster of H. armigera. a Positions of the two sgRNAs (sgRNA14 and sgRNA12) and the four primer pairs for allele-specific PCR detection. b Target sequences of the two sgRNAs (blue), the PAM sequences (red), and a representative chromatogram of direct sequencing of PCR products of individuals from the SCD-d6AE strain with the primer pair 14F/12R. The cutting sites by the Cas9 protein are indicated with red triangles. c Genotyping of individual H. armigera for deletion of the CYP6AE cluster according to banding patterns of the PCR products amplified with a set of four primer pairs

Fig. 2

Responses to phytochemical toxins and insecticides (SCD-d6AE relative to SCD). The SCD-d6AE strain was derived from SCD by knocking out the CYP6AE cluster. Increased folds in susceptibility were calculated as LC₅₀ of SCD/LC₅₀ of SCD-d6AE. LC₅₀ values were considered significantly different if their fiducial limits did not overlap, and asterisks indicate statistical significance of LC₅₀s between the two strains. Phytochemical toxins: xanthotoxin, gossypol acetate, 2-tridecanone, nicotine, and coumarin. Insecticides: esfenvalerate, indoxacarb, emamectin benzoate, and chlorantraniliprole

Fig. 3

Metabolism of xanthotoxin, 2-tridecanone, and indoxacarb by recombinant P450s. CYP6AE subfamily enzymes were from H. armigera and CYP6B1 from Papilio polyxenes. Error bars represent mean values ± SEM (n = 4). Asterisk indicates that no significant metabolism was detected (limits of detection were 0.081, 0.033, and 0.016 pmol/min/pmol P450 for xanthotoxin, 2-tridecanone, and indoxacarb, respectively)

Fig. 4

Effects of diverse xenobiotics on mRNA levels of CYP6AE subfamily genes. The midgut and fat body of the H. armigera were from the SCD strain of H. armigera. The mRNA levels were determined by RT-qPCR. Each gene has 5 biological repeats. The heat map was generated using the log2-transformed expression ratios relative to the midgut mRNA expression of CYP6AE12 in the control group. Blue: low expression levels. Red: high expression levels