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The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Divergence of gene function is a hallmark of evolution, but assessing such divergence in one species or between species requires information on functional alterations of the alleles and homologs. Here, we explore the functional divergence of two paralogs, CYP6AE19 and CYP6AE20, from Helicoverpa armigera, and two close orthologs, CYP6B8 and CYP6B7, from two related species (Helicoverpa zea and H. armigera); although there is high sequence identity within each pair of enzymes, the latter P450 of each pair has lost metabolic competence towards the plant allelochemical xanthotoxin. Multiple chimeric and single/double site mutants were created by exchanging the diverse substrate recognition sites (SRSs) and amino acids within each pair of P450s. Heterologous expression in Sf9 cells and in vitro metabolism studies showed that the exchange of SRS4 swapped the activity of CYP6AE19 and CYP6AE20, and subsequent site-directed mutagenesis demonstrated that the CYP6AE20 V318M substitution causes a gain-of-function towards xanthotoxin. Meanwhile, a single amino acid substitution (L489P) in SRS6 was found to swap activity between the CYP6B orthologs. Sequence alignments of CYP6AE paralogs and all reported insect xanthotoxin-metabolizing P450s suggest M318 and P489 are essential for the catalytic activities of CYP6AE paralogs and CYP6B orthologs, respectively, but P450s in different subfamilies may have different mechanisms towards the same substrate. Our findings demonstrate that a single amino acid substitution can suffice to alter substrate metabolism and this functional divergence resulting from natural mutations will help to further our understanding of the process of natural selection of P450 genes and their role in insect-host plant interactions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Insect CYP2 and mitochondrial clan P450s are relatively conserved genes encoding enzymes generally thought to be involved in biosynthesis or metabolism of endobiotics. However, emerging evidence argues they have potential roles in chemical defense as well, but their actual detoxification functions remain largely unknown. Here, we focused on the full complement of 8 CYP2 and 10 mitochondrial P450s in the generalist herbivore, Helicoverpa armigera. Their varied spatiotemporal expression profiles were analyzed and reflected their specific functions. For functional study of the mitochondrial clan P450s, the redox partners, adrenodoxin reductase (AdR) and adrenodoxin (Adx), were identified from genomes of eight insects and an efficient in vitro electron transfer system of mitochondrial P450 was established by co-expression with Adx and AdR of H. armigera. All CYP2 clan P450s and 8 mitochondrial P450s were successfully expressed in Sf9 cells and compared functionally. In vitro metabolism assays showed that two CYP2 clan P450s (CYP305B1 and CYP18A1) and CYP333B3 (mito clan) could epoxidize aldrin to dieldrin, while CYP305B1 and CYP339A1 (mito clan) have limited but significant hydroxylation capacities to esfenvalerate. CYP303A1 of the CYP2 clan exhibits high metabolic efficiency to 2-tridecanone. Screening the xenobiotic metabolism competence of CYP2 and mitochondrial clan P450s not only provides new insights on insect chemical defense but also can give indications on their physiological functions in H. armigera and other insects., (Copyright © 2021 Elsevier Ltd. All rights reserved.)