Gene knockout of P450s CYP392A16 and CYP392D2 reveals unexpected in vivo effects and transcriptional rewiring in the extreme generalist Tetranychus urticae.
Aug 2026· Insect Biochemistry and Molecular Biology· pp.
104666
· 0 citations· 73 references
Medicine
TL;DR
It is demonstrated that even resistance-associated detoxification genes that are considered functionally validated in vitro, can have strong context-dependent phenotypic effects in vivo, underscoring the complexity of resistance gene networks and highlighting the need to interpret CRISPR/Cas9 knockout phenotypes within the broader genetic and regulatory context.
Abstract
The two-spotted spider mite Tetranychus urticae rapidly evolves resistance to acaricides, frequently through the upregulation of detoxification enzymes such as cytochrome P450 monooxygenases. Among these, CYP392A16 and CYP392D2 have repeatedly been implicated as major resistance determinants, yet their in vivo contributions remain incompletely resolved. Here, we used efficient CRISPR/Cas9-mediated genome editing to generate stable knockouts of CYP392A16 and CYP392D2 in a multi-resistant genetic background. Contrary to expectations, loss of either gene resulted in surprisingly limited effects on acaricide susceptibility, with increased sensitivity observed only for a subset of compounds. Experimental evolution assays revealed a moderate, host-dependent fitness cost associated with disruption of CYP392D2, but not CYP392A16. Transcriptomic analyses further uncovered stark contrasts between the knockouts, with CYP392D2 loss triggering extensive regulatory reprogramming suggestive of compensatory detoxification pathways. Together, our results demonstrate that even resistance-associated detoxification genes that are considered functionally validated in vitro, can have strong context-dependent phenotypic effects in vivo, underscoring the complexity of resistance gene networks and highlighting the need to interpret CRISPR/Cas9 knockout phenotypes within the broader genetic and regulatory context.
Abstract It is well established that cytochrome P450 monooxygenases (P450s) play a crucial role in herbicide metabolism and resistance evolution in weeds. In most documented cases, P450-mediated resistance is primarily conferred through the overexpression of P450 enzymes. However, the regulatory mechanisms underlying this overexpression remain poorly understood. In insects, amino acid substitutions that enhance P450-mediated metabolic detoxification have been clearly demonstrated as a key mechanism of insecticide resistance. In contrast, their potential role in herbicide resistance in weeds remains unclear. In this study, two CYP96A146 variants from flixweed [Descurainia sophia (L.) Webb ex Prantl], designated CYP96A146-S and CYP96A146-R, were heterologously expressed in Saccharomyces cerevisiae. These variants, which differ by four amino acid residues, were examined for their ability to metabolize model substrates and herbicides. The results indicated that both variants exhibited catalytic activity toward model substrates of p-nitroanisole, methoxyresorufin, ethoxyresorufin, 7-ethoxycoumarin, and benzo[a]pyrene, as well as toward the herbicides tribenuron-methyl, bensulfuron-methyl, and carfentrazone-ethyl. Notably, CYP96A146-R showed significantly higher catalytic activity than CYP96A146-S to both the model substrates (p-nitroanisole, methoxyresorufin, ethoxyresorufin, and 7-ethoxycoumarin) and the herbicides (tribenuron-methyl and carfentrazone-ethyl). These findings suggest that the amino acid substitutions are likely responsible for the enhanced metabolic capability of CYP96A146-R. Such mutations may induce conformational changes of CYP96A146 enzyme, facilitating more frequent molecular collisions between CYP96A146 and the substrates or herbicides, thereby improving catalytic efficiency.
Zhiying Chen, Fan Xu, Yuxin Han et al.· Weed science· 0 citations
Pyridalyl is a novel insecticide with a unique mode of action, but resistance in Plutella xylostella is a significant challenge. To elucidate the resistance mechanism, we compared transcriptomes of susceptible (XY-PS) and laboratory-selected resistant (XY-PR) strains. The mitochondrial cytochrome P450 gene CYP12A2 emerged as a key candidate. RT-qPCR confirmed CYP12A2 was upregulated approximately 8-fold in XY-PR and 9-fold in a field-collected resistant strain (ZL-PR). Furthermore, RNA interference (RNAi) targeting CYP12A2 significantly increased larval susceptibility to pyridalyl. Recombinant CYP12A2 expressed in Sf9 cells specifically metabolized pyridalyl, producing a distinct hydroxylated metabolite identified via UPLC-MS/MS. These findings represent direct evidence of P450-mediated metabolic detoxification of pyridalyl. Ultimately, this unveils a novel biochemical adaptation in P. xylostella, establishing CYP12A2 as a critical biomarker for field resistance monitoring and a promising target for integrated insecticide resistance management (IRM) strategies.
Falong Wang, Chunyan Yin, Zhiyuan Qin et al.· Journal of Agricultural and...· 0 citations
Simple Summary Cytochrome P450s are crucial for insecticide detoxification, yet the molecular mechanism underlying P450-mediated pyrethroid detoxification in S. frugiperda remains largely unclear. Enzyme activity assays suggested the involvement of P450s in pyrethroid detoxification, with CYP9A58 and CYP9A59 being significantly induced by bifenthrin and lambda-cyhalothrin. RNAi-mediated knockdown of these genes significantly increased larval mortality upon lambda-cyhalothrin exposure. Homology modeling and molecular docking revealed that CYP9A58 and CYP9A59 can form stable complexes with both insecticides, showing notably stronger binding affinity for lambda-cyhalothrin. These findings enhance our understanding of P450 genes in insecticide stress responses and offer valuable insights into S. frugiperda control.
Dong-Liang Su, Wei-Fang Yang, Ya-Nan Fan et al.· Insects· 0 citations
Cytochrome P450 monooxygenases (P450s) core catalytic regulators of secondary metabolism and biotic stress defense across plants and fungi. The fungivore mite Tyrophagus putrescentiae is a major pest of cultivated mushrooms, but the molecular basis of fungal defense against this fungivore remains poorly understood. Here, we functionally identified and systematically characterized PoCYP5152C8, a CYP504 subfamily P450 gene from Pleurotus ostreatus that is transcriptionally responsive to mite infestation stress. Overexpression of PoCYP5152C8 in P. ostreatus increased mite survival, egg hatching, and population growth, while RNAi-mediated silencing enhanced fungal resistance. Integrated transcriptomic and metabolomic profiling revealed that PoCYP5152C8 specifically modulates the ubiquinone and other terpenoid-quinone biosynthesis pathway. In vitro enzymatic assays demonstrate that PoCYP5152C8 requires the reductase PoCPA15 to convert terpene substrates (FPP, GPP, and GGPP) into a suite of volatile terpenoids, including β-caryophyllene oxide, α-terpineol, and Z-citral. Behavioral assays identified several of these compounds as potent attractants for T. putrescentiae, with α-terpineol showing strong repellent activity at high concentrations. Our findings suggest that PoCYP5152C8 as a key metabolic node that redirects terpenoid flux toward attractant compounds, thereby reducing fungal defense. This study uncovers the enzymatic and molecular mechanism of a fungal neofunctionalized P450-mediated fungus-fungivore interactions, providing novel genetic targets for sustainable pest biocontrol in edible mushroom cultivation.
L. Yuan, S. Qu, Mengnan Niu et al.· Microbiology Research· 0 citations
Spider mites are globally important agricultural pests, many of which exhibit red body coloration associated with carotenoid accumulation. Although previous studies have implicated the cytochrome P450 gene CYP389B1 in pigmentation, its biochemical function remains unclear. Here, we functionally characterized CYP389B1 in Tetranychus pueraricola using an integrative approach combining heterologous expression, pigment profiling, and RNA interference. Heterologous expression of CYP389B1 in a ¦Â-carotene-producing Escherichia coli system resulted in increased levels of zeaxanthin, suggesting a role in carotenoid hydroxylation. Developmental expression analysis revealed that CYP389B1 level increased progressively and peaked in adults, coinciding with changes in carotenoid composition. RNAi-mediated knockdown of CYP389B1 led to the accumulation of ¦Â-carotene and a reduction in downstream zeaxanthin, supporting its involvement in carotenoid conversion. In addition, gene silencing was associated with reduced fecundity and survival. Together, these results suggest that CYP389B1 contributes to carotenoid metabolism and pigmentation in T. pueraricola, and may play a role in physiological processes linked to fitness. This study expands our understanding of the potential roles of cytochrome P450 enzymes in carotenoid-based coloration in arthropods, and offers a promising molecular target for RNAi-based pest management strategies.
Shi-Mei Huo, Xiao-Jin Zhang, Qiming Zhang et al.· Systematic and Applied Acaro...· 0 citations
Neonicotinoid insecticides are widely used to control sap-feeding pests, but their effectiveness is increasingly limited by cytochrome P450-mediated metabolic resistance. Here, we investigated the role of CYP4C64 in nitenpyram (NIT) detoxification in Bemisia tabaci through expression analyses, bioassays, heterologous expression, and in vitro metabolism assays. CYP4C64 was functionally expressed in insect cells using a Bac-to-Bac baculovirus system. UPLC–MS analysis showed that CYP4C64 catalyzed the conversion of NIT to IM-1-4, the major metabolite identified under the experimental conditions, which exhibited substantially lower toxicity than the parent compound. Enzyme kinetic analysis further confirmed the metabolic activity of CYP4C64 toward NIT. These findings provide direct biochemical evidence that CYP4C64 contributes to nitenpyram detoxification in B. tabaci and demonstrate that the Bac-to-Bac expression system is an effective platform for functional characterization of insect P450 enzymes involved in insecticide metabolism.
Wenhan Cheng, Hantang Lu, Chengjia Zhang et al.· Journal of Agricultural and...· 0 citations
