Award details

Understanding the evolution and function of xenobiotic detoxification enzymes in a global crop pest

Reference	BB/X010058/1
Principal Investigator / Supervisor	Dr Bartlomiej Troczka
Co-Investigators / Co-Supervisors
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	394,633
Status	Current
Type	Fellowships
Start date	01/03/2023
End date	28/02/2026
Duration	36 months

Abstract

Cytochrome P450s are a superfamily of metabolic enzymes which play an important role in the detoxification of xenobiotics. In insect pests P450s play a key role in the metabolism of insecticides leading to the development of resistance. Thanks to the expansion in insect genomic resources, a large number of insect P450 genes have been identified. However, due to the time and effort required to express these genes, the function of the majority of insect P450s remains unknown, and our ability to predict which P450s have the capacity to detoxify insecticides remains poor. Using an economically important pest species, Myzus persicae as a case study, I will exploit recent breakthroughs in computational protein structure prediction and cell-free expression of recombinant protein to address these knowledge gaps. In the first objective of the project, I will gain insight into the function of individual aphid P450s by establishing when and where they are expressed using transcriptome profiling. I will then use novel tools (AlphaFold) to create 3D models of each protein and predict their ability to metabolise model substrates and insecticides. These predictions will be experimentally tested using cell-free expression systems to individually express each aphid P450 and biochemical assays to profile their metabolic activity. In the second objective I will leverage a new population genomic dataset for M. persicae to characterise standing genetic variation in P450 genes in this species and use 3D modelling and functional expression to understand the potential for this variation to be co-opted in the evolution of resistance. The data generated in this analysis and in objective 1 will be exploited to identify key structure-function determinants of P450-mediiated insecticide metabolism. In the final objective, I will translate the knowledge generated in the project into a molecular toolkit that will provide a powerful resource to identify resistance-breaking chemistry.

Summary

Detoxification enzymes play an important role in the ability of insects to neutralise threats caused by foreign chemicals (xenobiotics), such as plant secondary metabolites and synthetic pesticides. Indeed, insect pests, such as the aphid Myzus persicae, frequently adapt to novel host plants or develop resistance to synthetic insecticides via quantitative or qualitative changes in these enzymes. One of the most important superfamilies of enzymes involved in the evolution of insect resistance to plant-derived and human-made toxins are cytochrome P450s (or CYPs). M. persicae has over 60 individual genes encoding P450 enzymes (the CYPome), however, only a handful of these have been functionally characterised to date. This is a reflection of the time and effort required to produce functional protein for analysis in the lab. As a consequence, while we have a good understanding of the type and number of P450 genes in insects we know very little about what they actually do. In the context of pest species, key questions remain on which P450s have the capacity to detoxify insecticides, the extent to which genetic variation in these P450s in insect populations can influence insecticide metabolism and which of the building blocks (amino acids) that make up P450 proteins are particularly important in interacting with insecticides and breaking them down (structure-function determinants). In this project I aim to exploit a unique set of genomic resources recently created for M. persicae, in combination with recent advances in synthetic biology and modelling of protein structure, to understand how global diversity in an entire gene superfamily impacts the ability of M. persicae to adapt to natural and synthetic xenobiotics. In the first objective of the project, I will investigate where and when all the individual P450s in M. persicae are present through the insect body and life cycle in order to gain insight into their potential function. I will then use state of the art modelling tools to create 3D structures of each protein to gain an intimate understanding of how they interact with small molecules such as toxins and predict which P450s break down insecticides. These computational predictions will be tested in the laboratory utilizing recent advances in protein production to individually express the entire complement of P450 enzymes in M. persicae and examine their ability to metabolise insecticides and other model substrates using biochemical approaches. This will provide the first ever functional dataset on the complete suite of P450s in an insect, and unprecedented insights into the biological function of each member of the CYPome including those that are key actors in xenobiotic metabolism. In the second objective of the project, I will leverage a global sample of >110 re-sequenced clones of M. persicae, in combination with 3D modelling and functional expression to understand the potential for standing genetic variation in CYP genes in aphid populations to be co-opted in the evolution of resistance. The data obtained from these analyses, together with that derived from Objective 1, will allow me to identify key structure-function determinants of P450-mediated insecticide metabolism. The results obtained from the first two objectives of this project will provide fundamental insights into the evolution and function of an important group of enzymes that are found in all kingdoms of life, however, they also have the potential for significant applied impact. To realise this impact I will translate the knowledge gained into a set of molecular tools that can be used to rapidly screen compounds against aphid pest P450s and so identify resistance-breaking chemistry. In summary, the knowledge and tools generated in this study will provide i) a holistic understanding of the function and diversity of the insect CYPome and its relevance for the evolution of insecticide resistance; ii) a toolkit to study and combat P450-mediated resistance.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Fellowship - David Phillips Fellowship (DF) [1995-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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