Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - P450RESIST (Understanding and exploiting the insect P450 resistome)
Teaser
Around 20% of crops worldwide are lost to insect pests and their effective management is essential to ensure food security for present and future generations. One of the most tangible threats to the sustainability of crop protection is insect resistance to natural and...
Summary
Around 20% of crops worldwide are lost to insect pests and their effective management is essential to ensure food security for present and future generations. One of the most tangible threats to the sustainability of crop protection is insect resistance to natural and synthetic xenobiotics such as insecticides and plant defence chemicals. The evolution of resistance to plant defence chemistry may allow insects to become crop pests, can pre-adapt them to resist insecticides (see description of our recent work below) or may be rapidly recruited to do so. Insect resistance to insecticides can result in dramatic crop yield losses, lead to wasteful and ineffective insecticide treatments and may prompt a return to older chemicals with less favourable environmental profiles. The cytochrome P450s are a superfamily of enzymes that are ubiquitous in nature, and one of the most important enzyme families used by insects to defend themselves against natural and synthetic xenobiotics. Insects have been shown to evolve resistance through quantitative changes in P450 expression or via qualitative changes in P450s that alter metabolic activity. Despite their importance in conferring resistance the variety of regulatory changes that modulate P450 expression in resistant insects and their relative frequency/impact is not fully understood. Furthermore, although qualitative changes in insect P450s associated with resistance are relatively rare they represent a unique opportunity to characterise insecticide/toxin binding and identify the critical structure/function determinants of the P450/insect toxin interaction.
The aim of the P450RESIST project is to exploit recent advances in genomics, epigenetics and transgenics to study the insect P450 resistome in two economically important insect crop pests, the peach potato aphid, Myzus pericae and the brown planthopper, Nilaparvata lugens, in three main workpackages:
WP-1: Will identify the molecular drivers of quantitative changes to insect P450s.
WP-2: Will explore the role of qualitative changes in insect P450s in mediating resistance and identify structure/function determinants of insecticide metabolism.
WP-3: Will exploit the knowledge gained in WP1/2 and from previous research to deliver a ‘P450 toolkit’ consisting of in vitro and in vivo screening tools, with which to identify resistance breaking chemistry, and high-throughput diagnostics for use in resistance management.
In summary this project will provide novel insights into this important enzyme family and provide tools that can be used to develop new products and strategies that slow, prevent, or overcome resistance and so ensure sustainable crop protection.
Work performed
Our work to date has characterised how cytochrome P450s confer resistance to natural and synthetic insecticides in both brown planthopper and peach potato aphid. In both cases we find that gene duplication/amplification plays a major role in co-opting P450 genes to resist insecticides.
Gene duplication is a major source of genetic variation and has been implicated in the evolution of a range of adaptive traits. In our work we observed profound genetic variation in the loci encoding CYP6ER1, a P450 enzyme, in field strains of the brown planthopper that are resistant to the insecticide imidacloprid. We used a range of modelling, biochemical, and transgenic approaches to show that this P450 is duplicated in resistant strains. Remarkably we found that one of the copies has acquired mutations that confer a novel function on the encoded enzyme – the ability to metabolise the insecticide imidacloprid (a major means of hopper control throughout Asia). This is a compelling example of neofunctionalization – a process by which a gene acquires a new function after a gene duplication event. Interestingly this appears to have arisen independently in different geographic populations of brown planthopper in Southeast Asia and India. Although individual resistant hoppers carry a copy of CYP6ER1 with the gain-of-function mutations and one without they only overexpress the resistant copy. We used a novel gene capture approach to explore the genomic architecture of different ER1 copies and identify the promoter sequences (the region of DNA that directs transcription of a gene) of each. This revealed a clear breakpoint in the sequence upstream of the major resistant CYP6ER1 variant with the sequence completely diverging after this point, strongly suggesting the resistant paralog occurs in a novel genomic context. We explored the effect of this using reporter gene assays and showed that cis-acting elements in this region result in a ~10-fold increase in expression. Thus our work provides a novel example of the evolution of metabolic resistance by gene duplication and neofunctionalization and highlights the versatility of gene duplication in providing dual opportunities for both functional and regulatory innovation during the evolution of key adaptive traits.
Our parallel work on the peach potato aphid has also demonstrated the role of gene duplication in resistance in this species. We found that several P450s are amplified in a tandem array in clones of aphids that are resistant to nicotine a potent natural insecticide produced by certain plants and neonicotinoids (synthetic derivatives of nicotine). In contrast to P450 duplication in hoppers, all P450 copies are identical and resistance results from increased production of the protein and the resulting increased metabolism of the insecticide. Additional mutational events have increased the expression of these resistance genes to even higher levels and we are currently characterising the sequence of these adaptive events and so tracing the evolutionary steps involved.
The key findings detailed above are already being translated into tools that can be used to overcome resistance. These comprise an in vitro and in vivo P450 screening toolkit which will allow the testing of future insecticidal compounds and P450 inhibitors in order to identify resistance breaking chemistry, and 2) high throughput DNA-based diagnostics which can be used to monitor the frequency and distribution of resistance as part of strategies to slow or delay the development and spread of resistance.
Final results
Our work has characterised P450 genes which play a key role in the resistance of two global crop pests to insecticides while providing fundamental insights into rapid adaptive evolution. Over the remainder of the project we plan to examine in more detail the mutations involved. We will also explore the role of epigenetic mechanisms (heritable changes in gene expression that do not involve changes to the underlying DNA sequence) in resistance using whole genome approaches. Furthermore, in addition to characterising the role of P450s in resistance, the large scale genomic and transcriptomic datasets we have generated provide an exceptional opportunity to explore what other genes play a role in resistance in these species and characterise at a genomic scale the mutational events involved.