Periodic Reporting for period 3 - P450RESIST (Understanding and exploiting the insect P450 resistome)
Reporting period: 2019-01-01 to 2020-06-30
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.
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.