Determine the primary responses of target parasites to xenobiotic compounds utilising the model organism C. elegans

Organisms have mechanisms to detoxify exposure to many compounds that are foreign and might be detrimental to them (xenobiotics). Understanding this mechanism is central to defining interactions between plant products ingested by pests and herbivores and also to the rational design pesticides. It is the second research field that is the focus of the current proposal for an industrial partner award. The project focuses on specific compounds with different modes of action and seeks to determine the pathways that metabolise each example. Each pathway is likely to operate in a hierarchical nature with a few key genes, termed transcription factors, being turned on after exposure to one or more xenobiotics. These genes will then in turn switch on those that encode products that metabolise the xenobiotic. We have already determined the likely identity of some of these transcription factors for this project. We will establish the involvement of particular transcription factors in assays using the small, free-living nematode Caenorhabditis elegans. We will knock-out the function of the transcription factors and determine if this enhances toxicity of particular xenobiotics. This will demonstrate that the ablated gene's function had a role in coordinating the metabolism of that chemical. We will then screen all the genes in the worm with microarray technology to identify those that are regulated by these upstream, transcription factors. This part of the work has substantial support from Syngenta. We will generate reporter lines of C. elegans that respond to specific xenobiotic(s) in real time to evaluate the time course of responses. Such nematodes have high potential for use later in rapid throughput screens of lead compounds and synthesised variants of them. The transcription factors must somehow perceive the xenobiotics to which they respond. This interaction could be direct and there are examples of this in the literature. We will clone our transcription factor genes and generate their protein products in bacteria before determining if the transcription factor protein does binds to the xenobiotic. We will also look at a greater diversity of compounds that are related to our focus chemicals. These compounds will by synthesised by Syngenta to have key chemical group substitutions. This will allow us to determine the nature of the chemical structure that elicits a particular metabolic response. The work centres on C. elegans. It is a useful model organism because of detailed genetic and other knowledge accumulated about it. It is used for these reasons by Syngenta in its search for lead compounds for pesticide development. Its use in this work will assure rapid progress but we will also carry out translational research to a pest with high economic impact in UK agriculture. We have chosen Globodera pallida as potato cyst nematodes are the major nematode pest of UK agriculture costing our potato industry an estimated £50 m/year. Nematicides are the largest variable cost of production for UK potato growers. Their potential global market is appreciable given nematodes cause estimated annual losses to global agriculture of $125b. Current nematicides are harmful both to the environment and possibly human health and several have already been removed from the market for these reasons. There is a large market opportunity for effective but environmentally benign compounds. The work is timely as it will be linked directly to the BBSRC-funded G. pallida sequencing project led by the PI of this proposal in collaboration with Sanger Centre, RES and SCRI. The work will provide information that will be valuable in the design and evaluation of not just nematicides but also insecticides given the central role of C. elegans in product development. These new products will help deliver food security and safeguard the environment.

This project will determine the primary responses of nematodes to xenobiotics utilising the model organism Caenorhabditis elegans. This underpins both understanding of the metabolism of xenobiotics by other target and non-target animals and the design of new pesticides. We will determine those transcription factors required by C. elegans to initiate metabolic detoxification of xenobiotics. Foundation studies have defined a set of xenobiotics with dissimilar modes of action and the candidate transcription factors they induce. We will also study further examples from the literature. Their involvement will be confirmed from altered toxicity of a xenobiotic in bioassays with C. elegans that are null mutants for the transcription factor (available from reference stocks). The metabolic genes that the transcription factors regulate will be identified using comparative microarray analysis of wild-type and the mutant C. elegans lacking expression of a transcription factor gene. The literature describes transcription factors that bind directly to the chemicals for which they coordinate a response. Recombinant bacteria will be used to obtain heterologously-expressed transcription factors and their binding to the xenobiotic determined by Surface Plasmon Resonance. Structural modifications the xenobiotic provided by our industrial partner will enable the chemical specificity of any binding and recognition to be defined. The information gained from C. elegans will be used in translational research to characterise the nature of xenobiotic responses in a target organism, the potato cyst nematode Globodera pallida. We will identify orthologues of the C. elegans genes and characterise their function in the pest species drawing on information from our current BBSRC-funded G. pallida genome sequencing project.