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

Lead Research Organisation: University of Leeds
Department Name: Ctr for Plant Sciences

Abstract

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.

Technical Summary

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.
 
Description 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 the rational design of pesticides. This project focused on specific compounds with different modes of action and sought to determine the pathways that metabolise each example in the model nematode species Caenorhabditis elegans. 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 then in turn switch on those that encode products that metabolise the xenobiotic. We had already determined the likely identity of some of these transcription factors in previous work. We established the involvement of particular transcription factors in assays using the small, free-living nematode Caenorhabditis elegans. Mutant strains that each lacked a specific transcription factor were exposed to a range of compounds to determine if they exhibited enhanced susceptibility to particular xenobiotics. A wide-scale RNAi screen was also used to knock out over 300 transcription factors. These assays identified 7 key transcription factors that regulated responses to particular xenobiotics.

We generated twelve GFP reporter strains of C. elegans that responded to specific xenobiotics. These nematodes were used in rapid throughput RNAi screens to identify additional transcription factors that controlled each response. Twelve transcription factors were identified, that all belong to a group of supplementary nuclear hormone receptors found only in nematodes. Nematodes that lack these genes have no reported phenotypes under standard conditions. RNAi knock down of one of these transcription factors resulted in enhanced susceptibility to thiabendazole. We then used microarray technology to identify downstream metabolic genes regulated by each of four C. elegans transcription factors.

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 cloned eight transcription factor genes into expression vectors and generated purified protein products from bacteria for two transcription factors in order to determine if the transcription factor protein does binds to the xenobiotic. These recombinant transcription factors will be also used to assess DNA binding.

The work centred on C. elegans. It is a useful model organism because of detailed genetic and other knowledge accumulated about it. Its use in this work assured rapid progress but we also carried out translational research to a pest with high economic impact in UK agriculture. We chose the potato cyst nematode Globodera pallida as this is the major nematode pest of UK agriculture costing our potato industry an estimated £50 m/year. The work was timely as it linked directly to the BBSRC-funded G. pallida sequencing project led by the PI of this proposal in collaboration with the Sanger Institute. Homologues of genes involved in cellular metabolism in C. elegans were identified in G. pallida by sequence similarity and their pattern of expression during the life-cycle was assessed from newly acquired transcriptome sequence data. G. pallida contains a vast reduction in genes encoding cellular metabolism but an expansion in some oxidation genes, indicating that this organism uses alternative pathways to C. elegans for metabolism of xenobiotics. Comparative transcript profiling of G. pallida exposed to xenobiotics was also carried out to identify genes that are regulated in response to xenobiotic treatment. The work has provided information that will be valuable in the design and evaluation of not just nematicides but also insecticides given the central role that C. elegans plays as a model in product development.
Exploitation Route The research has a number of potential non-academic applications, particularly within the crop protection industry. Understanding the mechanism by which compounds are metabolised by target species is central to the rational design of new pesticides. The new knowledge gained from this research provides important insights into the pathways by which nematodes metabolise and detoxify a range of compounds including known pesticides. The work has provided information that will be valuable in the design and evaluation of not just nematicides but also insecticides given the central role that C. elegans plays as a model in product development.
Sectors Agriculture, Food and Drink,Chemicals,Environment

 
Description Determining the primary responses of plant parasitic nematodes to xenobiotic compounds has opened up the opportunity to use the information to design new control strategies. EU legislation has already resulted in the recent loss of two nematicides in response to environmental concerns. Our research has generated technology that may allow the design of new chemicals that do not harm the environment. The reporter strains generated in the project could be used to provide a panel of reporters in high throughput screens of potential new active compounds. This possibility is currently being explored with a large chemical company. Assays developed as part of this research have been used for investigating the efficacy and mode of action of potential nematicidal compounds in work for two companies. The reporter strains of C. elegans that were produced are being used to assess the mode of action of plant-derived compounds in a current project.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Chemicals,Environment
Impact Types Economic

 
Description Xenobiotic responses
Amount £51,300 (GBP)
Organisation Syngenta International AG 
Department Syngenta Ltd (Bracknell)
Sector Private
Country United Kingdom
Start 11/2010 
End 10/2012
 
Description Globodera rostochiensis genome annotation 
Organisation Agriculture and Agri-Food Canada
Country Canada 
Sector Public 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation French National Institute of Agricultural Research
Department INRA Rennes Centre
Country France 
Sector Public 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation French National Institute of Agricultural Research
Department INRA Sophia Antipolis
Country France 
Sector Public 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation Government of Canada
Country Canada 
Sector Public 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation James Hutton Institute
Country United Kingdom 
Sector Public 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation Oregon State University
Country United States 
Sector Academic/University 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Globodera rostochiensis genome annotation 
Organisation University of Wageningen
Country Netherlands 
Sector Academic/University 
PI Contribution The collaboration was set up to analyse and annotate the genome of the golden potato cyst nematode Globodera rostochiensis. The foundation for this was a genome assembly produced as part of our BBSRC grant BB/F000642/1 for which the focus was the sister species G. pallida. Transcriptome data for two life stages was also contributed by our team. One member of our team initiated and coordinates the collaboration and took a major role in preparing the genome assembly for group manual annotation. Four members of our research team have been actively involved in the specialist annotation of the genome and the correction of gene models.
Collaborator Contribution Partners at the University of Edinburgh have contributed bioinformatics expertise, use of computing facilities and hosted a group annotation event attended by all members of the collaboration. Partners in Canada have contributed genome and transcriptome data and taken part in genome annotation. All other partners have contributed their specific expertise in manual annotation of particular gene families.
Impact Transcriptome data has been mapped to the genome assembly. Almost 1500 gene models (>10% of the total) have been manually curated and corrected by the collaborators, to act as a training set for improved gene predictions. A promoter motif has been identified that is associated with expression in the main, effector-producing gland cell of potato cyst nematodes. This provides a new route for prediction of novel effectors in cyst nematodes and has formed the basis for further studies by collaborating partners. A manuscript describing the genome and particular features of interest has been published in Genome Biology.
Start Year 2013
 
Description Yorkshire Show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact All members of the research team participated in preparation and delivery of an interactive display at The Great Yorkshire Show. The display explained the problem of plant parasitic nematodes in agriculture and introduced the research being done in our group to both the general public and members of the agricultural community. Members of the group engaged in numerous discussions about our research with a wide audience.

Our exhibits at the Great Yorkshire Show have resulted in an interview on Radio York and also interviews with a number of freelance journalists who provide articles for publications such as the Farmers Guardian.
Year(s) Of Engagement Activity 2012