Characterisation of cue-dependent behaviour in plant parasitic nematodes (PPNs); the neurobiology of host plant invasion

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

Abstract

Nematodes are rounds worms that live in many habitats ranging from the highest mountains to the deepest seas. There are a number of different nematodes that have evolved for distinct life-styles. They encompass both non-parasitic and parasitic worms. Amongst the latter are parasitic nematodes that use agriculturally important crops and animals as hosts often affecting their vitality and/or viability. Such parasites lead to disease processes that cost billions of pounds and reduce the capability for food self-sufficiency and security. In the UK and across the world we face an increasing problem from nematodes that infect animals and also plant parasitic nematodes that infest crops. For the latter, this is worsened by the growing realization that current treatments are becoming increasingly unacceptable due to environmental and human health implications and their withdrawal from use. This places farming in a very vulnerable position. The plant parasitic nematodes (PPNs) directly cause damage and also act as secondary carriers for diseases. In the UK they place a severe burden on potato crops. The life cycle of the PPNs involves the maturation of the worm in the host's root where it diverts plant nutrients to its own development and reproduction. This is followed by release of a free living form, from resource exhausted infected roots, that goes onto re-invade a different root. Some of these nematodes show a selective taste for one kind of plant while others can target a range of hosts. In either case the parasites often infect and reduce yields from intensively farmed and economically important crops. An essential aspect of the successful completion of the PPN life cycle is movement through the soil from the point of release to locate and reinvade new host roots. This is achieved through a simple three step process. Firstly, the free living worm detects signals from the root. Secondly it uses its sensor organs to detect these tell-tale host cues. Thirdly it uses these cues to track towards the new root. The details of what the worm's sensors are and how they guide its movement to an appropriate host are poorly understood but clearly very important for maintaining the parasite's life cycle. This is in complete contrast to the detailed understanding we have for another species of nematode, the so-called 'model' organism, C. elegans. This non-parasitic worm has been extensively studied by biologists since the 1960s and its success as a biological model is manifest by the fact that it was the first animal to have its genome sequenced (1998). Indeed, it has been instrumental in the award of three Nobel prizes. Neurobiologists have defined in precise detail the molecular, cellular and behavioural mechanisms through which this worm senses food and moves towards it. Furthermore, chemicals that act against parasitic nematodes also have effects on C. elegans consistent with the view that the latter provide a good model for understanding signalling and behaviour in PPNs. We will use the techniques, expertise and understanding of C. elegans and translate these to a detailed analysis of host location behaviour in PPNs. We will use laboratory based investigations in which root extracts from host plants modify PPN behaviour and investigate chemicals that could act against PPNs to prevent their ability to locate and/or move towards the host plant. This will be facilitated by ongoing genome sequencing for PPNs which will reveal the molecular identity of new targets through which nematicidal chemicals act. This will be done with a view to identifying new chemical targets for nematicides. These efforts will be facilitated by a supply of chemicals from collaborating industrial colleagues that have the potential to provide PPN selective nematicides that act by impairing the parasite's ability to find its host plant. In this way the project will address the threat to farming posed by the lack of effective, environmentally safe nematicides.

Technical Summary

Understanding of C. elegans neurobiology will be leveraged to provide insight into the neural mechanisms underpinning host location by plant parasitic nematodes (PPN). We will develop new assays for measuring cue-dependent behaviour in two species of PPN. Preliminary assays indicate PPNs exhibit a similar repertoire of locomotory behaviours as C. elegans comprising reversals, turns, speed of motility, direction of motility, and in a similar fashion to C. elegans, these behaviours are modified by chemosensory cues. Thus we will adapt video-based analysis designed for C. elegans to a discrete analysis of PPN behaviour and parameterise sub-components of locomotion that together enable the worm to localise its host. We will investigate the role of key signalling molecules, i.e. neurotransmitters and receptors, in mediating these behaviours. The work will be facilitated by maturing genome sequencing projects for PPNs permitting identification of PPN orthologues of key molecular components e.g. neurotransmitter receptors, involved in PPN navigation towards its host. These will be targeted by RNAi and the impact of their reduced expression on PPN plant cue-dependent responses will thus delineate potential nematicide targets. In addition, by testing the effects of compounds, encompassing known nematicides, anthelmintics and novel neuroactive chemicals we will quantify potential efficacy against PPN host location behaviour. Subsequent heterologous expression of the PPN receptors will build on the organism-based approaches and allow a more direct comparison of chemical action on the C. elegans and PPN targets. As this derives from a comparative approach it can contribute to the design of PPN selective compounds. This effort will be facilitated by an ongoing industrial collaboration in the field of crop protection that will provide access to compound libraries representing modifications of established nematicide chemistry or compounds with unknown mode of action.

Planned Impact

A: Beneficiaries from this research
A1: Commercial private sector
Crop protection industry: There is a need for basic research that will inform development of the next generation of crop protection products with the required specificity and lack of non-target effects. This is currently hampered by a relative lack of knowledge on the physiology and pharmacology of plant parasitic nematodes.
UK potato growers: The British Potato Council (BPC) estimates the UK potato production, processing and retail market has an annual value of c. £3 billion. This research will, in the long term, support potato producers who need approaches to control potato cyst nematode, particularly the cyst nematode species Globodera pallida. The pesticides used to control this nematode are the largest variable cost of the growers who use them. This represents 23% of the UK potato acreage being treated each year. The proposed project will demonstrate that fundamental research can support their agronomic needs.
International growers: Nematodes are responsible for worldwide crop losses of c. $ 125 billion per annum. This research will investigate a key aspect of plant-nematode interactions in two of the most economically important genera of plant parasitic nematodes. The information gained is likely to be relevant to a wide range of growers in both intensive and subsistence agriculture.
Supermarkets: Some of the major supermarkets see value in removing even the theoretical risk of pesticide residues from the potatoes they sell. The outputs of this project may in the longer term support that policy.
A2: Policy-makers
DEFRA and The Scottish Government (SG) must implement the EU's amendment Directive 91/414/EEC that seeks to reduce use of hazardous crop protection chemicals in EU agriculture. The Directive involves the abrupt or gradual withdrawal of nematicides from the UK market. This is a challenge for UK potato production as it receives more crop protection treatments than other broad acre crps. DEFRA and SG need evidence of effective alternatives for potato cyst nematode control as they must also support an EU Directive specifically aimed at potato cyst nematodes (2007/33/EC) that came into force in July 2010. The research addresses basic aspects of nematode behaviour that could culminate in improved pathogen management in the longer term.
A3: General Public
Most of the UK population consumes potatoes and there is a need to maintain potatoes as a nutritious and inexpensive staple food. This work will support that need in the long term.

B: Nature of benefits from this research
B1: Benefits for UK economic competitiveness
The prevalence of G. pallida contributed to a decline in the UK potato crop of 11.9% between 1999 and 2003. This is a larger fall than that of other major EU producers that lack this pest. This work would build a platform to address the problem of PCN control that faces many UK producers and is one cause of their move away from this crop.
B2: Increasing the effectiveness of public services and policy
It is relevant for those who seek to implement change in policies such as an amendment to Directive 91/414/EEC and implementation of 2007/33/EC to be aware that the UK science base supports its agricultural sector and is working to develop measures to address new legislation.

B3: Enhancing cultural enrichment, quality of life and health?
Reducing reliance on currently used hazardous pesticides carries a benefit for UK biodiversity and sustainability of food production.
A recent report from the Royal Society identified the need for training in agricultural sciences and related topics. The Postdoctoral scientist will therefore be trained in an area of considerable importance and recent neglect.
Impact activities will raise awareness in the general public of some of the problems associated with food production and the role science can play in addressing these issues.
 
Description Plant parasitic nematodes are microscopic worms that infect the roots of their host plants. Mounting concerns over the ecotoxicity of currently used chemical control agents, nematicides, and their subsequent regulation means that these pests present an increasing threat to food security. This project has brought together a group in Leeds with expertise in plant parasitic nematodes with neuroscientists based at the University of Southampton to tackle this problem from a new angle. The rationale is that a deeper understanding of the neural mechanisms in the nematode that enable it to recognise and invade its host plant will provide a better informed route to new approaches for chemical control. The Southampton group brought expertise in understanding the neural mechanisms of cue-dependent behaviour in the model genetic organism C. elegans to the project whilst the Leeds group brought expertise in the biology of plant parasitic nematodes and a very timely knowledge of the genome of the potato cyst nematode G. pallida through their key involvement in the sequencing project. We conducted a bioinformatics screen in G. pallida to identify orthologues of components of signalling pathways known to regulate cue-dependent behaviour in C. elegans. We identified more than 30 candidates for investigation and selected those most likely to impact on host plant invasion behaviour for further analysis including nicotinic acetylcholine receptor subunits and components of the serotonergic signalling pathways. We established the function of the G. pallida genes by showing that their expression in a C. elegans knockout genetic background could fully rescue the C. elegans phenotype. We have characterised key receptor signalling pathways involved in the behaviour of the plant parasitic nematode Globodera pallida, a major pest of potatoes in the UK, and found new chemical approaches to preventing them from invading the host plant root.
Exploitation Route The project opens the way for a more targeted approach to preventing plant parasitic nematodes from invading their host plants.
Sectors Agriculture, Food and Drink

 
Description The project, and in particular the fruitful collaboration between the Leeds Group and Southampton, has facilitated interactions with industry for chemical testing and mode of action studies. This collaboration is ongoing through a fully funded PhD student with Adama Agricultural Solutions. The collaboration sponsored by this project between Leeds and Southampton was also instrumental in the development of a novel platform, StyletChip, for testing the effect of chemicals on host plant invasion behaviour, which has further contributed to the launch of an enterprise unit, Neurivo, based at Southampton with a goal of further translating advances in nematode neurobiology to an industrial setting. The project has provided leverage to add value in the form of two PhD studentships; including a BBSRC CASE studentship with Bayer CropScience (Southampton) with the potential to deliver further impact in the arena of crop protection. Our discovery that serotonin signalling plays a key role in the invasion behaviour of potato cyst nematodes has led to a patent application based around the use of a natural plant compound as a seed treatment to protect crops from damage by plant parasitic nematodes.
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description An integrated strategy for control of animal and plant parasitic nematodes through targeting a 5-HT-gated chloride channel MOD-1
Amount £158,960 (GBP)
Funding ID BB/T001097/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2022
 
Description Bayer Grants4Targets
Amount € 30,000 (EUR)
Organisation Bayer 
Department Bayer CropScience Ltd
Sector Private
Country United Kingdom
Start 09/2016 
End 05/2017
 
Description Globodera acetylcholine receptors 
Organisation French National Institute of Agricultural Research
Department INRA Loire Valley Centre
Country France 
Sector Public 
PI Contribution Our team has contributed cloned genes for potato cyst nematode acetylcholine receptors, data concerning their function and expertise, to facilitate a collaborative investigation of the function of plant parasitic nematode receptors using heterologous expression in a Xenopus oocyte system.
Collaborator Contribution Our collaborators have contributed vectors, cloned genes, expertise in oocyte expression and analysis and access to specialist equipment and facilities for undertaking the experiments. A member of our research team has been hosted by the collaborators at INRA to undertake experiments.
Impact Data has been obtained that will provide insights into the composition and function of nicotinic acetylcholine receptors of the potato cyst nematode Globodera pallida. These receptors are targets for common drugs and pesticides and the information will help us to understand the differing sensitivity of nematodes to such chemicals. A joint publication describing the collaborative work is planned.
Start Year 2014
 
Title The use of reserpine as a seed coating 
Description The present invention relates to the use of reserpine as a seed coating, in particular to deter nematodes from damaging plants. The present invention further relates to a seed coating comprising reserpine. 
IP Reference GB1710057.9 
Protection Patent application published
Year Protection Granted
Licensed No
Impact None yet
 
Description NFU meeting (Stoneleigh) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentations allowed decisions to be made with respect to new biotechnological options

NFU had information to help set national priorities
Year(s) Of Engagement Activity 2013
 
Description School visit (Honley) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk and display of plants introduced Reception and Year 1-2 children to a range of crops and their different uses. This stimulated children to think about where food items and other products come from.
In 2015, older children were led in discussions and took part in demonstrations and practical expereiments designed to show the importance and various functions of plant roots. This was related to the groups research on root pathogens.

The first visit to this school was enjoyed by both children and staff and led to requests for repeat visits.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
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