Evolution of pathogen effectors to carry out novel functions in planta

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


The British Potato Council estimates the UK potato production, processing and retail markets to be worth around £3 billion per annum. The potato cyst nematodes Globodera rostochiensis and G. pallida, commonly known as eel worms, are important soil pests that cause major economic losses to potato growers. Globodera pallida is the most prevalent species in the UK and its control is the most problematic. A lack of commercially favoured resistant potato varieties and concerns surrounding the use of chemical control measures have resulted in G. pallida being an intractable problem to farmers both in the UK and in many other countries. G. pallida lives as a parasite and must complete the majority of its life-cycle in potato roots. It has a complex interaction with its plant host. Juvenile nematodes are microscopic worms that hatch from eggs in the soil upon detecting a host plant growing nearby, then locate and subsequently invade the roots of the host. The nematode migrates inside the root and selects a single root cell that it transforms into a large specialised feeding cell. Profound changes in plant cell structure and gene expression are induced by the nematode in establishing the feeding cell. These changes are mediated by proteins from the nematode, called 'effectors', directly injected into the host plant through a needle-like mouthpart.
Surprisingly little is known about the majority of these effectors; most have no ascribed function and no similarity to other proteins. Furthermore, even those with similarity to proteins of known function appear to have diversified. Recently we have accumulated strong evidence that a group of proteins, similar to glutathione synthetase (GS), have evolved to carry out a new function, and are deployed as effectors by potato cyst nematodes. Given that these appear to be specific to this lineage of plant-parasitic nematodes, we aim to characterise their new function in the hope that they will lead to a new target, specific to these pests.
In the first instance, we will determine how important these "GS-like effectors" are to the nematode, by utilising a highly targeted technique, known as RNA-interference. This will knock-down all these genes simultaneously and stop the proteins being delivered to the plant, allowing us to determine what large-scale effect these GS-like effectors have on the ability of the animal to parasitise the plant. Secondly, to gain insights into their function we will determine exactly which GS-like effectors are injected into the plant and where using a combination of antibody recognition and protein purification techniques. These data will allow us to focus on the most abundant GS-like effectors, and determine whether or not the expression of those genes in the plant alters the physiology of the plant and its ability to sustain a nematode infection, i.e. harbour more or less nematodes than control plants. Finally, we will use a range of techniques to elucidate the novel enzymatic process catalysed in plants by the G. pallida glutathione synthetase-like effectors so providing insight into their evolved role. The characterisation of a novel enzyme activity unique to plant parasitic nematodes, which is essential for their success as a parasite, may provide a foundation for the rational design of a chemical inhibitor that will afford control of these economically important parasites, without the off-target effects common to many pesticides.

Technical Summary

Plant parasitism by nematodes is a major threat to global food security with damage to crops valued at over £75 billion each year. Sedentary endoparasitic nematodes secrete effectors into their plant host to induce and maintain a specialised feeding site that supports development of the nematode over several weeks. Surprisingly little is known about the majority of putative effectors; most have no ascribed function and no similarity to any database sequence. Preliminary work for this application underlies our hypothesis that a massive expansion of genes with similarity to glutathione synthetase (GS) enzymes gave rise to nematode effectors that catalyse a novel biosynthetic pathway. We have evidence that these GS-like effectors, which are unique to certain plant parasitic nematodes, have lost the ability to synthesis glutathione but have retained catalytic activity. The proposed project, focussing on the economically important potato cyst nematode Globodera pallida, aims to provide evidence that GS proteins have evolved to acquire the capacity for secretion and a new function, both previously unreported in any other eukaryote. We will use immunolocalisation and also exploit the dimerization properties of the GS-like effectors to demonstrate their introduction into the host plant. The importance of the effectors in the plant-nematode interaction will be investigated using host-delivered RNA interference to target groups of genes. We will gain insights into their function through in planta expression and analysis of effects on both the host plant and nematode susceptibility. Finally, we will use a range of techniques to elucidate the novel biosynthetic process catalysed in plants by the G. pallida GS-like effectors so providing insight into their evolved role that may be a highly specific target for control of cyst and reniform nematodes.

Planned Impact

Plant pathogens cause catastrophic losses to crops worldwide. A wide range of plant pathogens, including bacteria, fungi, oomycetes and nematodes produce effector proteins that facilitate infection. The proposed research focuses on a class of enzymes that has expanded, evolved and diversified in plant parasitic nematodes, so gaining a novel function. This allows them to act as putative effectors that are introduced into the host plant and are necessary for successful parasitism. A better understanding of the mechanisms by which these novel effectors act and their role in nematode parasitism has the potential to make a significant contribution to global food security and the UK bioeconomy. The evolved enzymatic function of these effectors is unique to plant parasitic nematodes and thus represents a potent target for future strategic research aimed at developing specific inhibitors with agronomic applications. The strategic impact of the research will realistically be achieved at a more distant point in the future. The current proposal seeds the pipeline of discovery that can then be translated into strategic outputs. Details of academic beneficiaries are provided in the eponymously named section. Here we describe the wider potential impact of the research.

UK FARMERS: They urgently need approaches to control pests. The pesticides used to control nematodes are the largest variable cost of the growers who use them. This represents 23% of the UK potato acreage being treated each year. We have wide support from UK growers for our efforts to provide new bases for nematode control. There is considerable anxiety among UK potato growers about the economic future of UK potato growing following the loss from the market of a number of effective nematicides and the threat of withdrawal of those remaining treatments.

POLICY-MAKERS: DEFRA must implement EU Regulation EC 1107/2009 that seeks to reduce use of crop protection chemicals in EU agriculture. The Directive involves the withdrawal of pesticides from the UK market. This is a challenge for UK potato production as it receives more crop protection treatments than other broad acre crops. Effective alternatives for nematode control are required. DEFRA must also support an EU Directive specifically aimed at potato cyst nematodes (2007/33/EC) which came into force from July 2010.
UK ENVIRONMENT: Nematicides are among the most environmentally harmful agrochemicals currently used in UK agriculture. Particular compounds are targeted by environmentalists and listed in the "dirty dozen" of pesticides. There is now an opportunity to create new environmentally benign methods to control plant-parasitic nematodes.

GENERAL PUBLIC: Most of the UK population consumes the potatoes affected by the particular nematode pest that is the focus of the proposed research. There is also a need to maintain potatoes as a nutritious and inexpensive staple food. In the longer term, this work will support that need. In a broader context, potatoes for example are one of the world's most important food crops and potato cyst nematodes cause problems in almost all countries where potato is grown. Research that underpins improved control of these pathogens at some point in the future therefore has the potential to impact on food security.

INTERNATIONAL CONTEXTS: We continually use BBSRC-funded outputs to underpin our work for resource poor farmers (DFID, USAID, See BBSRC booklet Focus on the Developing World). Our effort is currently centred on potato, banana and rice. This work emphasises needs in Africa (NARO, Uganda; IITA, Kenya) and India (Indian Agricultural Research Institute). The research that we propose is not only relevant to UK crops. The novel effector family that we will study is also found in other cyst nematode species that infect sugar beet, soybeans and cereals such as wheat and the reniform nematode that has a wide host range and is a pathogen of crops worldwide.
Description The potato cyst nematode parasite Globodera pallida completes the majority of its life-cycle in host potato roots where it induces and feeds from a large specialised feeding cell. Profound changes in plant cell structure and gene expression are induced by the nematode in establishing the feeding cell. These changes are mediated by proteins from the nematode, called 'effectors', which are directly injected into the host plant through a needle-like mouthpart.
We have characterised a highly expanded gene family in G. pallida and shown that evolution and functional diversification of this family of glutathione-synthestase (GS)-like genes has led to a group of novel effectors that are important for successful parasitism.
We have confirmed, using an antibody specifically recognising the "GS-like" effector proteins, that these are synthesised in the large dorsal gland cell of the nematode, from where proteins are secreted into the host root. We visualised the effectors as they moved towards the nematode stylet prior to secretion. Importantly we have also shown accumulation of effector GS proteins within the feeding cell - one of few such direct examples for plant parasitic nematode effectors.
We designed plant transformation constructs that allowed us to simultaneously target almost all "GS-like effectors" for knock-down by RNA-interference delivered via the feeding cell. The resulting loss of effector gene function led to a significant reduction in the number of nematodes that were able to establish a successful interaction with host potato plants. Those surviving nematodes were also developmentally compromised, highlighting the importance of the novel GS effectors for nematode parasitism.
We used the model plant Arabidopsis as a tool for further functional characterisation of the expanded GS-like gene family. None of the GS-like effectors were able to rescue the lethal phenotype of the Arabidopsis GS mutant, confirming that they have lost their canonical GS-synthetic function and also that the alternative reaction product was unable to compensate for glutathione. Wild-type Arabidopsis plants over-expressing a range of single GS-like effectors, were no more or less susceptible than wild-type plants to cyst nematodes. This suggests that multiple effector genes are required to significantly affect the ability of the plant to sustain a nematode infection. The lack of canonical GS activity is not related to a reduction in plant glutathione, which is often associated with susceptibility to pathogens. We showed that plant-derived glutathione is a positive regulator of cyst nematode parasitism.
We have implicated novel thiol biology in plant-nematode parasitism. We discovered that thiols are abundant in, and largely localised to, the cytoplasm of cyst nematode feeding cells throughout infection. This is not explained by an increase in glutathione, but a series of other low-molecular weight thiols. Some of these novel thiols, which are more hydrophobic than glutathione, are only present in roots harbouring feeding cells and could represent reaction products of the GS-like effectors.
Exploitation Route We have demonstrated the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors.
Many aspects of the evolution of novel function that we discovered for these GS-like effectors will likely be common to the genesis of other plant-parasitic nematode effectors. Given that parasitic nematodes deploy a battery of effectors, many arising from the adaptation of either endogenous genes or loci acquired by horizontal gene transfer, this paradigm will have substantial impact on the effort to understand and ultimately undermine their parasitism.
The novel effector proteins that we have characterised appear to be specific to one lineage of plant-parasitic nematodes. This raises the possibility that they could be new targets for control measures, which would be specific to these pests.
For example, our findings provide a foundation for the rational design of a chemical inhibitor that will afford control of these economically important parasites, without the off-target effects common to many pesticides.
Sectors Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology

Description GS effectors 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Shared data and ideas
Collaborator Contribution Partners provided data and intellectual input in the form of technical expertise and ideas. This led to a joint publication.
Impact https://doi.org/10.1371/journal.pgen.1007310
Start Year 2017
Description Discovery Zone 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The whole research group was involved in preparing and carrying out an activity-based display at the "Discovery Zone" event for local schools held at the University of Leeds. The event took place over 2 days and more than four hundred school children from Key Stages 2 and 3 engaged with the interactive exhibit "Getting to the root of the matter". We saw over 490 pupils in total - around 260 primary school children, and 230 secondary school children. Age appropriate activities and information relating to crop plants, the importance of roots and root pests and diseases were provided in small group sessions.
Year(s) Of Engagement Activity 2016,2017,2018
Description University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The work undertaken by the research team in developing nematode resistant crops was demonstrated to visitors. Visitors were engaged in discussions about the work and GM technology in general. Postgraduate students, postdocs and technicians associated with the grants all took part in either preparing or demonstrating the events.

Prospective students encouraged to apply for courses with an applied biology focus
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019