Functional characterisation of novel pathogenicity genes of the parasitic nematode Globodera pallida

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

Abstract

The aim of this project is to characterise those genes that are responsible for the inception of pathogenicity by Globodera pallida. The British Potato Council estimates the UK potato production, processing and retail markets to be worth c. £3 billion p.a. and the potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are the most economically important nematode problems of this industry. They occur in 65% of UK potato land with G. pallida present at 92% of these sites. PCN impose an annual cost in excess of £50 million on UK potato growers and threaten the future of the crop for many growers. Breeding for resistance since the mid 1950s has produced few commercially acceptable varieties with resistance to G. pallida. Currently used chemical control methods are under increasing pressure due to cost, environmental and health concerns and there are no benign alternatives to the currently used compounds. Control of G. pallida is an essential requirement to maintain the competitiveness of U.K. production. For example, the consumer demand for food with no pesticide residues has resulted in Waitrose sourcing all its potatoes from crops that have not received a nematicide treatment (www.waitrose.com). This requires imports from countries with a lower PCN incidence or requires a more extensive agricultural system in the UK. Consumer support is likely for UK produce that avoids pesticide residues or environmental harm and is soundly based on a sustainable approach. This proposal underpins the innovation needed to reach that outcome. G. pallida must live as a parasite in plants. It has a complex interaction with its plant host. Second stage juvenile nematodes (J2) hatch from eggs in the soil, upon detecting a host growing nearby, then locate and subsequently invade the roots of the host. The J2 migrates inside the root and selects a single cell that it transforms into a large multinucleate feeding cell. Profound changes in plant cell structure and gene expreson are induced by the nematode in establishing the feeding site. The nematode is known to spit into the cell. A few components of this spit are known to alter plant cellular development. In this proposal we aim to undertake a broad characterisation of putative pathogenicity proteins that cause the changes in plant physiology and that are therefore responsible for feeding site induction. We will determine if the putative pathogenicity proteins are produced in the glands of the nematode that secrete their 'spit'. The timing of the proteins' manufacture relative to the lifecycle of the nematode and its interaction with the plant will be measured to determine if they are required at the beginning of the interaction between the nematode and the plant, or continuously throughout the interaction. We will utilise high throughput fluorescent assays to determine if the putative pathogenicity proteins cause the nuclei of plant cells to increase in size - a common observable phenomenon in nematode feeding sites. We will also determine if the proteins can suppress host defence responses. Analysis will reveal what components of the plant cell the putative pathogenicity proteins interact with and then to ensure that the interaction has biological relevance components will be linked to one half of a fluorescent marker protein and co-transformed into plants. The marker protein does not produce fluorescence when it is split into N and C-terminal halves. Each half will be fused to one of the two putative interacting partners. This will lead to restoration of fluorescence within a cell if the nematode and plant proteins interact and reconstitute the split fluorescent protein. The advantage of this technique over other methods of visualizing protein-protein interactions is that it gives an indication of cellular localization of the complex, as well as interaction.

Technical Summary

The aim of this project is to characterise those genes that are responsible for the inception of pathogenicity by Globodera pallida. Globodera pallida is the most economically important nematode problem of U.K. potato production. Control of G. pallida is an essential requirement to maintain the competitiveness of U.K. production. This application will provide useful information that can be translated into new biotechnological parasite-control solutions. Second stage juvenile nematodes (J2) hatch upon detecting a host growing nearby, locate and subsequently invade the roots of the host. The J2 migrates inside the root and selects a cell, then causes cell wall dissolution to form a large multinucleate feeding cell, termed a syncytium. Profound changes in plant cell structure and gene expression are induced by the nematode in establishing the syncytium. The nematode is known to secrete proteins that alter plant cellular development into the initial feeding cell. In this proposal we aim to undertake a broad characterisation of putative pathogenicity proteins that cause the changes in plant physiology and that are therefore responsible for feeding site induction. In situ hybridisation will be used to ensure putative pathogenicity proteins are synthesised in the pharyngeal glands. Gene expression will be measured, relative to the lifecycle of the nematode and its interaction with the plant. We will utilise high throughput fluorescent assays to determine if the putative pathogenicity proteins cause the plant cell nuclei to enlarge - a common observable phenomenon in nematode feeding sites. We will also determine if the proteins can suppress host defence responses. Yeast-2-hybrid analysis will reveal those components of the plant cell that interact with the putative pathogenicity proteins and we will then ensure that any interactions detected by this method have biological relevance by confirmation with Bimolecular fluorescence complementation i.e. split-YFP analysis.
 
Description The potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are the most economically important nematode problems of the UK potato industry. They occur in 65% of UK potato land with G. pallida present at 92% of these sites. PCN causes an annual loss in excess of £3 billion to the on UK potato industry. Breeding for resistance since the mid-1950s has produced few commercially acceptable varieties with resistance to G. pallida. Currently used chemical control methods are under increasing pressure due to cost, environmental and health concerns and there are no benign alternatives to the currently used compounds. Sustainable control measures are desperately needed and this research underpinned the innovation needed to reach that outcome.
Globodera pallida lives as a parasite in plant roots and has a complex interaction with its host. The nematode spits into the plant cell that it feeds from and some components of this spit, termed "effectors" or "effector proteins" are known to alter plant cellular development and trigger the changes that transform the root cell into a functional nematode feeding site. Other effectors probably ensure that the plant does not act against the nematode. The central aim of this project was to define and broadly characterise the effector proteins and the genes that encode them, as they are most likely responsible for inducing and maintaining parasitism by the potato cyst nematode. We have used information from the G. pallida genome sequencing project to identify more than 250 likely effectors and analyse their production throughout the nematode lifecycle. Some effectors are only produced by nematodes as they invade the plant root and others are produced once the nematode has started to feed. This gave us some clues about the functions of the different effectors.
One way that the effectors may cause changes in the plant cell is by switching potato genes on or off. We have shown, by linking effector proteins to a green fluorescent marker, that some effectors are transported into the nucleus of the plant cell where regulation of gene expression is controlled. These effectors may exert their effect by binding to important potato regulatory proteins called transcription factors and changing their activity. We carried out a screening experiment to identify the potato proteins that interact with different nematode effectors. Several G. pallida effectors did bind to potato transcription factors and so these effectors may act by changing potato gene expression. Significantly, these particular transcription factors are known to also be the targets of other, very different pathogens such as bacteria and potato blight.
Many plant pathogens suppress host defences but little was known about the proteins that cyst nematodes use to achieve this. We have screened many of the putative nematode effectors using a range of suppression assays and identified at least four that play a role in this process. Interestingly, a number of the G. pallida effectors interact with a plant protein which, in the soybean, plays a role in resistance against soybean cyst nematode. These interactions may play a similar role in determining host susceptibility to cyst nematodes in potato. We have confirmed that some of the interactions between effectors and potato proteins occur within the plant cell and not just in a test system. When one nematode effector binds to its target potato protein, it relocates within the plant cell from the cytoplasm to the nucleus.
Transgenic potato and Arabidopsis plants have been produced that express 19 of the putative effectors. Comprehensive phenotypic analysis of the plants highlights some effectors that cause morphological and/or developmental changes in the plants. For instance, expression of one effector results in shorter plants with delayed flowering and also increases susceptibility of the potato plants to the pathogen that causes potato blight.
Exploitation Route A number of resources were generated from this fundamental research that will be of lasting value to the research community.
Panels of nematode effector clones for future analysis: more than 40 G. pallida effector sequences were amplified and cloned into a Gateway entry vector as a primary resource. All the effectors are available in a range of plant transformation vectors that allow stable or transient expression of nematode effectors in plants for functional studies. Effectors are additionally cloned with a HA-tag to facilitate detection and purification strategies or as GFP fusions for subcellular localisation. Effectors are also present in specialist vectors for RNAi, screening of yeast-2-hybrid (Y2H) libraries and bimolecular fluorescence complementation studies.
Analysis and annotation of effectors in the G. pallida genome sequence: The genome sequence of G. pallida was used for the first complete analysis of any cyst nematode effector complement. This global analysis identified 129 homologues of effectors from other plant parasitic nematodes. A bioinformatic pipeline was employed to identify a further 117 novel, secreted putative effectors with little homology to proteins from other species. Many of these effector candidates are now being investigated in other projects. Such genes will be important targets for the disruption of this parasitic interaction that could lead to plant resistance. One route for achieving this would be to develop plants that produce molecules to specifically target and suppress these genes in the nematode.
Sectors Agriculture, Food and Drink,Environment

 
Description The project documented the molecular interactions that the nematode pathogen induces with the plant host. Other pathogens have analogous interactions and we identified likely common host targets of pathogen effectors. In the future this may allow common control measures for different pathogens but at present the long term impacts of the research are yet to be realised. Those involved with the work have since established careers in the plant pathology community. The findings of the project led to a researcher associated with the work securing a BBSRC Anniversary Future Leader Fellowship.
First Year Of Impact 2014
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description China Scholarship
Amount £72,000 (GBP)
Organisation University of Leeds 
Department China Scholarship Council
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description David Miller Travel Award
Amount £250 (GBP)
Organisation Imperial College London 
Department Schistosomiasis Control Initiative (SCI)
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 09/2014
 
Description ESN Travel Bursary
Amount € 1,000 (EUR)
Organisation European Society of Nematologists 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 05/2014
 
Description MPMI Travel Bursary
Amount $1,000 (USD)
Organisation International Society for Molecular Plant-Microbe Interactions (IS-MPMI) 
Sector Charity/Non Profit
Country United States
Start 07/2014 
End 07/2014