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

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 expression 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.

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.