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

这个项目的目的是描述那些导致Globodera pallida致病的基因。英国土豆协会估计，英国土豆的生产、加工和零售市场每年价值30亿英镑。而马铃薯胞囊线虫(PCN)、罗氏球囊线虫(Globodera Rostochiens)和苍白球囊线虫(G.pallida)是该行业最重要的经济问题。它们出现在英国65%的土豆土地上，其中92%的地点出现了苍白球虫。PCN给英国马铃薯种植者带来了超过5000万英镑的年成本，并威胁到许多种植者的作物未来。自20世纪50年代中期以来，抗病育种几乎没有产生出商业上可以接受的抗灰霉病品种。由于成本、环境和健康问题，目前使用的化学控制方法面临越来越大的压力，目前使用的化合物没有无害的替代品。控制苍白球虫是维持英国生产竞争力的基本要求。例如，消费者对不含农药残留的食品的需求导致WaitRose从没有接受过杀线虫处理的作物中采购所有土豆(www.waitrose.com)。这需要从PCN发病率较低的国家进口，或者需要在英国建立更广泛的农业体系。消费者可能会支持英国的产品，避免农药残留或环境损害，并牢固地基于可持续的方法。这一提议为实现这一结果所需的创新奠定了基础。苍白球虫必须以寄生虫的形式生活在植物中。它与植物寄主之间存在着复杂的相互作用。第二阶段幼虫(J2)从土壤中的卵孵化，在检测到附近生长的宿主后，定位并随后入侵宿主的根。J2在根部内迁移，并选择一个细胞，然后将其转变为一个大型的多核摄食细胞。线虫在建立取食部位的过程中，会引起植物细胞结构和基因表达的深刻变化。众所周知，这种线虫会向细胞内吐痰。已知这种唾液的一些成分可以改变植物细胞的发育。在这项建议中，我们的目标是对可能的致病蛋白进行广泛的表征，这些蛋白导致植物生理的变化，因此负责诱导取食部位。我们将确定假定的致病蛋白是否在线虫的腺体中产生，该腺体分泌线虫的“唾液”。将测量相对于线虫生命周期和它与植物相互作用的蛋白质制造的时间，以确定它们是在线虫与植物相互作用的开始时需要的，还是在整个相互作用过程中持续需要的。我们将利用高通量荧光分析来确定可能的致病蛋白是否会导致植物细胞的细胞核增大--这是线虫摄食地点常见的可观察到的现象。我们还将确定这些蛋白质是否可以抑制宿主的防御反应。分析将揭示推测的致病蛋白与植物细胞的哪些成分相互作用，然后确保这种相互作用具有生物相关性，这些成分将连接到一半的荧光标记蛋白，并共同转化到植物中。当标记蛋白分裂成N端和C端的一半时，它不会产生荧光。每一半将被融合到两个假定的互动伙伴中的一个。如果线虫和植物蛋白相互作用并重组分裂的荧光蛋白，这将导致细胞内荧光的恢复。与其他可视化蛋白质-蛋白质相互作用的方法相比，这项技术的优势是它给出了复合体的细胞定位以及相互作用的指示。

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.