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

该项目的目的是描述那些负责苍白球孢致病性开始的基因的特征。英国马铃薯协会估计，英国马铃薯生产、加工和零售市场每年价值约30亿英镑，马铃薯囊肿线虫（PCN）、罗斯托奇Globodera rostochiensis和G. pallida是该行业最重要的经济线虫问题。它们出现在英国65%的马铃薯地，其中92%的马铃薯地有苍白藻。PCN每年给英国马铃薯种植者造成超过5000万英镑的损失，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，抗性育种已经产生了一些商业上可接受的抗苍白菌品种。由于成本、环境和健康方面的考虑，目前使用的化学控制方法面临越来越大的压力，而且目前使用的化合物没有良性替代品。控制苍白螺旋藻是保持英国生产竞争力的基本要求。例如，消费者对无农药残留食品的需求导致Waitrose采购了所有未使用杀线虫剂处理的土豆（www.waitrose.com）。这需要从PCN发病率较低的国家进口，或者需要英国更广泛的农业系统。消费者很可能支持避免农药残留或环境危害的英国产品，并以可持续发展的方式为基础。该提案支持实现这一成果所需的创新。苍白藻必须以寄生物的形式生活在植物体内。它与寄主植物有复杂的相互作用。第二阶段稚线虫（J2）在发现附近生长的寄主后，从土壤中的卵孵化，然后定位并随后侵入寄主的根部。J2在根内移动，选择一个细胞，并将其转化为一个大的多核喂养细胞。线虫在建立取食位点的过程中，诱导植物细胞结构和基因表达发生深刻变化。众所周知，线虫会向细胞内吐口水。已知这种唾液的一些成分可以改变植物细胞的发育。在本提案中，我们的目标是对引起植物生理变化并因此负责摄食部位诱导的推定致病性蛋白进行广泛的表征。我们将确定假定的致病性蛋白是否在分泌“唾液”的线虫腺体中产生。与线虫的生命周期及其与植物的相互作用相关的蛋白质制造时间将被测量，以确定它们是在线虫与植物相互作用开始时需要的，还是在相互作用的整个过程中持续需要。我们将利用高通量荧光分析来确定假定的致病性蛋白是否会导致植物细胞核的大小增加——这是线虫取食部位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。分析将揭示假定的致病性蛋白质与植物细胞的哪些组分相互作用，然后确保相互作用具有生物学相关性，组分将与荧光标记蛋白的一半连接并共同转化为植物。标记蛋白分裂成N端和c端两半时不产生荧光。每一半将被融合到两个假定的相互作用的伙伴之一。如果线虫和植物蛋白相互作用并重建分裂的荧光蛋白，这将导致细胞内荧光的恢复。这种技术相对于其他可视化蛋白质-蛋白质相互作用的方法的优势在于，它给出了复合物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.