Evolution of pathogen effectors to carry out novel functions in planta

英国马铃薯协会估计，英国马铃薯的生产、加工和零售市场每年价值约30亿英镑。马铃薯囊线虫Globodera rostochiensis和G. pallida，俗称鳗鱼蠕虫，是马铃薯种植者造成重大经济损失的重要土壤害虫。苍白球虱是英国最常见的物种，其控制是最成问题的。由于缺乏商业上受欢迎的抗性马铃薯品种，以及对使用化学控制措施的担忧，导致苍白马铃薯对英国和许多其他国家的农民来说都是一个棘手的问题。苍白菌以寄生物的形式生活，其大部分生命周期必须在马铃薯根部完成。它与寄主植物有复杂的相互作用。幼年线虫是一种微小的蠕虫，在发现附近生长的寄主植物后，从土壤中的卵孵化出来，然后定位并随后侵入寄主的根部。线虫在根内移动，选择一个根细胞，并将其转化为一个大型的专门的喂养细胞。在取食细胞的形成过程中，线虫诱导植物细胞结构和基因表达发生了深刻的变化。这些变化是由来自线虫的蛋白质介导的，称为“效应器”，通过针状口器直接注射到寄主植物中。令人惊讶的是，我们对这些效应物中的大多数知之甚少；大多数蛋白质没有特定的功能，也与其他蛋白质没有相似性。此外，即使是那些与已知功能的蛋白质相似的蛋白质也出现了多样化。最近，我们已经积累了强有力的证据，表明一组类似谷胱甘肽合成酶（GS）的蛋白质已经进化出一种新的功能，并被马铃薯囊肿线虫作为效应器部署。鉴于这些似乎是特定于植物寄生线虫的谱系，我们的目标是表征它们的新功能，希望它们将导致新的目标，特定于这些害虫。首先，我们将利用一种被称为rna干扰的高度靶向技术，确定这些“类gs效应物”对线虫的重要性。这将同时破坏所有这些基因，并阻止蛋白质被运送到植物中，使我们能够确定这些类gs效应物对动物寄生植物的能力有多大的大规模影响。其次，为了深入了解它们的功能，我们将利用抗体识别和蛋白质纯化技术的结合，确定哪些类gs效应物被注射到植物中，以及在哪里注射。这些数据将使我们能够专注于最丰富的gs样效应物，并确定这些基因在植物中的表达是否会改变植物的生理机能及其承受线虫感染的能力，即比对照植物携带更多或更少的线虫。最后，我们将使用一系列的技术来阐明由苍叶草谷胱甘肽合成酶样效应物在植物中催化的新型酶促过程，从而深入了解它们的进化作用。植物寄生线虫特有的一种新酶活性的表征，对它们作为寄生虫的成功至关重要，可能为合理设计一种化学抑制剂提供基础，这种抑制剂将能够控制这些经济上重要的寄生虫，而不会产生许多杀虫剂常见的脱靶效应。

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.