Exploitation of genomic knowledge for sustainable resistance to the crop pest Globodera pallida

据估计，植物寄生线虫每年给全球农业造成1250亿美元的损失。马铃薯囊肿线虫每年给英国马铃薯产业造成约5000万英镑的损失。防治工作往往依赖于杀线虫剂，这些杀线虫剂对环境有害，也可能对人体健康有害。在英国，它们通常是经济马铃薯种植所必需的。这项工作中使用的新技术可以在不破坏环境的情况下控制植物寄生线虫。如果提供指定蛋白质编码的基因变得不活跃，蛋白质及其在细胞中所起的作用就会消失。这是一种自然效应，可能保护细胞免受移动基因元素（如某些病毒）的侵害。它的基础是一些病毒产生双链而不是正常的单链中间分子（RNA）。当每一个新的蛋白质分子形成时，它都参与了基因密码的解读。通过将RNA双链形式引入细胞，可以通过实验诱导自然基因沉默效应。这可能会导致有机体沉默自己的一个基因。这是一种实验性操作，通过向细胞中引入与要沉默的基因精确匹配的序列来实现。这被称为RNA干扰（RNAi），这种效应首先在自由生活的秀丽隐杆线虫（Caenorhabditis elegans）吞下dsRNA后被证明。将dsRNA引入一系列生物体也会产生同样的效果。可以对植物进行修饰，使其产生感兴趣的dsRNA。RNAi提供了一个强大的实验工具。感兴趣的单个基因可以被特异性地沉默，并研究其效果。我们建立了利用RNAi分析植物寄生线虫特定基因功能的方法。我们已经确定了线虫在植物中有效取食所必须表达的基因。其他人则利用这种方法来抑制植物中单个基因的功能。与此同时，我们还使用了其他方法来确定哪些植物基因必须在根部局部表达才能使线虫成功取食。在这个提议中，我们将在马铃薯植物中表达少量已知的沉默特定线虫基因的dsRNA分子。当动物从植物中进食时，吞下dsRNA后会发生沉默。我们还将瞄准一些植物基因，这些基因必须是活跃的，才能使线虫正常进食。这种方法的主要优点是不会产生新的蛋白质。没有任何已知的依据表明，任何特定的dsRNA在人类饮食或对环境有害。因此，它比目前使用的成本效益较低的杀虫剂或控制程序更为可取。dsRNA将由一个基因开关（启动子）驱动，该基因开关在线虫取食的根部而不是块茎或叶片中自然活跃。安全的dsRNA甚至不会存在于我们吃的马铃薯块茎中。这些基因的沉默将阻止线虫的发展，从而阻止它对作物造成的损害。这项工作将提供一种方法，帮助满足英国马铃薯种植者减少农药使用的迫切需求。它将允许受消费者青睐但目前受到线虫损害的品种广泛种植，而不会对食品安全造成任何风险。这种方法不会对环境造成危害，并通过消除目前危害昆虫的杀虫剂，使昆虫得到自然控制。该方法的潜力比在该方法中开发的潜力更大。目前的应用代表了在改善无脊椎动物和那些在英国马铃薯田以它们为食的环境中有竞争力和可持续的英国马铃薯种植的基础。

Plant parasitic nematodes cause estimated annual losses to global agriculture of $125b . Potato cyst nematode costs the UK potato industry an estimated £50 m/year. Control often depends on nematicides that are both harmful to the environment and possibly human health. They are often essential for economic potato cropping in the UK. The new technology to be used in this work can control plant parasitic nematodes without harming the environment. A protein and the role it plays in a cell can be abolished if the gene that provides the code specifying it becomes inactive. This is a natural effect that probably protecting cells from mobile genetic elements such as certain viruses. Its basis is that the some viruses make a double not the normal single strand of the intermediary molecule (RNA). It is involved in the reading of the genetic code when each new protein molecule is made. The natural gene silencing effect can be induced experimentally by introducing the double stranded form of RNA to a cell. This can cause an organism to silence one of its own genes. This is experimental manipulation is achieved by introducing to a cell a sequence that matches precisely that of the gene to be silenced. This is termed RNA interference (RNAi) and the effect was first demonstrated for the free-living nematode Caenorhabditis elegans after it swallowed dsRNA. The same effect induced by introducing dsRNA to a range of organisms. Plants can be modified to make the dsRNA of interest. RNAi provides a powerful experimental tool. Individual genes of interest can be specifically silenced and the effects of this studied. We established the use of RNAi for analyses to function of particular genes of plant parasitic nematodes. We have identified genes the nematode must express to feed in plants effectively. Others have used the approach to silence the function of individual genes in plants. In parallel we have used other methods to define which plant genes must express locally in roots for a nematode to feed successfully. In this proposal we will express in the potato plant a small number of dsRNA molecules known to silence particular nematode genes. Silencing will occur after the animal has swallowed the dsRNA when feeding from the plant. We will also target a few plant genes that must be active for the nematode to feed normally. The key advantage of the approach is that no novel protein is made. There is no known basis for any particular dsRNA to be harmful in either the human diet or to the environment. It is therefore highly preferable to current use of pesticides or control procedures that are less cost effective. The dsRNA will be driven from a gene switch (promoter) that is naturally active just where the nematode feeds in the root and not in its tubers or leaves. The safe dsRNA will not even be in the potato tubers we eat. The silencing of these genes will prevent the development of the nematode and so stop the damage it causes to the crop. The work will provide an approach that helps meet the urgent need for UK potato growers to reduce pesticide use. It will allow cultivars favoured by consumers but currently damaged by nematode to be widely grown without any risk to food safety. The approach will have no environmental harm and allow natural control of insects to flourish by eliminating pesticides that currently harm them. The approach has wider potential than to be developed in the approach. The current application represents a basis for competitive and sustainable UK potato cropping in an improved environment for invertebrates and those that feed on them in UK potato fields.