Exploiting eIF4E-based and associated broad-spectrum recessive resistance to potyviruses in dicots and monocots

该项目是研究双子叶植物的Warwick HRI和研究单子叶植物大麦的Rothamsted Research之间的合作。主要目的是：利用这些植物对马铃薯Y病毒的隐性eIF4E抗性来源。植物基因eIF4E参与病毒生活史的研究最早是在芜菁花叶病毒(TuMV，一种马铃薯Y病毒)和拟南芥上的实验中发现的。随后，人们发现eIF4E或其亚型(eIF(Iso)4E)与多种作物对马铃薯Y病毒的天然抗性有关。这种抗性的基础是马铃薯Y病毒需要这些基因中的一个才能完成它们的生命周期。众所周知，这些基因发生特殊变化，影响其编码的植物蛋白与病毒蛋白(VPG)之间的结合，植物就能抵抗感染。我们已经证明eIF4E与油菜和大麦对马铃薯Y病毒的抗性有关。到目前为止，在仅涉及eif4E的其他植物类型中描述的抗性并不是对特定马铃薯Y病毒的所有菌株都有效。然而，我们已经证明，在甘蓝中，eIF4E与另一个基因(Retr01)相结合，对马铃薯Y病毒(TuMV)的所有株系都具有抗性。病毒VPG蛋白和eIF4E之间的相互作用还涉及其他基因，包括非常相似的eIF(Iso)4E基因和其他相关基因(eIF4G、eIF4A及其异构体)。通过研究油菜和大麦抗病品系和感病品系的eIF4E和eIF(Iso)4E基因的自然变异，旨在寻找新的抗源并了解其抗性机制。我们将筛选代表十字花科植物中95%遗传变异的植物集合，以及由多达800个地方品种组成的大麦多样性小组。那些eIF4E基因不同的植物将用提供信息的马铃薯Y病毒分离株进行测试，以检查抗性/敏感性。将对任何导致抗药性的eIF4E或eIF(Iso)4E变体进行测序。这将通过观察用化学物质突变的植物来识别eIF4E和eIF(Iso)4E等位基因的其他新变化来支持这一点。发生这种变化的品系将对一组BaMMV和BaYMV分离物进行抗性测试。目的是确定eIF4E的形式，使其对所有(或大多数)病毒分离株具有耐受性。利用eIF4E可用的晶体结构，将从大麦和白菜中生成eIF4E的模型。在大麦和白菜中，与病毒抗性相关的eIF4E的构建块(氨基酸)的自然和化学诱导的变化将被映射到正常eIF4E蛋白的3D模型上。然后将研究这些变化对大麦和白菜eIF4E与病毒VPG蛋白结合的潜在能力的影响。这项工作的结果将是预测大麦和油菜eIF4E的优势抗性基因，以便在育种中部署。一个基因retr01与eIF4E相结合，赋予白菜对TuMV的广谱抗性，将被精细定位以确定该基因在植物基因组中的位置。该基因的序列将被用于搜索数据库，以预测其功能，了解广谱抗性的机制，以及retr01可能如何与eif4E相互作用。根据油菜和大麦中最好的病毒抗性来源，与抗病基因相关的变化将被用于设计分析方法，育种者可以使用这些方法通过正常的育种方法(杂交)将抗性纳入他们的优良育种群体中。该项目将提供对广谱抗药性的科学基础的理解。一旦了解了这种机制，就应该有可能在广泛的经济重要作物品种中部署这种抗性。

The project is a collaboration between Warwick HRI working on the dicotyledonous brassica plants and Rothamsted Research working on the monocotylonenous plants barley. The main aim is to: exploit sources of recessive eIF4E-based resistance to potyviruses in these plants. The involvement of the plant gene eIF4E in the life-cycle of viruses was first discovered in experiments on Turnip mosaic virus (TuMV, a potyvirus) and Arabidopsis. Subsequently it was found that the genes eIF4E or its isoform (eIF(iso)4E) are responsible for natural resistance to potyviruses in a range of crop types. The basis of the resistance is that potyviruses require one of these genes in order to complete their life-cycle. It is known that plants with specific alterations in these genes that affect the binding between the plant protein it codes and a viral protein (VPg) are resistant to infection. We have shown that eIF4E is associated with resistance to potyviruses in brassica and barley. The resistances described so far in other plant types involving eIF4E alone are not effective against all strains of the particular potyvirus. However, we have shown that in a brassica, eIF4E in combination with another gene (retr01), confers resistance against all strains of the potyvirus (TuMV). Other genes are involved in the interaction between the viral VPg protein and eIF4E, these include a the very similar gene eIF(iso)4E and other associated genes (eIF4G, eIF4A and isoforms of these). By studying the natural variation in the eIF4E and eIF(iso)4E genes from resistant and susceptible Brassica rapa and barley lines we aim to find new sources of resistance and understand the mechanism of the resistance. We will screen a collection of plants representing 95% of the genetic variation available within the brassica species and a barley diversity panel consisting of up to 800 landraces. Those plants that differ within the eIF4E gene will be tested with informative isolates of the potyviruses to check for resistance/susceptibility. Any eIF4E or eIF(iso)4E variants conferring resistance will be sequenced. This will be supported by looking at plants that have been mutated with chemicals to identify other novel changes in eIF4E and eIF(iso)4E alleles. Lines with such changes will be tested for resistance to a panel of BaMMV and BaYMV isolates. The aim is to identify forms of eIF4E that confer resistance that is durable to all (or most) virus isolates. Using the crystal structures available for eIF4E a model will be generated for eIF4E from barley and B. rapa. Natural and chemically-induced changes in the building blocks (amino acids) of the eIF4E associated with virus resistance in barley and B. rapa will be mapped on the 3D model of the normal eIF4E proteins. The effect of the changes on the potential ability of barley and B. rapa eIF4E to bind to the viral VPg protein will then be investigated. The outcome of this work will be the prediction of superior resistance genes of barley and B. rapa eIF4E for deployment in breeding. A gene retr01 which in combination with eIF4E confers the broad-spectrum resistance to TuMV in B. rapa will be fine mapped to identify the gene's position in the plant genome. The sequence of the gene will be used to search databases to predict its function, understand the mechanism of the broad-spectrum resistance and how retr01 might interact with eIF4E. Based on the best sources of virus resistance in brassicas and barley, changes associated with the genes conferring resistance will be used to design assays which can be used by breeders to incorporate the resistances into their elite breeding stocks by normal breeding methods (crossing). The project will provide an understanding of the scientific basis of the broad-spectrum resistance. Once the mechanism is understood it should be possible to deploy such resistance in a wide range of economically important crop species.