Functional analysis of the Blumeria Haustoria - Barley interactome

安全、可靠、负担得起和充足的粮食和原料来源-粮食安全-对我们个人和社会的福祉以及全世界的发展至关重要。农业在实现这一目标方面取得了巨大的成功，但今后的挑战是如何维持农业，以满足不断增长的人口和对粮食安全的期望。迄今为止的成功在很大程度上要归功于减少病原体，特别是真菌损失的能力，通过将杀虫剂和抗病性结合起来培育成商业作物品种。问题是病原体的进化是为了克服这些障碍：新的病原体菌株打破了抗病能力，杀虫剂因人们担心其对健康和环境的影响而变得无效或过时。因此，我们必须进一步了解这些疾病，以保持和提高我们养活自己的能力。一些最成功和最具破坏性的病原体的主要作物是生物营养真菌，建立高度密切和复杂的相互作用与他们的主机。这些的例子是谷物霉菌。这些真菌能够穿透细胞壁屏障并形成高度复杂的细胞内进食结构-'吸器'。最近已经变得明显的是，除了营养摄取之外，病原体还能够控制宿主的代谢和免疫。这使它们能够克服抗性，抑制防御，并在植物组织内安然无恙地生存，直到繁殖和传播。在这个建议中，我们的目标是发现大麦白粉病通过吸器和所谓的效应物的作用建立对宿主细胞的控制的分子机制的细节：蛋白质被递送到宿主并且是实际影响这种控制的试剂。为了做到这一点，我们联合收割机在英国和美国的三个研究小组的专业知识，汇集了世界一流的能力，在白粉病基因组学，蛋白质分析和大麦抗病性。我们计划1）调查候选霉菌效应子，2）测试和验证它们在植物中调节疾病和抗性的功能，以及3）鉴定与植物中的效应子（即宿主中的效应子靶标）物理相互作用的分子。1)调查将包括利用和系统地扩展对吸器中霉菌实际产生的蛋白质的描述。在这些蛋白质中，将选择具有候选效应物的标志的那些蛋白质，例如包括靶向它们用于分泌的分子指纹。此外，我们将测试由基因编码的蛋白质，这些基因与已知影响抗病性和毒力的功能有关。2)候选效应子将通过将它们递送到大麦细胞并观察这如何改变对霉菌的易感性来进行测试。传递将通过修饰细菌以将特定蛋白质注入植物细胞或通过轰击涂覆有效应器编码DNA的微观颗粒来实现。然后用霉菌接种处理过的叶子，并对感染率进行评分。真正的效应蛋白是那些改变疾病的蛋白。3)然后，验证的效应物将用于“钓”它们结合并因此相互作用的宿主蛋白质-从而定义候选效应物靶标。将在植物本身和受感染植物的提取物中寻找相互作用。然后将表征分子相互作用及其生物化学。本研究的结果将为深入了解禾谷霉变的分子机制奠定基础。这有可能有助于未来抗病、管理方面的发展，促进后代的粮食安全和生产。

The safe, reliable, affordable and sufficient source of food and feedstock - food security- is essential for our well-being as individuals and society and for development world-wide. Agriculture has been hugely successful in delivering this but the challenge ahead is to sustain it for an ever increasing population and expectation of food security. Success so far has in great part been thanks to the ability to reduce loss to pathogens, especially fungi, by the combined us of pesticides and disease resistance bred into commercial crop varieties. The problem is that pathogens evolve to overcome these barriers: disease resistance is broken by new pathogen strains, pesticides become ineffective or obsolete as concerns emerge about their impact on health and the environment. It is therefore imperative to further our understanding of the diseases to maintain and improve our ability to feed ourselves. Some of the most successful and devastating pathogens of the main crops are biotrophic fungi which establish highly intimate and sophisticated interactions with their hosts. Examples of these are the cereal mildews. These fungi are able to penetrate the cell wall barrier and develop a highly complex intracellular feeding structure - the 'haustorium'. It has recently become apparent that in addition to nutrient uptake, pathogens are capable of taking control of the host's metabolism and immunity. This enables them to overcome resistance, suppress defence and survive unharmed inside the plant tissues for as long as it takes to reproduce and disseminate. In this proposal we aim at discovering details of the molecular mechanisms by which barley powdery mildew establishes control of the host cell through the action of the haustorium and so-called effectors: proteins that are delivered to the host and are the agents that actually effect this control. In order to do this we combine the expertise of three research groups in the UK and the USA to bring together world-class abilities in the powdery mildew genomics, protein analysis and disease resistance in barley. We plan to 1) survey candidate mildew effectors, 2) test and verify their function in modulating disease and resistance in the plant and 3) identify the molecules that interact physically with the effectors in the plant, i.e. the effector targets in the host. 1) The survey will include making use of and systematically extending the description of the proteins actually made by mildew in the haustorium. Of these a selection will be made of those proteins that bear the hall-marks of candidate effectors, for example include molecular fingerprints that target them for secretion. Additionally, we will test proteins encoded by genes that are linked to functions known to affect disease resistance and virulence. 2) The candidate effectors will be tested by delivering them to barley cells and observing how this alters the susceptibility to mildew. Delivery will be achieved either by modifying bacteria to inject specific proteins into the plant cell or by bombarding microscopic particles coated with effector-encoding DNA. The treated leaves will then be inoculated with mildew and the rates of infection scored. True effector proteins will be those that alter disease. 3) Validated effectors will then be used to 'fish' host proteins they bind to and therefore interact with - thus defining candidate effector targets. The interactions will be sought both in the plant itself and in extracts from infected plants. The molecular interactions and their biochemistry will then be characterised. The outcome of this research will lay the foundation of a detailed understanding of the molecular mechanisms underlying disease in the cereal mildews. This has the potential of aiding future developments in disease resistance, management, to promote food security and production for generations to come.