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成功，但未来的挑战是为不断增长的人口和对粮食安全的期望维持农业。到目前为止，成功在很大程度上要归功于减少病原体损失的能力，尤其是真菌，这在很大程度上要归功于商业作物品种中培育的杀虫剂和抗病能力的结合。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疾病的抵抗力，杀虫剂随着对健康和环境的影响而变得无效或过时。因此，当务之急是加深我们对疾病的了解，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主要作物病原体是生物营养真菌，它们与寄主建立了高度亲密和复杂的相互作用。谷类霉病就是这样的例子。这些真菌能够穿透细胞壁屏障，形成高度复杂的细胞内摄食结构--吸器。最近发现，除了营养吸收外，病原体还能够控制宿主的新陈代谢和免疫力。这使它们能够克服抗性，抑制防御，并在植物组织内毫发无损地存活，直到繁殖和传播。在这项提议中，我们旨在发现大麦白粉病通过吸器和所谓的效应器的作用建立对宿主细胞控制的分子机制的细节。吸器和所谓的效应器是传递给宿主的蛋白质，是实际影响这种控制的因素。为了做到这一点，我们结合了英国和美国三个研究小组的专业知识，将大麦白粉病基因组学、蛋白质分析和抗病方面的世界级能力结合在一起。我们计划1)调查候选霉菌效应物，2)测试和验证它们在植物中调节疾病和抗性的功能，3)确定与植物中效应物物理相互作用的分子，即寄主中的效应物靶标。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，将选择那些带有候选效应器霍尔标记的蛋白质，例如包括以它们为分泌目标的分子指纹。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和毒力的功能有关。2)将通过将候选效应器输送到大麦细胞并观察这如何改变对霉菌的敏感性来测试候选效应器。将通过修改细菌将特定蛋白质注入植物细胞或轰击包裹有效应器编码DNA的微观颗粒来实现传递。然后，将处理后的叶片接种霉菌，并对侵染率进行评分。真正的效应蛋白将是那些改变疾病的蛋白。3)经过验证的效应器将被用来‘钓鱼’它们结合的宿主蛋白，从而与之相互作用--从而确定候选效应器靶标。这种相互作用将在植物本身和受感染植物的提取物中寻找。然后将对分子相互作用及其生物化学进行描述。这项研究的结果将为详细了解谷类霉病潜在的分子机制奠定基础。这有可能有助于未来在抗病和管理方面的发展，以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.