Host translocation requirements of the cytoplasmic Crinkler (CRN) effector protein family in Phytophthora

卵菌形成了真核生物丝状病原体的一个独特的谱系，这些病原体会在对农业、林业和自然生态系统重要的广泛植物上造成毁灭性的疾病。例如，马铃薯和西红柿晚疫病的致病菌--致病疫霉继续在世界各地造成困难，每年造成数十亿英镑的损失。在过去的十年中，松材线虫病和核盘菌作为许多乔木和灌木的毁灭性病害迅速出现，引起了人们对寄主范围广泛的病原菌的经济和生态意义的关注。重要的是，辣椒疫霉是番茄、辣椒和葫芦科(南瓜、甜瓜等)的重要病原菌，最近在豆科植物和冷杉上引起了病害的暴发，是另一种新出现的病原菌，寄主范围跨越20多种植物。对可持续粮食供应的需求上升，加上疫霉引起的作物损失和环境破坏的持续威胁，需要采取有效措施保护作物生产和环境。了解疫霉的分子过程。因此，用来促进疾病的做法是制定持久和可持续控制做法的核心。对疫霉-寄主相互作用的细胞生物学研究表明，在感染的早期阶段，当丝状菌丝突破细胞壁屏障并与植物细胞膜接触时，形成称为吸器的特殊病原菌结构。然而，在敏感植物中，穿透寄主细胞的完整性没有受到影响，这表明疫霉阻止了对菌丝作为异物的感知，或抑制了随后的植物免疫反应。现在已经证明，疫霉通过分泌共同操纵宿主细胞的效应蛋白来抑制免疫反应。旨在了解效应器的努力已经确定了在宿主-病原体界面分泌的RXLR蛋白，并被转移到植物细胞中，在那里它们操纵宿主。我们认为，对介导效应器传递的分子组成的了解将为控制策略提供关键目标，因为它们的干扰可能会阻止疾病的发展。最近，一组新的细胞内效应蛋白被鉴定出来。Crinkler(CRN)形成了一个庞大而多样的分泌型模块蛋白家族，在广泛的已测序卵菌基因组中被发现。随后的研究表明，在疫霉侵染过程中，CRN效应器被转移到植物细胞中，在那里它们可能会干扰细胞过程。因此，这项工作的早期目标是确定CRN效应器在感染过程中分泌的确切位置，开发新的方法来报告和可视化宿主细胞内的易位，并使用这些分析来确定CRN效应器N端的氨基酸，这些氨基酸是宿主靶向和易位所必需的。通过宿主-病原体界面运送效应物必须将病原体的生长和发育与蛋白质分泌和运送结合起来，这一过程可能需要复杂的蛋白质-蛋白质相互作用。因此，一个关键的目标是鉴定和表征来自病原体和/或宿主的蛋白质，这些蛋白质与参与效应器递送的关键CRN N末端氨基酸物理上相互作用。这些蛋白质对易位的贡献将通过沉默相应的基因和使用该项目早期开发的报告系统调查效应器传递来测试。在这项工作中，我们将使用辣椒疫霉，它不仅在世界范围内成为一种日益重要的病原体，而且由于其基因组序列的可用性以及最近开发的高效快速转化系统，它还作为一种关键的卵菌模式，用于详细研究支撑毒力的基本过程。

Oomycetes form a distinct lineage of eukaryote filamentous pathogens that cause devastating diseases on a wide range of plants important to agriculture, forestry and natural ecosystems. For example, Phytophthora infestans, the causal agent of late blight on potato and tomato, continues to cause hardship throughout the world with multibillion pound losses each year. In the past decade, the rapid emergence of P. ramorum and P. kernoviae as devastating diseases of many tree and shrub species has brought to attention the economic and ecological significance of broad-host-range pathogens. Importantly, P. capsici, an economically important pathogen of tomato, (chili) pepper and cucurbits (squash, melon etc), has recently caused disease outbreaks on legumes and Fraser fir, and is thus another emerging pathogen with a host range that spans over 20 plant species. The rising demand for sustainable food supplies, coupled with the continued threat of Phytophthora-incited crop losses and environmental damage, require effective measures to protect crop production and the environment. Understanding the molecular processes that Phytophthora spp. employ to promote disease is therefore central to developing durable and sustainable control practices. Cell-biological studies on Phytophthora-host interactions reveal that, in the early infection stages, specialized pathogen structures called haustoria are formed when filamentous hyphae breach the cell wall barrier and interface with the plant cell membrane. Integrity of the penetrated host cell in susceptible plants is not affected, however, suggesting that Phytophthora prevents perception of hyphae as foreign bodies, or suppresses subsequent plant immune responses. It has now been shown that Phytophthora suppresses immune responses through secretion of effector proteins that collectively manipulate host cells. Efforts aimed at understanding effectors have identified RXLR proteins that are secreted at the host-pathogen interface and are translocated into plant cells where they manipulate the host. We propose that an understanding of the molecular components mediating effector delivery will provide key targets for control strategies, as their disruption may prevent disease development. Recently, a new set of intracellular effector proteins was identified. The Crinklers (CRNs) form a large and diverse family of secreted modular proteins that have been identified in a wide range of sequenced oomycete genomes. Subsequent studies have demonstrated that CRN effectors are translocated into plant cells during Phytophthora infection, where they may interfere with cellular processes. Early aims of this work are therefore to define the exact sites of CRN effector secretion during infection, to develop novel approaches to report on and to visualize translocation inside host cells; and to use these assays to determine the amino acids within the N-terminus of CRN effectors that are required for host targeting and translocation. Delivery of effectors to and across the host-pathogen interface must integrate pathogen growth and development with protein secretion and delivery, processes that are likely to require complex protein-protein interactions. A key aim is thus to identify and characterize proteins from pathogen and/or host that physically interact with critical CRN N-terminal amino acids involved in effector delivery. The contribution of these proteins to translocation will be tested by silencing the corresponding genes and investigating effector delivery using the reporter systems developed earlier in the project. For this work, we will use P. capsici which is emerging not only as an increasingly significant pathogen worldwide, but also as a key model oomycete for detailed investigation of basic processes that underpin virulence, due to the availability of its genome sequence combined with the recent development of an efficient and rapid transformation system.