Phosphatidylinositides defining effector protein delivery in Phytophthora

磷脂酰肌醇定义了疫霉菌中效应蛋白的传递

• 批准号: BB/X015920/1
• 负责人: Stephen Whisson
• 金额: $ 61.85万
• 依托单位: James Hutton Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX015920%2F1
• 关键词: Phosphatidylinositides; defining; effector; protein; delivery

Up to 23% of the five major food crops can be lost to pests and disease, representing a threat to global food security. The potato late blight pathogen, Phytophthora infestans, alone can cause $10bn of crop losses annually. Plant pathogens cause disease by delivering an arsenal of virulence proteins, called effectors, into plant tissues during infection. A major scientific challenge in the plant-microbe interaction field is to understand how effectors are secreted and translocated into host cells.Breakthroughs in our laboratory have revealed that effectors from P. infestans may be delivered via distinct secretion pathways. Secretion pathways involve the movement of vesicles between different subcellular locations and the vesicles are modified during these transitions. Compartment identity is in part specified by membrane composition and phospholipids known as phosphatidylinositides (PtdIns) play an important role in this. In eukaryotes, there are up to seven specific forms of PtdIns present in subcellular membranes and they are essential regulators that can define and change membrane properties. These are converted from one form to another by enzymes called lipid kinases and phosphatases. Little is known about which PtdIns are present in Phytophthora, where they are localised in the cell, and which lipid kinases/phosphatases convert one form of PtdIns into another. We also do not know the major sites of PtdIns synthesis during infection and whether the PtdIns and associated lipid kinases/phosphatases contribute significantly to Phytophthora effector secretion and thus to pathogenicity. In this Industrial Partnership Award proposal, supported by industrial partner Syngenta, we will examine the presence and synthesis of PtdIns in secretory pathways in P. infestans, and the importance of these phospholipids to secretion of specific classes of effector proteins. We envisage four inter-related objectives.First, we will determine which PtdIns are present in secretory pathways in P. infestans. We will use PtdIns biosensors made by fusing fluorescent proteins to PtdIns-binding proteins, express them in P. infestans, and localise them using confocal and super-resolution microscopy.Second, focusing on PtdIns that are known to be involved in protein secretion in other organisms, we will localise the lipid kinases and phosphatases that are up-regulated during plant infection, to identify the major sites of PtdIns transformation. We will use fusions of kinases/phosphatases to fluorescent proteins in P. infestans to localise their sites of action and their association with secretory pathways during infection.Third, since effector proteins determine the success of pathogen infection, and components of protein secretion pathways are likely to be enriched for specific PtdIns, we will use chemical inhibitors of lipid kinases to determine if effector protein secretion can be directly disrupted, or indirectly through alteration of cellular membrane trafficking.Fourth, we will use gene silencing in P. infestans to remove the activity of specific lipid kinases and phosphatases. We will test the silenced strains for their ability to infect plants, revealing the importance of PtdIns for this process. We will also assess whether silencing specific lipid kinases/phosphatases leads to disruption of different effector protein secretion pathways in Phytophthora.Our findings will demonstrate the critical involvement of PtdIns, lipid kinases and phosphatases, and PtdIns metabolism in P. infestans infection, and how these can be exploited for targeted agrochemical control of Phytophthora diseases.

在五种主要食品作物中，多达23％可能会因害虫和疾病而丢失，这代表了对全球粮食安全的威胁。马铃薯晚期的病原体，单独的疫霉菌Infestans每年就会造成100亿美元的作物损失。植物病原体通过在感染期间将毒力蛋白（称为效应子）的武器纳入植物组织来引起疾病。植物 - 微生物相互作用领域的一个主要科学挑战是了解如何将效应子分泌和易位到宿主细胞中。我们实验室中的破裂表明，来自P. Infestans的效应子可以通过不同的分泌途径传递。分泌途径涉及在这些过渡期间修改不同亚细胞位置和囊泡之间囊泡的运动。隔室的身份部分由膜组成和称为磷脂酰肌醇（PTDINS）的磷脂指定，在此中起重要作用。在真核生物中，亚细胞膜中最多有七种特定形式的PTDIN，它们是可以定义和改变膜特性的必不可少的调节剂。这些被称为脂质激酶和磷酸酶从一种形式转化为另一种形式。鲜为人知的ptdins中存在哪些ptdins，它们位于细胞中，哪些脂质激酶/磷酸酶将一种形式的ptdins转化为另一种脂质激酶/磷酸酶。我们也不知道感染过程中PTDINS合成的主要部位，以及PTDINS和相关的脂质激酶/磷酸酶是否对phytophthora效应子分泌以及因此对致病性有显着贡献。在该工业合作伙伴先正达的支持下，我们将研究PTDINS在分泌途径中的存在和合成，以及这些磷脂对分泌特定类别的效应蛋白的重要性。我们设想了四个相关的目标。首先，我们将确定哪些PTDIN存在于P. Infestans的分泌途径中。我们将使用通过将荧光蛋白融合到ptdins结合蛋白的pTDINS生物传感器，在炎症芽孢杆菌中表达它们，并使用共同体和超分辨率显微镜进行定位，并将其定位。 PTDINS转换的位点。 We will use fusions of kinases/phosphatases to fluorescent proteins in P. infestans to localise their sites of action and their association with secretory pathways during infection.Third, since effector proteins determine the success of pathogen infection, and components of protein secretion pathways are likely to be enriched for specific PtdIns, we will use chemical inhibitors of lipid kinases to determine if effector protein secretion can be directly通过改变细胞膜运输的改变，或间接地破坏。我们将测试沉默的菌株感染植物的能力，揭示PTDINS对此过程的重要性。 We will also assess whether silencing specific lipid kinases/phosphatases leads to disruption of different effector protein secretion pathways in Phytophthora.Our findings will demonstrate the critical involvement of PtdIns, lipid kinases and phosphatases, and PtdIns metabolism in P. infestans infection, and how these can be exploited for targeted agrochemical control of Phytophthora疾病。