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.

五种主要粮食作物中多达 23% 可能因病虫害而损失，对全球粮食安全构成威胁。仅马铃薯晚疫病病原体致病疫霉每年就可造成 100 亿美元的农作物损失。植物病原体在感染过程中通过将毒力蛋白库（称为效应子）传递到植物组织中来引起疾病。植物-微生物相互作用领域的一个主要科学挑战是了解效应子如何分泌并转移到宿主细胞中。我们实验室的突破表明，来自致病疫霉的效应子可能通过不同的分泌途径传递。分泌途径涉及囊泡在不同亚细胞位置之间的运动，并且囊泡在这些转变过程中发生改变。区室特性部分由膜组成决定，磷脂酰肌醇（PtdIns）在其中发挥着重要作用。在真核生物中，亚细胞膜中存在多达七种特定形式的 PtdIn，它们是可以定义和改变膜特性的重要调节因子。它们通过脂质激酶和磷酸酶从一种形式转化为另一种形式。对于疫霉属中存在哪些 PtdIn、它们位于细胞中以及哪些脂质激酶/磷酸酶将一种形式的 PtdIn 转化为另一种形式，人们知之甚少。我们也不知道感染过程中 PtdIns 合成的主要位点，也不知道 PtdIns 和相关的脂质激酶/磷酸酶是否对疫霉效应子分泌有显着贡献，从而对致病性有显着贡献。在工业合作伙伴先正达支持的这项工业合作奖提案中，我们将研究致病疫霉分泌途径中 PtdIn 的存在和合成，以及这些磷脂对特定类别效应蛋白分泌的重要性。我们设想了四个相互关联的目标。首先，我们将确定哪些 PtdIn 存在于致病疫霉的分泌途径中。我们将使用通过将荧光蛋白与 PtdIns 结合蛋白融合而制成的 PtdIns 生物传感器，在致病疫霉中表达它们，并使用共焦和超分辨率显微镜对它们进行定位。其次，重点关注已知参与其他动物中蛋白质分泌的 PtdIns。生物体中，我们将定位植物感染期间上调的脂质激酶和磷酸酶，以确定 PtdIns 转化的主要位点。我们将使用激酶/磷酸酶与致病疫霉中荧光蛋白的融合来定位其作用位点及其与感染过程中分泌途径的关联。第三，由于效应蛋白决定病原体感染的成功，并且蛋白质分泌途径的组成部分很可能为了富集特定的 PtdIns，我们将使用脂质激酶的化学​​抑制剂来确定是否可以直接破坏效应蛋白分泌，或通过改变细胞膜运输间接破坏效应蛋白分泌。第四，我们将使用基因沉默致病疫霉以消除特定脂质激酶和磷酸酶的活性。我们将测试沉默菌株感染植物的能力，揭示 PtdIns 在此过程中的重要性。我们还将评估沉默特定脂质激酶/磷酸酶是否会导致疫霉中不同效应蛋白分泌途径的破坏。我们的研究结果将证明 PtdIns、脂质激酶和磷酸酶以及 PtdIns 代谢在致病疫霉感染中的关键参与，以及这些如何可用于有针对性的农业化学防治疫霉病害。