Phosphatidylinositides defining effector protein delivery in Phytophthora

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（Phytophthora infestans）每年就可造成100亿美元的作物损失。植物病原体通过在感染过程中向植物组织输送一系列称为效应子的毒力蛋白来致病。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物是如何分泌和转运到宿主细胞中的。我们实验室的突破性研究表明，致病疫霉的效应物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移动，并且囊泡在这些转变期间被修饰。生物膜的特性部分由膜组成决定，磷脂（称为磷脂酰肌醇（PtdIns））在其中起着重要作用。在真核生物中，亚细胞膜中存在多达七种特定形式的PtdIns，它们是可以定义和改变膜特性的重要调节剂。它们通过称为脂质激酶和磷酸酶的酶从一种形式转化为另一种形式。很少有人知道哪些PtdIns存在于疫霉属中，它们在细胞中的位置，以及哪些脂质激酶/磷酸酶将一种形式的PtdIns转化为另一种形式。我们也不知道PtdIns的合成在感染过程中的主要网站，以及是否PtdIns和相关的脂质激酶/磷酸酶的疫霉效应分泌，从而致病性显着贡献。在这个由工业合作伙伴先正达支持的工业合作奖提案中，我们将研究P. infestans分泌途径中PtdIns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PtdIns存在于致病疫霉的分泌途径中。我们将利用荧光蛋白与PtdIns结合蛋白融合制成PtdIns生物传感器，在致病疫霉中表达PtdIns，并使用共聚焦和超分辨率显微镜对其进行定位。其次，我们将关注已知参与其他生物体中蛋白质分泌的PtdIns，定位在植物感染过程中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物来定位它们的作用位点以及它们在感染期间与分泌途径的关联。第三，由于效应蛋白决定病原体感染的成功，并且蛋白分泌途径的组分可能富集特异性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的分泌，或者通过改变细胞膜运输来间接破坏。第四，我们将在致病疫霉中使用基因沉默来去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在这一过程中的重要性。我们还将评估沉默特定的脂质激酶/磷酸酶是否会导致不同的效应蛋白分泌途径的破坏Phytophthora.Our研究结果将证明PtdIns，脂质激酶和磷酸酶，以及PtdIns代谢在P. infestans感染中的关键参与，以及如何利用这些用于有针对性的农业化学品控制疫霉属疾病。

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.