Phosphatidylinositides defining effector protein delivery in Phytophthora

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.

Oomycete plant pathogens, exemplified by the potato and tomato late blight pathogen Phytophthora infestans, cause some of the most destructive crop diseases. Infection is driven by delivery of effector proteins into host tissues. In Phytophthora, effectors can be secreted via at least two different pathways. Protein secretion involves coordination of specialised membranous organelles and vesicles. Membranes can be differentiated by the presence of specific phosphatidylinositol phosphates (PtdIns) that are interconverted by lipid kinases and phosphatases. Inhibition of the kinases and phosphatases that synthesise PtdIns can potentially disrupt membrane trafficking, block effector protein secretion and control disease. The occurrence and localisations of PtdIns and their associated PtdIns kinases (PIKs) and phosphatases are largely unknown in Phytophthora.

We propose to use PtdIns binding proteins, fused to fluorescent proteins, as biosensors in P. infestans to reveal which PtdIns are present in secretory pathways. We will localise infection-up-regulated lipid kinases/phosphatases to reveal major sites of PtdIns modification during infection, and their association with secretory pathways. PtdIns-4-kinases (PI4Ks) and PI3Ks are potentially associated with secretory pathways, thus we will use pan-PI4K/PI3K and Phytophthora-specific PI4K inhibitors to determine their impact on effector secretion pathways in P. infestans. Finally, we will use gene silencing to remove the activity of specific infection up-regulated PI4Ks, PI3K and phosphatases to determine their importance for pathogenicity and effector secretion in P. infestans, thus providing potential targets for agrochemical control.