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

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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.