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

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.

With the emerging view that pathogen effectors are the principal determinants of epidemics, an understanding of the means by which they are delivered inside host cells will be fundamental to preventing disease and securing food production. This is particularly true of emerging, broad-host-range pathogens, such as Phytophthora ramorum, P. kernoviae, and P. capsici, where the development of resistant host plants is, in many cases, not a viable strategy. In Phytophthora, in addition to the RxLR proteins, 'the Crinklers' (CRNs) have recently been implicated as a second class of intracellular effectors. Unlike RXLRs, CRNs are present in all plant pathogenic oomycetes examined. The aim of this proposal is to identify the processes that underlie secretion and delivery of cytoplasmic CRN effector proteins during infection. We will identify and characterize P. capsici and/or host proteins involved in CRN targeting, secretion and translocation by: (1) detailed expression analyses, across the infection cycle, of CRN-coding and co-expressed genes; (2) identifying the site of CRN secretion and translocation; (3) mutational determination of the N-terminal CRN translocation region; (4) conventional and split ubiquitin yeast two hybrid analyses as well as immuno-precipitation experiments to identify host and pathogen translocation factors; and (5) Localization and characterization, using gene silencing in combination with available and novel translocation assays, to assess the roles of CRN-interacting proteins in targeting and secretion of these effectors during infection. This work will reveal the processes that are required for secretion and translocation of Crinkler effectors, providing potential key molecular targets to control disease, and yielding a critical understanding of how eukaryotic plant pathogens infect their hosts.

The proposed project is relevant to the BBSRC mission and its strategic priorities of 'living with environmental change', 'systems biology' and 'crop science'. We expect that the findings from this research will aid the development of strategies for controlling Phytophthora and other plant pathogens to improve crop yields and improve food security both in the UK and, importantly, in developing countries worldwide. Improved yields will also impact the development of, for example, biofuels technologies, which to a significant extent, will depend on efficient and sustainable agricultural practices that allow production of biomass. This work will have an immediate impact on biologists studying Phytophthora biology, Plant biologists and stakeholders in industry as well as society by providing new avenues of disease control. This proposal addresses a basic and pivotal process in a group of devastating pathogens. Protein translocation, i.e. transport of proteins secreted from a pathogen, across the host membrane, is a fundamental process in eukaryote and prokaryote organisms that remains understudied. Information emanating from this work is therefore likely to result in advances that apply to other biological systems and questions. Consequently, the PI expects to initiate interdisciplinary collaborations with effector and plant biologists and groups focusing on protein trafficking, to address fundamental unknowns. With the emerging view that pathogen effectors are the principal determinants of epidemics, an understanding of the means by which they are delivered inside host cells will be fundamental to preventing disease and securing food production. This is particularly true of emerging, broad-host-range pathogens, such as Phytophthora ramorum, P. kernoviae, and P. capsici, where the development of resistant host plants is, in many cases, not a viable strategy. This work will therefore provide alternative avenues for disease control that is sustainable, broad in its scope and has a wide and positive impact on food security and relieving food scarcity in developing regions. The anticipated outputs of this research are therefore likely to have benefits to society in the longer term. This work addresses fundamental processes in an economically relevant group of pathogens. Research findings (e.g. identification of essential components for parasitism) can in principle be applied to a wider range of crop plants and Phytophthora pathogens. We therefore expect to contribute to wealth creation, new products, public health. For example, Phytophthora proteins that are deemed essential for infection are attractive targets for development of new chemical compounds that (1) limit losses and (2) are specific inhibitors of infection thus potentially reducing environmental pollution. Alternatively, plant factors involved in translocation could be modified or inhibited in order to perturb pathogen protein translocation. The Huitema Lab aims to make a positive impact on attitudes towards the (Life) Sciences. The PI will directly engage with high schools and train students with an interest in the life sciences in his lab. The PI strongly believes that exposing students to a research setting at a centre of excellence will motivate and increase interest in the sciences. In addition, the PI commits to training and improving the careers of (young) (international) early to mid-stage career scientists.