Molecular mechanisms of virulence and avirulence in the Avr3a family of Phytophthora.

The oomycetes are fungus-like eukaryotic microorganisms. Several species of the oomycetes are devastating pathogens that are of great importance to world agriculture and food security. In essence, this means that they destroy crops that are critical sources of food. The word 'Phytophthora', which is a genus with the oomycete lineage means 'plant destroyer'. Phytophthora species include the organism responsible for the Irish potato famine, Phytophthora infestans, which causes a disease commonly know as 'potato blight' or 'late blight' (it also causes tomato blight), as well as pepper blight (Phytophthora capsici) and soybean root rot (Phytophthora sojae). Recently, Phytophthora ramorum has gained considerable press in the UK as it is threatening iconic trees, such as oak. Phytophthora infestans remains the best-known and, arguably, the most important oomycete pathogen. It continues to cost modern agriculture billions of pounds annually and also impacts subsistence farming in developing countries. With potato now ranked the third most important crop in the world, P. infestans is an important biotic threat to global food security. Damage caused by other Phytophthora species is also severe, on a global scale. Our long-term objective is to understand how oomycetes, particularly Phytophthora, successfully infect plants and dissect the plant processes that are affected by these pathogens. It is now well established that, like other pathogens, Phytophthora species secrete a number of proteins, termed 'effectors', that modulate the immune response of plants and enable host colonization. Deciphering the biochemical activities of effectors is critical to understanding mechanisms of pathogenesis. Among these proteins, RxLR-type effectors, named after an amino acid sequence present in the protein, are targeted to the inside of plant cells. In this proposal, we focus on a particular group of RxLR effectors that is found in the Phytophthora species mentioned above. These are all members of the Avr3a family, as they share amino acid sequence homology to the Phytophthora infestans effector Avr3a. Building on preliminary data we have already obtained, we aim to define what conserved and divergent functions these proteins have in suppressing the plant immune system. Through host factors, members of this protein family can also initiate a programmed cell-death response in plants designed to limit pathogen growth. We also aim to understand some details of how early steps in this response occur, specifically how these effectors are detected by host cell factors. This study will help to establish functional connections between pathogen proteins and plant processes. Detailed knowledge of how the Avr3a family is able to manipulate certain aspects of plant immunity and be recognized by others will improve our understanding of the infection process and enable novel strategies for engineering resistance to Phytophthora pathogens.

This research proposal aims to characterize the conserved and divergent virulence activities of the Avr3a family of Phytophthora RxLR-type effectors. It will also provide insights into the molecular recognition of RxLR effectors by resistance (R)-proteins. These studies will show how Phytophthora spp. attempt to manipulate host cell processes and how the plant responds to these challenges, with the potential to deliver novel approaches to control important plant diseases. Building on our preliminary data we will use in planta expression assays to provide a comprehensive analysis of Avr3a family members' activities in interfering with pathogen-triggered-immunity (PTI) signaling pathways elicited by known PAMPs of diverse origin. We will link these activities with host protein interactions using standard biochemical approaches. We will then use structure-based mutagenesis of sites on the protein surface, predicted to encode function (based on our crystal structure of Avr3a11 from P. capsici), to dissect the functions of the Avr3a family and map them to structure. We will also build on our preliminary data to define the molecular determinants that allow recognition of different Avr3a family members by a library of variant R3a resistance proteins. Firstly, using agrobacterium-mediated co-expression in N. benthamiana we will establish which residues in variant R3a's are responsible for expanding recognition to include P. capsici Avr3a11. Secondly, we will use structure-based mutants of P. capsici Avr3a11 to define the residues in the effector that are important for R3a interaction. We will then screen the variant R3a library to discover new resistances against other Avr3a family members. Finally, we will generate transgenic N. benthamiana expressing variant R3a proteins and test whether they confer resistance to pathogens from which the cognate effector is derived.

Plant diseases represent a significant threat to human welfare resulting in economic hardship, malnutrition, famine, and environmental degradation. Oomycetes are the most devastating pathogens of dicot plants, causing vast economic losses on important crop species and environmental damage in natural ecosystems. With the oomycetes, species are the most significant pathogens of plants. P. infestans is considered one of the most important biotic threats to global food security and annual losses in potato production caused by late blight are conservatively estimated at $6.7 billion. Management of Phytophthora spp. in the field is difficult and requires repeated applications of chemicals, some of which might be banned in the near future due to concerns over environmental damage. The breeding of resistant cultivars has seen limited success, but these resistances are often quickly overcome. Despite this, breeding resistant crops, through breeding programs or GM approaches, remains a critical approach for combating disease. The advantages conferred by engineered R-proteins with extended recognition specificities is one example of an exciting new approach that may deliver more durable resistances. Non-academic groups who would benefit from the research proposed, and be potential users of its outputs include: plant biotechnology companies; the agricultural community, including farmers (both in the UK and worldwide); policy makers; the general public (local interest groups, home gardeners, schools etc.). The PIs will take the lead on managing the impact plan. Both the PIs have excellent track records in communicating the outcomes of their research to a broad audience. Primarily this is through publication in academic journals, but also increasingly in open-source forums. We are also invited speak about our research at national/international meetings. The PIs have prior experience at writing scientific and general articles, as well as developing websites. The project offers unique training opportunities in multiple disciplines. An individual training plan will be implemented for the RAs and they will attend regular group meetings in the PI's laboratories to present their data and interact formally with colleagues. They will have weekly one-on-one meetings with their respective PI to discuss progress and direction. In addition, monthly project meetings involving the PIs, RAs and other relevant scientists will be conducted. The PDRA will also participate in relevant seminar series and present a seminar to the whole Department once a year. The RAs will also be able to interact with outstanding collaborators and meet the frequent national/international visitors to the site. The PIs regard these activities as an important part of mentorship and professional development. TSL and JIC have a Technology Transfer policy based on maintaining close links with those who are able to make use of discoveries for the benefit of society. Discoveries at TSL/JIC are monitored to establish whether they present opportunities to obtain Intellectual Property Protection (IPP). This is typically through patenting. Where impact activities include technology transfer or outreach/press releases the relevant offices at TSL/JIC will be involved. Throughout his career Kamoun has received funding from the plant biotechnology sector. We also engage with non-expert non-academic groups. For instance, Banfield is involved with initiatives to enthuse school children about science including the SAW (Science, Art and Writing) and TSN (Teacher-Scientist Network) initiatives. The JIC and TSL also engage with the local community thorough the 'Friends of the John Innes Centre' and events such as the 'Discovery Day'; Kamoun recently delivered a talk to > 200 members of the general public at a Food Security event at JIC. TSL/JIC has a dedicated communications office for release of information to the general public through websites and the media.