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.

The research in this proposal will be of significant interest to others investigating the function of oomycete effectors, in particular those of plant pathogens. Such end-users will directly benefit from the knowledge our novel approach to the study of effector biology delivers: using protein structure to guide further experiments to address function. However, interest in our work will not be limited to the oomycete field and will also impact research into other plant diseases caused by eukaryotic pathogens (such as fungi and rusts); some of the principles will also likely be applicable to bacterial diseases of both plants and mammals. The program will also be of significant interest to researchers engaged in understanding mechanisms of plant immunity, specifically how effectors are recognized by NBS-LRR R-proteins to initiate signaling, and how these might be engineered to extend their activity. As there are members of the NBS-LRR protein family that are also involved in mammalian innate immunity, our results should also be of interest to researchers active within this area. Management of Phytophthora spp. in the field 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. Therefore, we expect the research described in this proposal to benefit the industrial plant biotechnology sector by providing a proof-of-principle example of how this technology can work. In the long term this could have impact: within the agricultural community, including farmers (both in the UK and worldwide); with policy makers, to demonstrate alternatives to current approaches; for the general public (local interest groups, home gardeners, schools etc.). The proposal fits squarely with the remit of 'food security', and as such could contribute to ensuring a stable supply of food into the future. The project also offers exciting opportunities to build/develop experimental skills in multiple disciplines, within a stimulating research environment. It will therefore benefit the RAs employed, preparing them for the next stage of their careers. As the RAs will also be responsible for maintaining accurate records of their work, and have the opportunity to present their work in Lab./Dept. meetings, the project offers training in generic 'transferable' skills that would be applicable in any employment area.