An effector-detector domain in a rice immune receptor: towards structure-guided design of new disease resistance proteins.

Every growing season, significant losses are realised to many of the world's most important crop harvests because of diseases caused by pathogenic micro-organisms, such as fungi, oomycetes and bacteria. This is against the backdrop of increasing demand for food, which continues to rise as the world's population grows and there are changes in diets. Many scientific resources, from traditional plant breeding to chemical control (e.g. fungicides/pesticides), are available to help limit the impact of pathogens on crop yield. However, many of these approaches may have only short-term effects or are environmentally unsustainable. New ways to control plant diseases are required. One way to develop novel control strategies is by understanding the intricate mechanisms of how pathogens cause disease or evade detection by the plant immune system. By understanding these processes we can develop ways to engineer plants to help them fight infection.

One mechanism used by plants to fight infection is to detect pathogen agents that are attempting alter plant cells for the benefit of the pathogen. These agents, known as "effectors", give away the presence of the pathogen and plants have evolved to sense these. This triggers a response by the plant that helps stop the infection spreading. The plant sensors can work by directly contacting the effectors, like a handshake, but the important details of how this actually occurs are not known. The plant has to be very precise about knowing if a pathogen molecule is present, and all it may have to go on is the shape of the "hand" (imagine trying to identify one person in a room of 10,000 only by the shape of their hand). The plant cell will induce death of the cell if it senses the effectors, so it has to get it right.

We have been studying the interactions of a set of pathogen effectors from a microorganism (fungus) that produces a devastating disease of rice, a major food crop that many people rely on for calories. We have been able to define the nature of the "handshake" between one of these effectors and a plant sensing protein. This is just a snapshot of the interaction, and we need to understand its implications using biochemistry and biological studies in plants. To do this we will use a number of experimental approaches. Firstly, we will define how strong the handshake is between the pathogen effector and plant sensor. Secondly, we will make small changes to the shape of the molecules and see how this affects the strength of the handshake. We will then investigate some small changes that have happened in these pathogen and plant molecules during evolution in nature, using the picture of the handshake we have determined to help us. We also wish to understand why the pathogen is producing the molecules it does, and we will ask questions about what these molecules are doing. Finally, after we understand the interactions formed by the various handshakes in this system, we will engineer a plant sensor protein that can recognise more than one pathogen molecule - which could give the plant a better chance of fighting off infection. We will also see if we can get this to work in a plant other than rice, to see if our new knowledge can help other crops recognise their pathogens.

The aims of this proposal are to define the molecular mechanisms underlying recognition of a rice blast effector protein family (AVR-Pik) by an unconventional domain incorporated into rice resistance proteins (Pik-HMAs) during evolution. Arms-race co-evolution, developed through direct protein-protein interaction, has resulted in an allelic series of AVR-Pik effectors and Pik resistance proteins that show deferential recognition patterns. This affects the capability of rice cultivars to respond to infection. Understanding the structural basis of recognition between these effectors and plant resistance proteins presents opportunities to engineer novel disease resistance specificities in rice, and perhaps other plant species.

To deliver on our objectives, we propose a novel multi-disciplinary approach combining biochemistry, structural biology and plant biology, with the latter directly in the host pathosystem. Building on our preliminary data, which includes the first example of a structure of a plant pathogen effector bound to a plant intracellular immune receptor, we will interrogate the interactions between AVR-Pik effectors and Pik resistance protein domains in vitro and in planta. This will include both mutational analysis based on our structural work and also natural variants of AVR-Pik and Pik-HMA domains. Further, we have identified proteins called s-HMAs as putative susceptibility factors targeted by AVR-Pik effectors. Fascinatingly, these s-HMAs are sequence (and presumably structurally)-related to the resistance protein HMA domains. We will also characterise the interaction and activity of AVR-Pik effectors with these s-HMAs. Finally, we will use structure-guided mutagenesis to engineer Pik resistance proteins with novel, extended recognition specificities (to include as-yet unrecognised AVR-Pik alleles) and also transfer HMA-mediated recognition to NLRs of other plants.

Who will benefit from this research and how will they benefit?

This project will contribute knowledge and resources of relevance to the Food Security Strategic Priority area of the BBSRC.

Academics: Details of academic beneficiaries (including details of staff training) are given in the appropriate section.

The Ag-biotech and Agrichemical industries: Despite the importance of understanding the molecular mechanisms underlying plant innate immunity and how pathogens manipulate this to cause disease, little research is conducted by industry in this area. Current disease management strategies for fungi from industry mainly focus on the use of chemical interventions (fungicides). Into the future this will be unsustainable, especially as some chemicals are due to be banned. Novel genetic strategies are required to deliver plant disease resistance in important food crops. Therefore, industry will benefit from this research through an increased knowledge of disease processes that may present new opportunities for disease management. Industry could also benefit from technological developments, such as the application of structural biology and biophysical approaches to understanding plant diseases, running alongside in planta work.

Farmers: Although a challenging aim and a long-term goal, the research in this project (alongside other studies) has the potential to impact disease management strategies in the field. This may benefit commercial farmers by, for example, limiting the need for repeated spraying of fungicides on crops. It may also benefit subsistence farming, where growers do not have access to means of chemical disease control.

Policy Makers and Regulators: Government Departments and senior academics will benefit from new perspectives on opportunities for management of important crop diseases of relevance to Food Security. This will help them to formulate strategies to fast-track adoption of new/emerging technologies.

The general public: Diseases such as potato and tomato late-blight are well-known to the UK public, but perhaps other important diseases that affect global food security are not so well known. As part of our research we will aim to better present such diseases through public engagement with the research. The general public will largely benefit through knowledge exchange (local interest groups, home-growers), but there is also potential for benefit of new resources.

Education: Through engagement with schools, the education sector has the potential to benefit from the research through knowledge exchange. Especially relevant is the long-term potential of the research to contribute to food security, global sustainability (limiting potentially damaging chemical inputs into the environment) and to enthusing the next generation of scientists.

Staff training: This project will employ a PDRA and a PGRA. As part of the research they will receive specific training in diverse experimental techniques that could benefit a future career in research. They will also receive training in generic 'transferable' skills such as writing, oral presentation and outreach to the public and schools. These latter skills would be beneficial in any employment sector.