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

Lead Research Organisation: John Innes Centre
Department Name: Biological Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Microbial pathogens deliver protein molecules called "effectors" to the inside of plant cells to re-program the host and make it a better environment for the invader, leading to disease. However, these effectors are also a pathogen's Achiles' heel as their presence can be sensed by host immune receptors leading to the plant fighting back, mounting a defence response. The research funded on this grant focused on a plant immune receptor from rice, and effector proteins from the pathogen that causes the most devastating fungal disease of rice (rice blast). Our main key findings are:

1. Activation of signaling by a plant intracellular immune receptor can be mediated by direct binding via an integrated domain (like a molecular handshake)
2. The strength of signaling in planta is directly linked to the strength of binding between the receptor and the effector (measured using purified proteins)
3. The effectors studied here bind to other related host proteins in the same way, these are the proteins through which the effectors promote disease. This establishes an evolutionary relationship between effector host targets and immune perception.
4. We can generate variation in the immune receptors that enhances effector binding and strengthens the signalling response in plants, opening opportunities to improve the utility of these receptors in agriculture through plant breeding.








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 todefine 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.
Award Abstract

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.
Exploitation Route I anticipate that we and others will build on our findings in this system and extend them to other related areas. I also anticipate that through collaboration we will breed new rice cultivars with bespoke disease resistance properties.
Sectors Agriculture, Food and Drink

 
Description ERC Advanced Investigator
Amount € 2,500,000 (EUR)
Funding ID BLASTOFF 743165 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2017 
End 08/2022
 
Description The Royal Society International Exchanges Cost Share 2017 Japan (JSPS) award for overseas travel between collaborators in the UK and Japan
Amount £50,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 03/2020
 
Description Collaboration with Chatchawan Jantasuriyarat, Kasetart University, Thailand 
Organisation Kasetsart University
Country Thailand 
Sector Academic/University 
PI Contribution Shared research project, joint publications, grant applications, student exchange
Collaborator Contribution Shared research project, joint publications, grant applications, student exchange
Impact student exchange, joint publication
Start Year 2017
 
Description Collaboration with Hiromasa Saitoh, Tokyo University of Agriculture 
Organisation Tokyo University of Agriculture
Country Japan 
Sector Academic/University 
PI Contribution Joint publications, sharing research materials, intellectual contribution, grant applications
Collaborator Contribution Joint publications, sharing research materials, intellectual contribution, grant applications
Impact This is a new collaboration, developed from a previous collaboration. No direct outcomes yet.
Start Year 2017
 
Description Collaboration with Lab of Ryohei Terauchi at IBRC in Iwate and Kyoto University, Japan 
Organisation Iwate Biotechnology Research Centre
Country Japan 
Sector Public 
PI Contribution Leading collaborative research project on Structure/function studies of rice blast disease and host resistance.
Collaborator Contribution Collaborative work on research project.
Impact Research publications and BBSRC grant funded (M02198X).
Start Year 2011
 
Description Press release for research paper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release about a research publications
Year(s) Of Engagement Activity 2018
 
Description VISCEA - Plant Biotic Stresses and Resistance Mechanisms III, International organising committee 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact VISCEA - Plant Biotic Stresses and Resistance Mechanisms III, International conference
Year(s) Of Engagement Activity 2018