Engineering cereal immunity using structure-guided design of effector/host interactions.

Every year, significant yields of our key global food crops are lost to pre-harvest plant disease. These diseases are caused by pathogenic micro-organisms, such as fungi, oomycetes and bacteria. These yield losses are set against the world's increasing demands for food, which continue to rise as the world's population grows and there are changes in dietary habits. This is also set against the backdrop of the impact of climate change on plant growth.

Traditional plant breeding approaches and chemical control (via fungicides and pesticides), can help limit the impact of pathogens on pre-harvest crop yield. However, many of these approaches may have only short-term effects, or are unsustainable due to the environmental impact of production and use. New ways to control plant diseases are required, and genetic forms of disease resistance offer the potential for environmentally friendly, sustainable agriculture. 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.

Pathogens use agents that they deploy into plant cells to alter the environment for the benefit of the pathogen, usually by interacting with plant cell components. These agents, known as "effectors", can also give away the presence of the pathogen as the plant immune system has evolved receptors to sense the presence and/or activity of these agents. These plant sensors can work by directly contacting the effectors and their interactions with host cell components, a bit like a handshake, but the details of how this actually occurs are not well 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 an effector, 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. This pathogen can also cause serious disease of barley and wheat crops, so is a major concern around the world. We have defined a picture of the "handshake" between one of these effectors and a plant cell component, which is then sensed by the plant immune system. But this is just a snapshot of the interaction, and we need to understand the implications of the interaction much better using biochemistry and biological studies in plants.

To do this we will use a number of experimental approaches. Firstly, we will define how strong and specific the handshake is between the pathogen effector and the plant cell component it targets, and how important this is for immunity. As part of this we will make small changes to the shape of the molecules and see how this affects the strength of the interaction. Then we will perform experiments to ask questions about the diversity of activity of the family of effectors we have been studying as there maybe additional targets in plant cells. Finally, after we understand the interactions formed by the handshakes in this system, we will seek to engineer these interactions and observe whether this improves the robustness of the plant immune system in rice, but to also see if we can transfer the disease fighting capability to barley and, in the longer term, wheat.

One strategy to limit pre-harvest yield loss to disease is to understand how the plant immune system works to recognise pathogens at the biochemical and structural level and apply this information in plants. Our aims in this proposal are to (i) define the molecular mechanisms that underlie immune recognition of the rice blast effector AVR-Pii by the NLR Pii, through the interactions of this effector with homologs of the host target Exo70, a component of the Exocyst complex, (ii) investigate extended interaction profiles of a new family of effectors (ZiFs, to which AVR-Pii belongs); (iii) engineer Exo70 proteins and NLRs for improved recognition capabilities and transfer this resistance to barley and wheat.

To deliver on our aims, we propose a multi-disciplinary approach combining biochemistry, structural biology, genetics and plant pathology, with the latter directly in the relevant host pathosystems. Building on our preliminary data, which includes the structure of the AVR-Pii effector in complex with a rice Exo70 homolog, we will interrogate the interactions between this effector and Exo70 homologs to understand specificity of immune activation by the rice NLR receptor Pii both in vitro (ITC, Y2H) and in planta (co-IP, cell death and disease resistance assays). This will include both mutational analysis and use of natural effector variants. Further, we will study the interactions of AVR-Pii and variants with NLR immune receptors that contain integrated Exo70 domains in their architecture using the same techniques. We will then use both these approaches to inform engineering studies to transfer AVR-Pii (and variant) effector recognition into barley, and in the longer term, wheat. Finally, we will use Y2H and co-IP/mass spec approaches to study the extended interactome of the newly discovered ZiF effector family of Magnaporthe, of which AVR-Pii is a member.