Interplay between phosphorylation and ubiquitination in plant immune signaling homeostasis

Plant diseases contribute greatly to annual crop losses and pose a real threat to food security world-wide. Over a million people died during the Great Irish Famine in the 19th century as the result of a potato blight epidemic. Currently, the world's most popular fruit, the Cavendish banana, is under threat of extinction due to infection by highly virulent fungal pathogens [1]. Indeed, many other food- and cash-crops such as wheat, rice, maize, soybean, barley, potato, cotton, canola, and others are susceptible to many different types of diseases. There are >80 million Ash trees growing in UK forests and along neighbourhood roads currently under threat of Ash dieback disease, caused by a relentless fungal pathogen [2]. Recent estimates project that 75% of Ash trees in the south and east of England will be infected by this disease by 2018 [2]. Battling diseases that affect our crops and trees is a global challenge requiring the work of scientists in both academia and industry, as well as the work of policy-makers and government.

Pathogens are capable of infecting plants and causing disease largely because they can suppress plant immune systems. Thus, only when we clearly understand plant immunity will we be able to offer sustainable solutions to diseases that affect our crops.

The plant immune system is multi-faceted and composed of many different proteins with broad functions. The aim of this work is to find out exactly how key immune proteins work at the molecular level, how they are activated and repressed, and how they influence normal growth and development. Because pathogen responses necessarily re-direct plant energy away from growth, a major challenge is how to boost plant immune systems without affecting development. To address these important questions, I will study host proteins involved in the interaction between the model plant Arabidopsis thaliana and some of its natural pathogens. Working with a model plant offers many advantages over directly studying crop plants, the most important being the wealth of genetic and technological tools available (fully sequenced and annotated genome, thousands of indexed mutants, world-wide data repositories) and the general ease of experimentation (small stature, fast growing time, convenient breeding techniques). The project will be undertaken at The Sainsbury Laboratory in Norwich [3], a world-leading research institute dedicated to work on plant-microbe interactions, and will involve collaborative work with laboratories in Germany. Knowledge gained from this project will advance our understanding of how plants defend against pathogen infection and may inform agricultural practices to improve crop yield.

REFERENCES
[1] 'Yes, we have no bananas' The Economist (1 March 2014); [2] 'Ash dieback 'could affect 75% of trees worst hit areas'' The Guardian (30 April 2014); [3] www.tsl.ac.uk.

Plants perceive pathogens through receptor kinases located at the cell surface [1]. The kinase BIK1 is phosphorylated (and activated) by several receptors and is a key component of the plant immune system [2]. My recent work has shown that the amplitude of immune output positively correlates to the amount of BIK1 available. Plants maintain an optimal level of BIK1 through a process of continuous proteasomal degradation. This is influenced by the Ca+2-dependent protein kinase CPK28, which interacts with and phosphorylates BIK1. Thus, it seems that BIK1 is both positively and negatively regulated by phosphorylation, raising the intriguing possibility that residue-specific or quantitative differences contribute to the two states. Interestingly, both CPK28 and BIK1 also function in other stress and developmental pathways [2,3]. Differential and/or quantitative protein modifications may establish specificity between these pathways, possibly by affecting sub-cellular localization, binding partners, or enzymatic activity.

The primary goal of the proposed research is to reveal the detailed mechanisms that regulate BIK1 and CPK28. To this end, I will characterize CPK28-mediated phosphorylation and subsequent ubiquitination events on BIK1 and how these events control its continual turnover. Furthermore, I aim to identify the E3 ligase(s) responsible for BIK1 degradation. To understand the role of CPK28 in plant immunity and development, I will characterize CPK28-associated proteins that we recently identified by mass-spectrometry. Because of the need to identify novel proteins involved in plant immune signaling, a secondary goal of the work is to screen for such proteins using forward-genetics.

This work is state-of-the art and will advance our understanding of the plant immune system at the molecular level.

REFERENCES
[1] Macho & Zipfel, Mol Cell (2014); [2] Lin et al, J Integr Plant Biol (2013); [3] Matschi et al, Plant J (2013).

WHO WILL BENEFIT FROM THIS RESEARCH?
The findings of this research will be of interest to and benefit many groups of people. Plant diseases pose a real threat to food security, and a molecular understanding of plant immunity is absolutely necessary if we are to develop solutions and preventative measures to keep our crops healthy. Understanding how plants defend against pathogens while maintaining optimal growth is of great interest to the UK/EU agribiotech industry both in terms of herbicide chemistry and genetically modified (GM) crops. Translational work stemming from this project could contribute toward environmentally sustainable crops with a boosted immune system. This topic therefore has the potential to be of interest to UK/EU policy makers refining current laws regarding GM crops and chemical use in our fields, and to the general public. Continuing the tradition of world-class research at these institutes therefore contributes to the economic wealth of Norwich and the UK.

HOW WILL THEY BENEFIT FROM THIS RESEARCH?
Although often overlooked, plants are extremely important to the well-being of the UK and the world. Not only are plants a major source of our food, clothing, and building materials, they are increasingly becoming a major source of fuel as well. Microbial diseases and pests place major constraints on agriculture and have significant economic and social impacts. One way to improve plant disease resistance is to enhance the capability of the plants' own innate immune system through genetic modification. However, enhancing immune responses without affecting plant yield is a difficult task. Research in genetically tractable model systems allows detailed biological mechanisms to be uncovered that may have far-reaching impacts in agriculture. It is worth noting that the host supervisor, Cyril Zipfel, is currently actively engaged with translational applications stemming from his work on model plants, and interacts with several non-academic groups and Agri-biotech companies that would benefit from the proposed research. Moreover, TSL is actively engaged in many translational projects with different industrial partners. However, any potential translational applications stemming from this work are likely to be realized in the future and are beyond the scope of this proposal.

Given my track record in public engagement and my dedication to teaching and outreach, it is expected that this project will increase public awareness and understanding of plant biology and GM technology. For example, I previously participated in Science in Norwich Day (SIND) where we engaged the public about the kind of plant research we do at TSL, including the controversial GM potato field trial. We set up a kiosk where interested people could come and infiltrate Nicotiana leaves and observe microbial infections on Arabidopsis plants. Furthermore, we used a light microscope to demonstrate what infected tissues look like close-up. Future participation in SIND and other outreach projects will ensure public engagement with the proposed research. For example, I will host summer students in the lab and engage budding scientists through online outreach programs such as 'I'm a scientist, get me out of here.' Furthermore, BBC East Anglia and iTV Anglia are based in Norwich, providing excellent opportunities for radio and/or television engagement.

In addition, I have experience writing short articles for non-specialist audiences and would be happy to write these sorts of articles about my work. As I gain more independence as a researcher, I think it is necessary to set up a personal scientific webpage to outline my research interested and how they impact society. Therefore, I will set up a Wordpress-hosted page within the first 6 months of the project.