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Uncovering how plant pathogens take control of chloroplast protein import to limit chloroplast-mediated immunity

Reference	BB/X000192/1
Principal Investigator / Supervisor	Professor Paul Jarvis
Co-Investigators / Co-Supervisors	Professor Gail Preston
Institution	University of Oxford
Department	Biology
Funding type	Research
Value (£)	602,516
Status	Current
Type	Research Grant
Start date	01/01/2023
End date	31/12/2025
Duration	36 months

Abstract

We previously discovered the chloroplast ubiquitin E3 ligase SP1, which acts in a proteolytic pathway named CHLORAD (Science, 2012, 2019). SP1 regulates the chloroplast protein import machinery to modify the organelle's biogenesis and functions; and it promotes abiotic stress tolerance by limiting protein import to suppress photosynthesis and avoid overproduction of harmful ROS (Curr. Biol., 2015). Here, our unpublished data (on bacterial infection of plants with manipulated SP1 expression) reveal a new role for SP1 in plant immunity, which may enable development of more disease-resistant crops. We propose that bacterial effector proteins subvert the aforementioned ROS attenuation mechanism to promote virulence. To test this idea, we will: 1. Define the role of chloroplast protein import, and its regulation by SP1, in virulence and immunity. Inoculated Arabidopsis plants (WT, sp1 mutant, SP1 overexpressor) will be studied to reveal how protein import is altered upon infection, and what the functional consequences are. 2. Define the role of effector proteins in SP1-mediated virulence. A library of bacterial effectors will be screened to identify those delivering an SP1-dependent effect on immunity; these will be studied with regard to function, in planta activity, and physical interaction with SP1. 3. Characterize SP1's role in virulence and immunity. To elucidate how SP1 is controlled on infection, its expression, post-translational modification, and activity will be assessed. Also, transcriptomics will reveal which downstream pathways are affected. 4. Investigate conservation of SP1's immunity role with different pathogens. The Arabidopsis genotypes in 1 will be challenged with four other pathogens (biotrophs and necrotrophs), and the responses studied. 5. Investigate conservation of SP1's immunity role in crops. Transgenics of two major crops (with manipulated SP1 expression) will be challenged with different pathogens, and the responses studied.

Summary

A key global challenge of our era is to deliver increased agricultural yields that have resilience to stress and disease. This imperative arises because of rapid human population growth (set to exceed 9 billion by 2050) and anthropogenic climate change, which together place ever increasing pressures on food security and natural resources. To meet this challenge, it will be crucial to develop improved crop varieties. Through research on the model plant Arabidopsis, we previously made important breakthroughs that are pertinent in this regard: We discovered a gene called SP1 that controls diverse aspects of plant growth via a process named "CHLORAD"; and we showed how SP1 is needed for plants to mount effective responses to adverse environmental conditions like drought and salinity (so-called abiotic stresses). Our latest results (which are unpublished but presented here) uncover another, vitally important function of SP1, in plant immunity - i.e., in the way plants defend themselves against the threat of disease posed by plant pathogens. In this project, we will perform experiments to understand this new function of SP1 in detail; and, in doing so, we will shed significant new light on the mechanisms of plant immunity. The SP1 gene controls the formation and operation of structures inside plant cells called chloroplasts. Chloroplasts are normal cellular constituents (i.e., organelles), and they define plants. They contain the green pigment chlorophyll and are responsible for photosynthesis, which harnesses sunlight to power the activities of the cell. As photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of huge importance, not just to plants but to all life on Earth. Chloroplasts also have vital roles in plant immunity, and so are ideal targets for engineering disease resistance in crops. Chloroplasts are composed of thousands of different proteins, most of which are encoded by genes in the cell's nucleus and so are synthesized outside of the chloroplast in the cellular matrix known as the cytosol. As chloroplasts are each surrounded by a double-membrane envelope, sophisticated machinery is needed to bring about the import of these proteins into the chloroplast. This comprises two molecular machines, one in each membrane, called TOC (for "Translocon at the Outer membrane of Chloroplasts") and TIC. Each machine is composed of several different proteins that work cooperatively. The SP1 gene encodes a type of regulatory factor called a "ubiquitin E3 ligase". Such regulators work by labelling-up unwanted proteins to target them for removal. The SP1 E3 ligase mediates the removal of certain TOC components, and this in turn influences which proteins are imported by chloroplasts. Such control enables the plant to alter its chloroplasts' functions during development and in adaptation to stress. During abiotic stress, SP1 inhibits the import of photosynthetic machinery components, which limits photosynthesis. This may seem counterproductive, but actually under stress conditions photosynthesis can overproduce toxic chemicals called "ROS". Thus, by turning down photosynthesis at these times, the plants are more likely to survive. While ROS are harmful if overproduced, they do have a beneficial role to play as signals during immunity, by orchestrating anti-pathogen defences. Our results reveal that pathogens have evolved mechanisms to promote SP1 activity during infection, to limit photosynthesis and so reduce synthesis of defence-promoting ROS. In effect, the pathogens subvert the plant's system for dealing with abiotic stress. We will study the role of SP1 in immunity, and elucidate how pathogens affect SP1 activity. We will also examine the immunity-related functions of SP1 with regard to different pathogens, and in crop plants. Overall, we will enhance our understanding of plant immunity, which is key to the development of crops with improved disease resistance.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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