Molecular engineering of the ubiquitin-proteasome system: A new approach to pathogen resistance in Plants

Barley (Hordeum vulgare) is the fourth most important crop worldwide and second in the UK. Diseases, including those caused by the brown rust pathogen, Puccinia hordei, cause barley yield losses of up to 40%. This project focuses on improving plant's own natural defences to alleviate disease-related losses as a cost-effective and environmentally

sustainable alternative to chemical treatments. In response to pathogen attack, plants initiate immune responses that are sufficient to fend off most pathogens. Adapted pathogens, however, produce effector proteins that are capable of suppressing the host immune response and promote successful infection,

causing severe crop damage. This project takes a novel engineering biology approach to re-purpose a plant's own enzymes to mark effector proteins for degradation. Addition of ubiquitin to a protein, via the K48 type linkage, targets that protein for degradation by the proteasome. UKRI BBSRC EASTBIO Doctoral Training Partnership 2022 Cohort: PhD Project Proposal

2 The goal of this PhD project is to re-engineer the substrate specificity of K48 type E3 ligases, and thus to create a synthetic ubiquitin pathway that targets specific pathogen effectors for proteasome-mediated degradation, thereby generating enhanced disease resistance. We seek a student who is interested in applying fundamental biological molecular

engineering to a real-world problem. The student will benefit from the combined mentorship of Regan (expert in protein engineering) and Orosa (expert in plant science), and of Syngenta, our industrial partner and a leading company in plant biotechnology and crop protection. Specifically, the goals of the PhD project are i) identify E3 ligases in barley that add ubiquitin

to substrates via K48 linkages; ii) identify and analyse their substrate-binding domains - with emphasis on the specificity of the E3 ligase-substrate interaction; iii) re-engineer chosen E3 ligases to bind and ubiquitinylate effector proteins of the target pathogen. iv) test the re-engineered E3 ligases in barley for increased resistance to the pathogen.

Goals i) and ii) will involve state of the art sequence and structural analysis to identify specificity-determining residues in substrate-binding domains. Goals iii) and iv) will involve wet-lab protein engineering and plant biotechnology work. The amino acids essential for substrate binding will be combinatorially mutated and the mutants screened using high

throughput methodologies developed by our teamto identify E3 ligase variants of the that are able to bind to and ubiquitinylate key effector(s) protein(s) of the target pathogen. Newly developed synthetic E3 ligases targeting pathogen effector(s) will be tested in planta, by stable overexpression in barley. The impact of the synthetic E3 ligase on the progression

of Puccinia induced disease will be tested by infection assays. An ultimate goal of the PhD project will the simultaneous targeting of several effectors to produce robust and durable disease resistance.

Through this project an innovative approach and new tools to manipulate plantpathogen interactions will be developed. The student will join a vibrant group if researchers, and will gain strong interdisciplinary training in addition to experience of both academic and company research. If successful, the technology will be transferable to

other crops and pathosystems.Reducing the impact of disease on crops, will contribute to addressing the United Nations Goal2: Zero Hunger programme