For whom the bell tolls: Linking protease degradomes with the proteasome through the N-end rule pathway to understand biological function.

Intracellular proteases cleave protein substrates to reveal polypeptide fragments. What are those fragments, do they have novel activities, and how does the cell regulate their stability? These questions are the focus of this proposal, that will attempt for the first time to systematically unite two different areas of plant protein biochemistry; the action of proteases and the ubiquitin-mediated degradation of polypeptides. It will address a novel hypothesis with important implications in both, that the N-end rule pathway is an undiscovered component controlling the biological function of plant proteases by linking protease targets to the ubiquitin proteasome system. The project will vastly increase the number of known plant N-end rule substrates, and will provide a biological understanding of the consequences of plant protease action. It will focus on the plant immune response and seek to uncover novel N-end rule substrate(s) that enhance the immune response.

Although plant genomes encode large numbers of proteases (over 700 in Arabidopsis) very few substrates have been identified. In the study of the N-end rule pathway, still very few substrates, and even fewer proteases that give rise to N-terminal destabilising residues have been identified. Therefore, key questions of these two fields represent two sides of the same coin, that can both be addressed following N-terminomics approaches. In this proposal we will focus on two approaches to unite these two areas of plant protein biochemistry, the first approach led by protease degradomes, the second approach led by biological process, identifying NTAQ substrates regulating the plant immune response:

Approach 1, protease degradomes (Objectives 1 and 2): We will use N-terminomics data derived from published and unpublished metacaspase Nt-degradomes. MC's represent a class of plant caspase-like proteases with proven functions in several plant developmental and environmental responses, including biotic interactions, and therefore represent good examplars on which to focus this grant.

Approach 2 biological process (Objectives 3 and 4): In collaboration with Professor Kris Gevaert (VIB Ghent) we have produced the first plant N-end rule N-terminome dataset, that has identified Arabidopsis NTAQ specific Gln Nt-polypeptides. We have shown that the NTAQ branch of the N-end rule pathway is a novel component of the plant immune response, regulating phytoalexin and defence protein gene expression. We are therefore poised to use this unpublished dataset to identity and characterise new N-end rule substrates that could be used to enhance the plant immune system.

Economic and Societal Impact: The project aims to provide greater understanding of plant protease biology by investigating the downstream consequences of protease action on protein targets. Our research will produce new knowledge and scientific advancements which will be published and therefore enhance UK and global knowledge economies. Development of crops in which N-end rule substrates that control the plant immune response have been enhanced has the potential to enhance food security and boost local economies. A surety of crop safety leads to better adoption of technology and directly contributes to poverty alleviation.

Collaborations to enhance the knowledge economy: This project will be a collaboration between the Holdsworth and Ray groups at Nottingham, and Dr. Frank van Breusegem and Professor Kris Gevaert at VIB, Gent. The collaboration enhances the work programme and thus the knowledge economy by bringing together experts in N-end rule pathway, plant pathology, plant protease biology and N-terminomics.

Training: The project creates an opportunity to train the PDRA in advanced aspects of plant protein biochemistry, plant pathology and plant protease/proteomics. The PDRA will have the opportunity of giving Guest Lectures in relevant modules at the University of Nottingham.

Communication and Engagement: A set of deliverables at specific timepoints within the project lifetime is provided in the detailed impact statement, however we provide a summary of it below;
Dissemination to the wider scientific community: The research will be published in relevant peer-reviewed international journals and presented at appropriate meetings.
Outreach and public engagement activities: To communicate directly with the relevant commercial and stakeholder audiences, we will showcase the data in a variety of outreach events in through their institutions to maximize knowledge transfer and public engagement.