Understanding the role of ubiquitin signalling in barley to improve crop health

Abstract

Barley (Hordeum vulgare) is the second most important crop in the UK and ranks fourth among cereal crops globally. Yet, it is highly vulnerable to diseases, including those caused by fungal pathogens, such as Puccinia hordei, which can lead to yield losses of up to 40%, posing a serious threat to food security. Understanding and enhancing plant immune responses against pests is therefore essential to safeguarding crop productivity, and there is a pressing need for innovative, mechanistically informed strategies.

Ubiquitination is a complex protein posttranslational modification, which serves various functions from modulating protein activity, localisation, and stability, to promoting protein degradation. Previous studies showed that it plays a pivotal role in regulating plant immune responses. Most of this work, however, is undertaken in model plant species, and little is known about immune-related ubiquitination in crops. This knowledge gap has hindered efforts to utilise the ubiquitin pathway as a strategy for enhancing pest resistance in crops. In this study, I provide a comprehensive profiling of the barley immune ubiquitome, to uncover molecular mechanisms that could be harnessed to enhance resistance to fungal pathogens.

This work has been divided into three chapters. Given the strict regulations on genetically modified or edited crops and the urgent need to reduce pesticide use, alternative strategies to enhance crop resilience are essential. In Chapter 3, I explore the use of arbuscular mycorrhizal fungi (AMF) as tools to prime barley immune responses. These results suggest, that AMF colonisation alters the barley transcriptome and modulates expression of ubiquitin-related genes during P. hordei infection. This suggests that AMF-mediated priming involves the ubiquitin system as part of a broader immune regulatory network.

Chapter 4 addresses the dynamic nature of ubiquitin signalling in barley during the immune response. Here, I perform an immune ubiquitome profiling by identifying ubiquitin-regulated proteome during (i) hormone-induced immune responses, (ii) pathogen infection in the lab, and (iii) in field conditions. The data reveal that the barley immune ubiquitome is signal-specific and dynamic, with a clear modulation of protein abundance and turnover. Common pathways and novel ubiquitinated targets have been identified, providing a comprehensive view of how ubiquitin signalling

contributes to barley immune responses and offering new candidate proteins for the improvement of crop resistance. E3 ubiquitin ligases are the central specificity determinants of the ubiquitination cascade. Identifying immune-related E3 ligases and their substrates is key to understanding the regulatory nodes within the ubiquitin system. In Chapter 5 I employed a substrate-trapping approach to identify substrates of HvRGLG2, a putative immune-related E3 ligase in barley. The captured targets were implicated in core immune processes, and I demonstrated that their ubiquitination directly modulates immune function at the molecular level.

Taken together, these findings highlight two complementary routes to strengthen barley immunity: (i) exploiting beneficial associations with arbuscular mycorrhizal fungi (AMF) as sustainable strategies to enhance crop health, and (ii) targeting ubiquitin-mediated immune regulation to uncover novel resistance mechanisms. The immune ubiquitome generated in this study provides a valuable resource for understanding stress-responsive pathways and identifying new targets for resistance breeding. Furthermore, the substrate-trapping method developed here offers a scalable approach for mapping E3 ligase–substrate interactions in crops. Collectively, this research lays the groundwork for innovative tools to improve crop health and resilience.