One of the biggest challenges of our time is to feed the growing global population. World arable land is practically at its maximum capacity, and will decrease in coming years due to climate change and urban development. Therefore, the productivity of our food system must be increased to tackle world hunger and increase food security. One exceptional strategy to achieve this goal is to reduce losses by pests. Barley (Hordeum vulgare) is the fourth most important crop worldwide and second in the UK. Diseases, including those caused by the brown rust Puccinia hordei and Ramulaira collo-cygni, represent the largest threat to barley, causing yield losses of up to 40%. Based on their lifestyle and interaction with the host, pathogenic fungi can be classified as either biotrophic or necrotrophic. The key difference between biotrophic and necrotrophic fungi is that biotrophic fungi derive nutrients from living plant cells, maintaining them alive, while necrotrophic fungi kill the plant tissues and then obtain nutrients from them. However, many pathogenic fungi use sequential biotrophic and necrotrophic infection phases. During the biotrophic phase they invade and extensively colonise the plant with minimum damage, before switching to the often fatalnecrotrophic phase. Importantly, fungicides are most effective at reducing disease when applied during the early biotrophic stages of infection, but since this phase cannot be visually identified, they are often applied too late when the pathogen is already well established. In response to pathogen attack, plants initiate immune responses that are sufficient to fend off most pathogens. However, adapted pathogens secret effector proteins that are capable of suppressing the host immune response and promote successful infection, causing severe crop damage. These effectors are constantly evolving, thereby avoiding existing host resistance or current plant protection strategies. Consequently, alternative methods to enhance crop resistance are required. The use of immune elicitors or protective biostimulants is a highly promising sustainable method for inducing long-lasting disease resistance, but the modes of action and plant cell targets of these chemicals remain unknown. Plants use the conserved protein ubiquitin to regulate immune responses. Ubiquitination is a fast and reversible protein modification that regulates the amplitude and intensity of the immune response. Using a proteomic pipeline developed in our laboratories, we discovered that protein ubiquitination is a far better biomarker for early biotrophic pathogen infection than currently available genomic and transcriptomic markers. Our results in barley cultivars on a field trial with P. hordei showed a general ubiquitin-mediated immune activation of all infected barley cultivars, implying that ubiquitin regulation of the immune system is a conserved mechanism across cultivars. Therefore, in this project we will investigate the ubiquitin-dependent response of barley to elicitors/biostimulants and economically relevant pathogens with changing lifestyles. These will be compared to the ubiquitin proteomes of plant immune hormones responsive to biotrophic and necrotrophic stages, revealing the modes of action of elicitors/biostimulants and precisely identifying when pathogens change their lifestyle, identifying new biomarkers for early detection of the 'invisible' biotrophic disease phase. In summary, this project will address a crucial gap in knowledge and uncover new fundamental insights into the activation and modulation of plant immunity during biotrophic and necrotophic fungal infections, and real the mode of action of elicitors and biostimulants, which will be used to enhance crop resistance. These novel, sustainable approaches will have a significant impact on global food security and drive innovation in the agrifood sector.