Dynamic ubiquitin signalling coordinates transcriptional reprogramming in plant immunity

Project Information

UbRegulate

Grant agreement ID: 101001137

DOI

10.3030/101001137

EC signature date 20 January 2021

Start date 1 September 2021

End date 31 August 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

THE UNIVERSITY OF EDINBURGH
United Kingdom

Periodic Reporting for period 2 - UbRegulate (Dynamic ubiquitin signalling coordinates transcriptional reprogramming in plant immunity)

Reporting period: 2023-03-01 to 2024-08-31

Summary of the context and overall objectives of the project

Human activities increasingly impose constraints on agriculture and threaten future food security. These constraints result in crops having to face a multitude of abiotic and biotic stresses. Consequently, crops either engage in a lengthy and costly battle against disease or succumb under disease pressure, both of which result in severely reduced crop yield. Indeed, a reduction in marketable crop yields cost the European Union and global economies billions every year and still renders much of agriculture subsidised. Resistance or tolerance against pathogenic attackers is provided by effective immune responses that are activated in a timely manner and are prioritised over normal cellular demands. Current biotechnological approaches to improve disease resistance in crops are often not broad spectrum, not durable, or lead to trade-offs with plant growth and yield. Thus, in accordance with the European Green Deal previously introduced by the European Union, new innovative approaches are required that are both environmentally sustainable and provide long-lasting resistance to a wide variety of attackers. The ground-breaking ‘UbRegulate’ project seeks to unravel how plants reprogram cellular gene expression to establish broad-spectrum, durable immunity.

To answer this question the team focusses on the poorly understood connection between the immune hormone salicylic acid (SA; of which the drug aspirin is a derivative) and the post-translational modifier ubiquitin, both of which are indispensable signals for cellular reprogramming of gene expression necessary for the establishment of durable disease resistance. Specifically this project will demonstrate how SA-induced ubiquitin signalling:
(i) engages the gene expression machinery and reveal its potential for exploitation in agriculture;
(ii) avoids genome instability triggered by excessive immune gene expression;
(iii) orchestrates selective access to the genome for the gene expression machinery;
(iv) and utilises diverse chain linkage types to establish durable, broad-spectrum immunity.

Together, these discoveries will revolutionise the development of new sustainable strategies for more effective crop protection.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Pathogen-induced accumulation of the immune hormone SA leads to dramatic reprogramming of cellular gene expression to prioritise immune response activation over other cellular household processes. SA triggers massive gene expression changes through its nuclear receptor protein, NPR1, a potent gene activator. SA-induced modification of NPR1 by the small post-translational modifier, ubiquitin, regulates its activity and lifetime, thereby functioning as an instruction manual for onset of immunity. Importantly, proteins such as NPR1 are modified by chains of interlinked ubiquitin of different structural topologies, but the biological effects of these chain topologies remain poorly understood. We investigated how SA orchestrates ubiquitin-mediated activation of NPR1. Surprisingly, we discovered that SA-induced NPR1 is subject to a relay of three different ubiquitin ligases that each have distinct effects on its activity. First, a modular Cullin3-RING Ligase (CRL3) modifies NPR1 with a short ubiquitin chain of a specific topology. This short ubiquitin chain is a platform for the recruitment of additional transcriptional regulators that together with NPR1 attract the necessary machinery for activation of immune genes. Unlike CRL3, the subsequent UBE4 ubiquitin ligase deactivates NPR1 by extending its ubiquitin chains with a different topology. This results in chains of mixed topologies that target NPR1 for degradation by the proteasome. Despite having already been recruited to the proteasome, NPR1 is subject to further ubiquitination by the proteasome-associated HECT-type ligases, UPL3 and UPL4. This ‘eleventh hour’ modification prevents stalling of the proteasome during the degradation of NPR1. Subsequently, we discovered that other gene activators are also regulates by ubiquitin ligase relays, suggesting they are a universal mechanism for regulating the lifetimes and activities of proteasome-targeted substrates.

So why is NPR1 subject to such complicated control by a relay of multiple different ubiquitin ligases? We hypothesised that NPR1 activity if tightly controlled by ubiquitin signalling, because its dysregulation leads to pathological conditions, including autoimmunity. Could it be NPR1 is such a potent gene activator that its uncontrolled activity risks genome instability caused by excessive demand for gene expression? In support of this notion, we discovered that mutants with enhanced NPR1 activity are sensitive to DNA damage and that NPR1 physically interacts with a number of DNA repair proteins. Thus, the project now explores if dynamic ubiquitin signalling curbs NPR1 activity to avoid genomic instability associated with excessive demands for gene expression.

To further explore how SA-induced ubiquitin signalling orchestrates activation of immunity, we established a proteomics pipeline for detection of protein ubiquitination. This has allowed us to define the immune-induced ubiquitin chain topology landscape and observe direct links between specific ubiquitin topologies and cellular processes. For example, we discovered that activation of immunity induces novel changes in the ubiquitination of histones that are key DNA packaging proteins for stabilisation of the genetic code. Our findings suggest that histone ubiquitination at previously unrecognised sites is critical for the activation of immune responses.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

To date the project has already discovered novel ways in which cellular gene activators are precisely regulated. Our findings that ubiquitin ligase relays orchestrate the activities and lifetimes of gene activators is likely a universal mechanism whereby eukaryotic cells control gene expression programmes. Moreover, we illustrate that ‘eleventh hour’ ubiquitination at the proteasome itself is necessary to prevent substrate stalling during degradation. These findings reveal a new level of complexity and cellular capability to fine-tune signalling by using the post-translational modifier, ubiquitin. In the next phase of the project, we are exploring if this understanding can be exploited to generate novel crop protection strategies for agriculture.

While excessive gene expression is widely recognised to trigger undesirable genome instability, how cells prevent this in times of high demand for gene expression (e.g. pathogen attack or other environmental challenges), remains largely unknown. Our findings now begin to reveal the elusive link between activation of gene expression by gene activators and genome instability. During the remainder of the project, we will seek to cement our hypothesis that ‘dangerous’ gene activators, like NPR1, use ubiquitin signalling to attract the DNA damage repair machinery to prevent genome instability when demands for gene expression are high. This is critical knowledge to avoid undesirable side-effects of newly engineered crop protection strategies.

Lastly, we are generating novel strategies for detection of diverse ubiquitin topologies in the plant cell proteome. This is revealing that plants utilise the full spectrum of known ubiquitin topologies and more, and allow us to associate these with specific cellular processes. Moreover, we can now assess how the deletion of a single ubiquitin ligase impacts the entire ubiquitin topology landscape of the cell. Together, these advancements enable us to dissect how and to what extend immune-activated plant cells utilise the complexity of ubiquitin signalling, and ultimately translate these findings into novel agri-tech solutions to combat devastating crop diseases.

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