Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - IMMUNE-EXPRESS (Proteasome-Mediated Gene Expression in Plant Immunity)

Teaser

SIGNIFICANCERecent concerns over food security are founded in the daunting prospect of feeding a rapidly growing world population that is predicted to reach ~9 billion in the next 30-40 years. Although this significant challenge can theoretically be met by sharply increasing...

Summary

SIGNIFICANCE
Recent concerns over food security are founded in the daunting prospect of feeding a rapidly growing world population that is predicted to reach ~9 billion in the next 30-40 years. Although this significant challenge can theoretically be met by sharply increasing global crop production, available agricultural lands and resources are in decline. Thus, innovative methods are required to increase food production per unit of land. The presence of many different crop diseases suppresses yields and qualities of agricultural crops. Reductions in marketable crop yields cost the European and global economies billions every year. To keep up with increasing food demands there is an overwhelming need for novel methodologies and practices that allow crops to better cope with disease causative agents. However, any new methods or practices should be sustainable, avoid any trade-offs with local biodiversity, and not pose a threat to human health. An attractive method for securing agricultural and industrial crop supplies is to chemically or genetically enhance the plant\'s own immune system. This method avoids the use of harmful pesticides that may pose a significant hazard to biological diversity and our own health, while optimizing crop productivity even in adverse pathological conditions.


BACKGROUND
Plants are continuously exposed to a large variety of pathogenic microbes, including bacteria, fungi, oomycetes, and viruses. To defend themselves, plants have evolved a sophisticated innate immune system that provides specificity, self-tolerance, and immune memory. These features are largely orchestrated by the immune hormone salicylic acid (SA), a derivative of the better known aspirin. Upon pathogen infection, SA accumulates at the site of infection where it is required for local resistance responses, such as programmed cell death to isolate the invading pathogen, and production of antimicrobial compounds. In addition, SA accumulates in distal tissues where it is involved in establishing a memory of the initial attack, providing broad-spectrum, long-lasting immunity to subsequent pathogen attack. Because of the importance of SA in establishing disease resistance, agribusinesses have previously developed SA mimics (e.g. Actigard® or Bion®) as well as compounds that activate SA signalling (e.g. probenazole and tiadinil), many of which are still heavily used as plant activators or fungicides in agriculture today.

To establish immunity, SA and SA mimics induce dramatic changes in the expression of thousands of genes. These changes are largely managed by the activator protein NPR1, a regulator of ~10% of genes in the genome of many plant species. Mutation of NPR1 results in severe susceptibility to a large variety of pathogens, demonstrating its importance in SA-mediated plant immunity. We showed previously that SA activates NPR1 in a two-step process. First, in resting cells NPR1 interacts with itself to form a high-molecular weight multimer. Pathogen-induced SA levels trigger the release of NPR1 monomer that can translocate into the plant cell nucleus to for activation of target genes. Second, SA unexpectedly signals for recruitment of a Cullin-RING E3 ligase that adds a chain of the small molecule ubiquitin to NPR1, thereby targeting it for degradation by the proteasome, a large multi-subunit complex that breaks down proteins into their smallest building blocks.

So why does SA signal for the degradation of NPR1? Surprisingly, we found that degradation of NPR1 is paradoxically necessary for the activation of its target genes. We suggested a model in which activation of target gene expression marks NPR1 as a ‘spent’ activator. Removal of ‘spent’ NPR1 may be necessary to allow ‘fresh’ NPR1 activator to reinitiate the expression of target genes (Fig. 1). This novel mode of gene expression by unstable activatorshas now been widely found in diverse eukaryotic organisms including humans, indicating its general importanc

Work performed

Objective 1:
Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target genes, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays a role in gene expression prior to its proteasomal turnover. We found that ubiquitination of NPR1 is a processive event in which initial modification by a Cullin-RING E3 ligase promotes its association with target genes and facilitates their expression. Only when the ubiquitin chain fused to NPR1 is elongated to longer chain lengths by another ubiquitin ligase, NPR1 is targeted for proteasomal degradation. Conversely,ubiquitin ligase activities may be opposed by deubiquitinases (DUBs) that can trim or entirely remove ubiquitin chains from proteins. We discovered that two proteasome-associated DUBs trim ubiquitin chains fused to NPR1, thereby enhancing NPR1 longevity. Our findings indicate that sequential actions of two ubiquitin ligases balanced by opposing DUBs fine-tune the ability of NPR1 to activate its target genes without strict requirement for sacrificial turnover. This dramatically changes the way we think about regulation of plant immune gene expression and more generally about gene expression in multicellular organisms including humans. Our work has also revealed new targets (i.e. two ubiquitin ligases and two DUBs) for crop improvement through chemical or genetic means.

Objective 2:
Regulated degradation of proteins by the proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-mediated proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. We discovered that specific members of the HECT domain-containing family of ubiquitin E3 ligases play important roles in proteasomal substrate processing during plant immunity. Mutations in three HECT-type E3 ligases significantly diminished immune responses activated by the immune hormone SA. In depth analyses of mutants in one of these HECT-type ligases indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. This HECT-type ligase was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-mediated proteasome pathway components. In agreement, we uncovered that HECT-type ligases enabled proteasomes to form ubiquitin chains, thereby regulating total cellular protein ubiquitination levels. Taken together, our findings suggest that proteasome-associated HECT-type ligase activity promotes proteasomal processivity and is indispensable for development of plant immunity. Thus, we identified HECT-type ligases and the proteasome as potential new targets for improvement of broad-spectrum crop immunity.

Objective 3:
So how does SA regulate the activity of E3 ligases that are essential for activation of plant immunity? We reported previously that SA induces changes in the cellular redox environment, resulting in oxidation and reduction reactions of proteins. We now discovered that the specific substrate adaptors of immune-related E3 ligases are modified by redox changes. Substrate adaptors ensure that E3 ligases recruit specific substrates for ubiquitination and subsequent degradation. We found that these substrate adaptors can be specifically modified by oxidative molecules such as nitric oxide (NO), which results in rapid multimerization. Conversely, we now demonstrate that antioxidant enzymes of the Thioredoxin (TRX) family reverse this process, thereby facil

Final results

Taken together, our findings so far demonstrate several new targets for potential exploitation in chemical or genetic agritechnological applications. Enzymes involved in ubiquitin signalling have been subject of interest to pharmacology and biomedicine but thus far have largely escaped the attention of the agrichemical and agritech industry. The discover of four new enzymes involved in the activation of broad-spectrum plant immunity therefore opens the door to the targeted design of new chemicals that activate or inhibit these enzymes. Moreover, the constitutive or regulated expression of these enzymes may be a genetic tool to generate enhanced disease resistance against a wide variety of pests. We also found that the proteasome plays intimate roles in regulating immunity that go well beyond just the destruction of substrates. This notion now opens up the possibility that specific immune-induced proteasomes exist and that their structural constituents may be of interest for crop improvement.

Website & more info

More info: http://spoel.bio.ed.ac.uk/.