Periodic Reporting for period 4 - IMMUNE-EXPRESS (Proteasome-Mediated Gene Expression in Plant Immunity)
Berichtszeitraum: 2020-09-01 bis 2021-08-31
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 while NPR1 is an activator of immunity, unexpectedly SA triggers its degradation. 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 importance to regulation of eukaryotic gene expression. Our contributions to understanding the perception of SA and downstream activation of SA-responsive immune genes represent major leaps forward in our understanding of plant immune responses. Nonetheless, it remains unknown what defines a ‘spent’ NPR1 activator and how quickly it is rendered in this inactive state. Moreover, SA regulates diverse E3 ligases that target NPR1 and other substrates for degradation, but the regulatory mechanisms and identity of these substrates remains elusive.
Objective 1: We aim to understand what defines the 'spent' state of NPR1, as this could open up new avenues to NPR1-based crop protection strategies.
Objective 2: Identify and characterise additional SA-induced ubiquitin E3 ligases and their substrates. This could reveal new targets for chemical or genetic crop innovation.
Objective 3: Elucidate how ubiquitin E3 ligases are regulated by the SA signal. Understanding how SA and active SA mimicking molecules regulate the activities of ubiquitin E3 ligases could offer the possibility to design improved agrichemicals.
Objective 1: We aim to understand what defines the 'spent' state of NPR1, as this could open up new avenues to NPR1-based crop protection strategies.
Objective 2: Identify and characterise additional SA-induced ubiquitin E3 ligases and their substrates. This could reveal new targets for chemical or genetic crop innovation.
Objective 3: Elucidate how ubiquitin E3 ligases are regulated by the SA signal. Understanding how SA and active SA mimicking molecules regulate the activities of ubiquitin E3 ligases could offer the possibility to design improved agrichemicals.
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 sequential actions of two ubiquitin ligases balanced by opposing deubiquitinases (DUBs) fine-tune the ability of NPR1 to activate its target genes. 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: 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. These HECT-type ligases were found to physically interact with the proteasome, enabling proteasomes to form ubiquitin chains. Moreover, we discovered that unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates. 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. Moreover, deubiquitinases (DUBs) that trim ubiquitin chains of substrates, are also subject to inhibitory oxidation. Conversely, we demonstrate that antioxidant enzymes of the Thioredoxin (TRX) family reverse this process, thereby facilitating the active states of ubiquitin modifying enzymes, indicating that TRX enzymes play a key role in regulating ubiquitin signalling in plant immunity. Thus, TRX enzymes represent new chemical and genetic targets from crop improvement.
Objective 2: 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. These HECT-type ligases were found to physically interact with the proteasome, enabling proteasomes to form ubiquitin chains. Moreover, we discovered that unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates. 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. Moreover, deubiquitinases (DUBs) that trim ubiquitin chains of substrates, are also subject to inhibitory oxidation. Conversely, we demonstrate that antioxidant enzymes of the Thioredoxin (TRX) family reverse this process, thereby facilitating the active states of ubiquitin modifying enzymes, indicating that TRX enzymes play a key role in regulating ubiquitin signalling in plant immunity. Thus, TRX enzymes represent new chemical and genetic targets from crop improvement.
Taken together, our findings 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 several 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.