PABP/purine-rich motif as an initiation module for cap-independent translation in pattern-triggered immunity.

Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.

Formation of NPR1 Condensates Promotes Cell Survival during the Plant Immune Response.

In plants, pathogen effector-triggered immunity (ETI) often leads to programmed cell death, which is restricted by NPR1, an activator of systemic acquired resistance. However, the biochemical activities of NPR1 enabling it to promote defense and restrict cell death remain unclear. Here we show that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through formation of salicylic acid-induced NPR1 condensates (SINCs). SINCs are enriched with stress response proteins, including nucleotide-binding leucine-rich repeat immune receptors, oxidative and DNA damage response proteins, and protein quality control machineries. Transition of NPR1 into condensates is required for formation of the NPR1-Cullin 3 E3 ligase complex to ubiquitinate SINC-localized substrates, such as EDS1 and specific WRKY transcription factors, and promote cell survival during ETI. Our analysis of SINCs suggests that NPR1 is centrally integrated into the cell death or survival decisions in plant immunity by modulating multiple stress-responsive processes in this quasi-organelle.

NPR1, a key immune regulator for plant survival under biotic and abiotic stresses.

Nonexpressor of pathogenesis-related genes 1 (NPR1) was discovered in Arabidopsis as an activator of salicylic acid (SA)-mediated immune responses nearly 30 years ago. How NPR1 confers resistance against a variety of pathogens and stresses has been extensively studied; however, only in recent years have the underlying molecular mechanisms been uncovered, particularly NPR1's role in SA-mediated transcriptional reprogramming, stress protein homeostasis, and cell survival. Structural analyses ultimately defined NPR1 and its paralogs as SA receptors. The SA-bound NPR1 dimer induces transcription by bridging two TGA transcription factor dimers, forming an enhanceosome. Moreover, NPR1 orchestrates its multiple functions through the formation of distinct nuclear and cytoplasmic biomolecular condensates. Furthermore, NPR1 plays a central role in plant health by regulating the crosstalk between SA and other defense and growth hormones. In this review, we focus on these recent advances and discuss how NPR1 can be utilized to engineer resistance against biotic and abiotic stresses.

Nuclear Pore Permeabilization Is a Convergent Signaling Event in Effector-Triggered Immunity.

Nuclear transport of immune receptors, signal transducers, and transcription factors is an essential regulatory mechanism for immune activation. Whether and how this process is regulated at the level of the nuclear pore complex (NPC) remains unclear. Here, we report that CPR5, which plays a key inhibitory role in effector-triggered immunity (ETI) and programmed cell death (PCD) in plants, is a novel transmembrane nucleoporin. CPR5 associates with anchors of the NPC selective barrier to constrain nuclear access of signaling cargos and sequesters cyclin-dependent kinase inhibitors (CKIs) involved in ETI signal transduction. Upon activation by immunoreceptors, CPR5 undergoes an oligomer to monomer conformational switch, which coordinates CKI release for ETI signaling and reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC. Consequently, these coordinated NPC actions result in simultaneous activation of diverse stress-related signaling pathways and constitute an essential regulatory mechanism specific for ETI/PCD induction.

Pervasive downstream RNA hairpins dynamically dictate start-codon selection.

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites1-4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.

Structural basis of NPR1 in activating plant immunity.

NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid1-4. Despite the important role of NPR1 in plant immunity5-7, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA32-NPR12-TGA32 complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.

Salicylic acid in plant immunity and beyond.

As the most widely used herbal medicine in human history and a major defence hormone in plants against a broad spectrum of pathogens and abiotic stresses, salicylic acid (SA) has attracted major research interest. With applications of modern technologies over the past 30 years, studies of the effects of SA on plant growth, development, and defence have revealed many new research frontiers and continue to deliver surprises. In this review, we provide an update on recent advances in our understanding of SA metabolism, perception, and signal transduction mechanisms in plant immunity. An overarching theme emerges that SA executes its many functions through intricate regulation at multiple steps: SA biosynthesis is regulated both locally and systemically, while its perception occurs through multiple cellular targets, including metabolic enzymes, redox regulators, transcription cofactors, and, most recently, an RNA-binding protein. Moreover, SA orchestrates a complex series of post-translational modifications of downstream signaling components and promotes the formation of biomolecular condensates that function as cellular signalling hubs. SA also impacts wider cellular functions through crosstalk with other plant hormones. Looking into the future, we propose new areas for exploration of SA functions, which will undoubtedly uncover more surprises for many years to come.

m6A modification plays an integral role in mRNA stability and translation during pattern-triggered immunity.

Plants employ distinct mechanisms to respond to environmental changes. Modification of mRNA by N 6-methyladenosine (m6A), known to affect the fate of mRNA, may be one such mechanism to reprogram mRNA processing and translatability upon stress. However, it is difficult to distinguish a direct role from a pleiotropic effect for this modification due to its prevalence in RNA. Through characterization of the transient knockdown-mutants of m6A writer components and mutants of specific m6A readers, we demonstrate the essential role that m6A plays in basal resistance and pattern-triggered immunity (PTI). A global m6A profiling of mock and PTI-induced Arabidopsis plants as well as formaldehyde fixation and cross-linking immunoprecipitation-sequencing of the m6A reader, EVOLUTIONARILY CONSERVED C-TERMINAL REGION2 (ECT2) showed that while dynamic changes in m6A modification and binding by ECT2 were detected upon PTI induction, most of the m6A sites and their association with ECT2 remained static. Interestingly, RNA degradation assay identified a dual role of m6A in stabilizing the overall transcriptome while facilitating rapid turnover of immune-induced mRNAs during PTI. Moreover, polysome profiling showed that m6A enhances immune-associated translation by binding to the ECT2/3/4 readers. We propose that m6A plays a positive role in plant immunity by destabilizing defense mRNAs while enhancing their translation efficiency to create a transient surge in the production of defense proteins.

Structural basis of salicylic acid perception by Arabidopsis NPR proteins.

Salicylic acid (SA) is a plant hormone that is critical for resistance to pathogens1-3. The NPR proteins have previously been identified as SA receptors4-10, although how they perceive SA and coordinate hormonal signalling remain unknown. Here we report the mapping of the SA-binding core of Arabidopsis thaliana NPR4 and its ligand-bound crystal structure. The SA-binding core domain of NPR4 refolded with SA adopts an α-helical fold that completely buries SA in its hydrophobic core. The lack of a ligand-entry pathway suggests that SA binding involves a major conformational remodelling of the SA-binding core of NPR4, which we validated using hydrogen-deuterium-exchange mass spectrometry analysis of the full-length protein and through SA-induced disruption of interactions between NPR1 and NPR4. We show that, despite the two proteins sharing nearly identical hormone-binding residues, NPR1 displays minimal SA-binding activity compared to NPR4. We further identify two surface residues of the SA-binding core, the mutation of which can alter the SA-binding ability of NPR4 and its interaction with NPR1. We also demonstrate that expressing a variant of NPR4 that is hypersensitive to SA could enhance SA-mediated basal immunity without compromising effector-triggered immunity, because the ability of this variant to re-associate with NPR1 at high levels of SA remains intact. By revealing the structural mechanisms of SA perception by NPR proteins, our work paves the way for future investigation of the specific roles of these proteins in SA signalling and their potential for engineering plant immunity.

Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity.

Systemic acquired resistance (SAR) is a broad-spectrum plant immune response involving profound transcriptional changes that are regulated by the coactivator NPR1. Nuclear translocation of NPR1 is a critical regulatory step, but how the protein is regulated in the nucleus is unknown. Here, we show that turnover of nuclear NPR1 protein plays an important role in modulating transcription of its target genes. In the absence of pathogen challenge, NPR1 is continuously cleared from the nucleus by the proteasome, which restricts its coactivator activity to prevent untimely activation of SAR. Surprisingly, inducers of SAR promote NPR1 phosphorylation at residues Ser11/Ser15, and then facilitate its recruitment to a Cullin3-based ubiquitin ligase. Turnover of phosphorylated NPR1 is required for full induction of target genes and establishment of SAR. These in vivo data demonstrate dual roles for coactivator turnover in both preventing and stimulating gene transcription to regulate plant immunity.

Global translational reprogramming is a fundamental layer of immune regulation in plants.

In the absence of specialized immune cells, the need for plants to reprogram transcription to transition from growth-related activities to defence is well understood. However, little is known about translational changes that occur during immune induction. Using ribosome footprinting, here we perform global translatome profiling on Arabidopsis exposed to the microbe-associated molecular pattern elf18. We find that during this pattern-triggered immunity, translation is tightly regulated and poorly correlated with transcription. Identification of genes with altered translational efficiency leads to the discovery of novel regulators of this immune response. Further investigation of these genes shows that messenger RNA sequence features are major determinants of the observed translational efficiency changes. In the 5' leader sequences of transcripts with increased translational efficiency, we find a highly enriched messenger RNA consensus sequence, R-motif, consisting of mostly purines. We show that R-motif regulates translation in response to pattern-triggered immunity induction through interaction with poly(A)-binding proteins. Therefore, this study provides not only strong evidence, but also a molecular mechanism, for global translational reprogramming during pattern-triggered immunity in plants.

uORF-mediated translation allows engineered plant disease resistance without fitness costs.

Controlling plant disease has been a struggle for humankind since the advent of agriculture. Studies of plant immune mechanisms have led to strategies of engineering resistant crops through ectopic transcription of plants' own defence genes, such as the master immune regulatory gene NPR1 (ref. 1). However, enhanced resistance obtained through such strategies is often associated with substantial penalties to fitness, making the resulting products undesirable for agricultural applications. To remedy this problem, we sought more stringent mechanisms of expressing defence proteins. On the basis of our latest finding that translation of key immune regulators, such as TBF1 (ref. 3), is rapidly and transiently induced upon pathogen challenge (see accompanying paper), we developed a 'TBF1-cassette' consisting of not only the immune-inducible promoter but also two pathogen-responsive upstream open reading frames (uORFsTBF1) of the TBF1 gene. Here we demonstrate that inclusion of uORFsTBF1-mediated translational control over the production of snc1-1 (an autoactivated immune receptor) in Arabidopsis thaliana and AtNPR1 in rice enables us to engineer broad-spectrum disease resistance without compromising plant fitness in the laboratory or in the field. This broadly applicable strategy may lead to decreased pesticide use and reduce the selective pressure for resistant pathogens.

Comprehensive mapping of abiotic stress inputs into the soybean circadian clock.

The plant circadian clock evolved to increase fitness by synchronizing physiological processes with environmental oscillations. Crop fitness was artificially selected through domestication and breeding, and the circadian clock was identified by both natural and artificial selections as a key to improved fitness. Despite progress in Arabidopsis, our understanding of the crop circadian clock is still limited, impeding its rational improvement for enhanced fitness. To unveil the interactions between the crop circadian clock and various environmental cues, we comprehensively mapped abiotic stress inputs to the soybean circadian clock using a 2-module discovery pipeline. Using the "molecular timetable" method, we computationally surveyed publicly available abiotic stress-related soybean transcriptomes to identify stresses that have strong impacts on the global rhythm. These findings were then experimentally confirmed using a multiplexed RNA sequencing technology. Specific clock components modulated by each stress were further identified. This comprehensive mapping uncovered inputs to the plant circadian clock such as alkaline stress. Moreover, short-term iron deficiency targeted different clock components in soybean and Arabidopsis and thus had opposite effects on the clocks of these 2 species. Comparing soybean varieties with different iron uptake efficiencies suggests that phase modulation might be a mechanism to alleviate iron deficiency symptoms in soybean. These unique responses in soybean demonstrate the need to directly study crop circadian clocks. Our discovery pipeline may serve as a broadly applicable tool to facilitate these explorations.

Redox rhythm reinforces the circadian clock to gate immune response.

Recent studies have shown that in addition to the transcriptional circadian clock, many organisms, including Arabidopsis, have a circadian redox rhythm driven by the organism's metabolic activities. It has been hypothesized that the redox rhythm is linked to the circadian clock, but the mechanism and the biological significance of this link have only begun to be investigated. Here we report that the master immune regulator NPR1 (non-expressor of pathogenesis-related gene 1) of Arabidopsis is a sensor of the plant's redox state and regulates transcription of core circadian clock genes even in the absence of pathogen challenge. Surprisingly, acute perturbation in the redox status triggered by the immune signal salicylic acid does not compromise the circadian clock but rather leads to its reinforcement. Mathematical modelling and subsequent experiments show that NPR1 reinforces the circadian clock without changing the period by regulating both the morning and the evening clock genes. This balanced network architecture helps plants gate their immune responses towards the morning and minimize costs on growth at night. Our study demonstrates how a sensitive redox rhythm interacts with a robust circadian clock to ensure proper responsiveness to environmental stimuli without compromising fitness of the organism.

Salicylic acid activates DNA damage responses to potentiate plant immunity.

DNA damage is normally detrimental to living organisms. Here we show that it can also serve as a signal to promote immune responses in plants. We found that the plant immune hormone salicylic acid (SA) can trigger DNA damage in the absence of a genotoxic agent. The DNA damage sensor proteins RAD17 and ATR are required for effective immune responses. These sensor proteins are negatively regulated by a key immune regulator, SNI1 (suppressor of npr1-1, inducible 1), which we found is a subunit of the structural maintenance of chromosome (SMC) 5/6 complex required for controlling DNA damage. Elevated DNA damage caused by the sni1 mutation or treatment with a DNA-damaging agent markedly enhances SA-mediated defense gene expression. Our study suggests that activation of DNA damage responses is an intrinsic component of the plant immune responses.

NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants.

Salicylic acid (SA) is a plant immune signal produced after pathogen challenge to induce systemic acquired resistance. It is the only major plant hormone for which the receptor has not been firmly identified. Systemic acquired resistance in Arabidopsis requires the transcription cofactor nonexpresser of PR genes 1 (NPR1), the degradation of which acts as a molecular switch. Here we show that the NPR1 paralogues NPR3 and NPR4 are SA receptors that bind SA with different affinities. NPR3 and NPR4 function as adaptors of the Cullin 3 ubiquitin E3 ligase to mediate NPR1 degradation in an SA-regulated manner. Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance. Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity. Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid.

The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-like 9 (NTL9) and CCA1 hiking expedition (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, phytoalexin-deficient 4 (PAD4) and enhanced disease susceptibility 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators calmodulin binding protein 60g (CBP60g) and systemic acquired resistance deficient 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production.

Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal.

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated ß-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd Moreover, the GPI modification signal from both Pd- and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.

Timing of plant immune responses by a central circadian regulator.

The principal immune mechanism against biotrophic pathogens in plants is the resistance (R)-gene-mediated defence. It was proposed to share components with the broad-spectrum basal defence machinery. However, the underlying molecular mechanism is largely unknown. Here we report the identification of novel genes involved in R-gene-mediated resistance against downy mildew in Arabidopsis and their regulatory control by the circadian regulator, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Numerical clustering based on phenotypes of these gene mutants revealed that programmed cell death (PCD) is the major contributor to resistance. Mutants compromised in the R-gene-mediated PCD were also defective in basal resistance, establishing an interconnection between these two distinct defence mechanisms. Surprisingly, we found that these new defence genes are under circadian control by CCA1, allowing plants to 'anticipate' infection at dawn when the pathogen normally disperses the spores and time immune responses according to the perception of different pathogenic signals upon infection. Temporal control of the defence genes by CCA1 differentiates their involvement in basal and R-gene-mediated defence. Our study has revealed a key functional link between the circadian clock and plant immunity.

A kiss of death--proteasome-mediated membrane fusion and programmed cell death in plant defense against bacterial infection.

Eukaryotes have evolved various means for controlled and organized cellular destruction, known as programmed cell death (PCD). In plants, PCD is a crucial regulatory mechanism in multiple physiological processes, including terminal differentiation, senescence, and disease resistance. In this issue of Genes & Development, Hatsugai and colleagues (pp. 2496-2506) demonstrate a novel plant defense strategy to trigger bacteria-induced PCD, involving proteasome-dependent tonoplast and plasma membrane fusion followed by discharge of vacuolar antimicrobial and death-inducing contents into the apoplast.