Immunity In Arabidopsis Research Articles

Reprogramming of flagellin receptor responses with surrogate ligands.

Receptor kinase (RK) families process information from small molecules, short peptides, or glycan ligands to regulate core cellular pathways in plants. To date, whether individual plant RKs are capable of processing signals from distinct types of ligands remains largely unexplored. Addressing this requires the discovery of structurally unrelated ligands that engage the same receptor. Here, we focus on FLAGELLIN-SENSING 2 (FLS2), an RK that senses a peptide of bacterial flagellin to activate antibacterial immunity in Arabidopsis. We interrogate >20,000 potential interactions between small molecules and the sensory domain of FLS2 using a large-scale reverse chemical screen. We discover two small molecules that interact with FLS2 in atypical ways. The surrogate ligands weakly activate the receptor to drive a functional antibacterial response channeled via unusual gene expression programs. Thus, chemical probes acting as biased ligands can be exploited to discover unexpected levels of output flexibility in RKs signal transduction.

A peptide-receptor module links cell wall integrity sensing to pattern-triggered immunity.

Plants employ cell-surface receptors to perceive non- or altered-self, including the integrity of their cell wall. Here we identify a specific ligand-receptor module responsive to cell wall damage that potentiates immunity in Arabidopsis. Disruption of cell wall integrity by inhibition of cellulose biosynthesis promotes pattern-triggered immunity transcriptionally in a manner dependent on the receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Notably, while MIK2 can perceive peptides of the large SERINE RICH ENDOGENOUS PEPTIDE family, a single member of this family, SCOOP18, is transcriptionally induced upon cell wall damage and is required for subsequent responses such as lignification and immunity potentiation. Collectively, our results identify the SCOOP18-MIK2 ligand-receptor module as an important central hub, connecting plant cell wall integrity sensing with immunity.

The pathogen-induced peptide CEP14 is perceived by the receptor-like kinase CEPR2 to promote systemic disease resistance in Arabidopsis.

Secreted plant peptides that trigger cellular signaling are crucial for plant growth, development, and adaptive responses to environmental stresses. In Arabidopsis (Arabidopsis thaliana), the C-TERMINALLY ENCODED PEPTIDE (CEP) family is a class of secreted signaling peptides that is phylogenetically divided into two groups: group I (CEP1-CEP12) and group II (CEP13-CEP15). Several group I CEP peptides regulate root architecture and nitrogen starvation responses, whereas the biological activity and roles of group II CEPs remain unknown. Here, we report that a group II CEP peptide, CEP14, functions as a pathogen-induced elicitor of Arabidopsis immunity. In response to infection by the bacterial pathogen Pseudomonas syringae, CEP14 expression was highly induced via the salicylic acid pathway in Arabidopsis leaves and roots. In the absence of pathogen attack, treatment of Arabidopsis plants with synthetic CEP14 peptides was sufficient to trigger immune responses. Genetic and biochemical analyses demonstrated that the receptor-like kinase CEP RECEPTOR 2 (CEPR2) perceives CEP14 to trigger plant immunity. The SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SERK4 also participated in CEP14 perception by forming CEP14-induced complexes with CEPR2. Overexpression of CEP14 largely enhanced Arabidopsis resistance to P. syringae, while CEP14 or CEPR2 mutation significantly attenuated Arabidopsis systemic resistance to P. syringae. Taken together, our data reveal that the pathogen-induced CEP14 peptide, which is perceived by the CEPR2-BAK1/SERK4 receptor complexes, acts as an endogenous elicitor to promote systemic disease resistance in Arabidopsis.

Phosphorylation of PIP2;7 by CPK28 or Phytophthora kinase effectors dampens pattern-triggered immunity in Arabidopsis

Plasma membrane intrinsic proteins (PIPs), a subclass of aquaporins, play an important role in plant immunity by acting as H2O2 transporters. Their homeostasis is mostly maintained by C-terminal serine phosphorylation. However, the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown. Here, we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H2O2. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation. During pathogen infection, CPK28 dissociates from PIP2;7 and destabilizes, leading to PIP2;7 accumulation. As a countermeasure, oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation. Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28. Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection. Accordingly, we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant. The strategy only allows accumulation of PIP2;7S273/276A during infection to limit potential side effects on normal plant growth.

Distinct profiles of plant immune resilience revealed by natural variation in warm temperature-modulated disease resistance among Arabidopsis accessions.

Elevated temperature suppresses the plant defence hormone salicylic acid (SA) by downregulating the expression of master immune regulatory genes CALMODULIN BINDING PROTEIN 60-LIKE G (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1). However, previous studies in Arabidopsis thaliana plants have primarily focused on the accession Columbia-0 (Col-0), while the genetic determinants of intraspecific variation in Arabidopsis immunity under elevated temperature remain unknown. Here we show that BASIC HELIX LOOP HELIX 059 (bHLH059), a thermosensitive SA regulator at nonstress temperatures, does not regulate immune suppression under warmer temperatures. In agreement, temperature-resilient and -sensitive Arabidopsis accessions based on disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 did not correlate with bHLH059 polymorphisms. Instead, we found that temperature-resilient accessions exhibit varying CBP60g and SARD1 expression profiles, potentially revealing CBP60g/SARD1-dependent and independent mechanisms of immune resilience to warming temperature. We identified thermoresilient accessions that exhibited either temperature-sensitive or -insensitive induction of the SA biosynthetic gene ICS1 (direct target gene of CBP60g and SARD1) and SA hormone levels. Collectively, this study has unveiled the intraspecific diversity of Arabidopsis immune responses under warm temperatures, which could aid in predicting plant responses to climate change and provide foundational knowledge for climate-resilient crop engineering.

Myosin XI-mediated BIK1 recruitment to nanodomains facilitates FLS2–BIK1 complex formation during innate immunity in Arabidopsis

Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various immune receptors and associated proteins are often dynamically distributed in specific nanodomains on the plasma membrane (PM). However, the exact molecular mechanism and functional relevance of this nanodomain targeting in plant immunity regulation remain largely unknown. By utilizing high spatiotemporal resolution imaging and single-particle tracking analysis, we show that myosin XIK interacts with remorin to recruit and stabilize PM-associated kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) within immune receptor FLAGELLIN SENSING 2 (FLS2)-containing nanodomains. This recruitment facilitates FLS2/BIK1 complex formation, leading to the full activation of BIK1-dependent defense responses upon ligand perception. Collectively, our findings provide compelling evidence that myosin XI functions as a molecular scaffold to enable a spatially confined complex assembly within nanodomains. This ensures the presence of a sufficient quantity of preformed immune receptor complex for efficient signaling transduction from the cell surface.

Increase of secondary metabolites in sweet basil (Ocimum basilicum L.) leaves by exposure to N2O5 with plasma technology

Exposure to N2O5 generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of N2O5 exposure on other plant species. Sweet basil synthesizes many valuable secondary metabolites in its leaves. Therefore, metabolomic analyses were performed at three different exposure levels [9.7 (Ex1), 19.4 (Ex2) and 29.1 (Ex3) μmol] to assess the effects of N2O5 on basil leaves. As a result, cinnamaldehyde and phenolic acids increased with increasing doses. Certain flavonoids, columbianetin, and caryophyllene oxide increased with lower Ex1 exposure, cineole and methyl eugenol increased with moderate Ex2 exposure and l-glutathione GSH also increased with higher Ex3 exposure. Furthermore, gene expression analysis by quantitative RT-PCR showed that certain genes involved in the syntheses of secondary metabolites and jasmonic acid were significantly up-regulated early after N2O5 exposure. These results suggest that N2O5 exposure increases several valuable secondary metabolites in sweet basil leaves via plant defense responses in a controllable system.

Cutin-derived oligomers induce hallmark plant immune responses.

The cuticle constitutes the outermost defensive barrier of most land plants. It comprises a polymeric matrix-cutin, surrounded by soluble waxes. Moreover, the cuticle constitutes the first line of defense against pathogen invasion, while also protecting the plant from many abiotic stresses. Aliphatic monomers in cutin have been suggested to act as immune elicitors in plants. This study analyses the potential of cutin oligomers to activate rapid signaling outputs reminiscent of pattern-triggered immunity in the model plant Arabidopsis. Cutin oligomeric mixtures led to Ca2+ influx and mitogen-activated protein kinase activation. Comparable responses were measured for cutin, which was also able to induce a reactive oxygen species burst. Furthermore, cutin oligomer treatment resulted in a unique transcriptional reprogramming profile, having many archetypal features of pattern-triggered immunity. Targeted spectroscopic and spectrometric analyses of the cutin oligomers suggest that the elicitor compounds consist mostly of two up to three 10,16-dihydroxyhexadecanoic acid monomers linked together through ester bonds. This study demonstrates that cutin breakdown products can act as inducers of early plant immune responses. Further investigation is needed to understand how cutin breakdowns are perceived and to explore their potential use in agriculture.

A root-knot nematode effector targets the Arabidopsis cysteine protease RD21A for degradation to suppress plant defense and promote parasitism.

Meloidogyne incognita is one of the most widely distributed plant-parasitic nematodes and causes severe economic losses annually. The parasite produces effector proteins that play essential roles in successful parasitism. Here, we identified one such effector named MiCE108, which is exclusively expressed within the nematode subventral esophageal gland cells and is upregulated in the early parasitic stage of M. incognita. A yeast signal sequence trap assay showed that MiCE108 contains a functional signal peptide for secretion. Virus-induced gene silencing of MiCE108 impaired the parasitism of M. incognita in Nicotiana benthamiana. The ectopic expression of MiCE108 in Arabidopsis suppressed the deposition of callose, the generation of reactive oxygen species, and the expression of marker genes for bacterial flagellin epitope flg22-triggered immunity, resulting in increased susceptibility to M. incognita, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst) DC3000. The MiCE108 protein physically associates with the plant defense protease RD21A and promotes its degradation via the endosomal-dependent pathway, or 26S proteasome. Consistent with this, knockout of RD21A compromises the innate immunity of Arabidopsis and increases its susceptibility to a broad range of pathogens, including M. incognita, strongly indicating a role in defense against this nematode. Together, our data suggest that M. incognita deploys the effector MiCE108 to target Arabidopsis cysteine protease RD21A and affect its stability, thereby suppressing plant innate immunity and facilitating parasitism.

Rice small secreted peptide, OsRALF26, recognized by FERONIA-like receptor 1 induces immunity in rice and Arabidopsis.

Rapid alkalinization factors (RALFs), belonging to a family of small secreted peptides, have been considered as important signaling molecules in diverse biological processes, including immunity. Current studies on RALF-modulated immunity mainly focus on Arabidopsis, but little is reported in crop plants. The rice immune receptor XA21 confers immunity to the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Here, we pursued functional characterization of rice RALF26 (OsRALF26) up-regulated by Xoo during XA21-mediated immune response. When applied exogenously as a recombinant peptide, OsRALF26 induced a series of immune responses, including pathogenesis-related genes (PRs) induction, reactive oxygen species (ROS) production, and callose deposition in rice and/or Arabidopsis. Transgenic rice and Arabidopsis overexpressing OsRALF26 exhibited significantly enhanced resistance to Xoo and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), respectively. In yeast two-hybrid, pull-down assays, and co-immunoprecipitation analyses, rice FER-like receptor 1 (OsFLR1) was identified as a receptor of OsRALF26. Transient expression of OsFLR1 in Nicotiana benthamiana leaves displayed significantly increased ROS production and callose deposition after OsRALF26 treatment. Together, we propose that OsRALF26 induced by Xoo in an XA21-dependent manner is perceived by OsFLR1 and may play a novel role in the enforcement of XA21-mediated immunity.

A novel lectin receptor kinase gene, AtG-LecRK-I.2, enhances bacterial pathogen resistance through regulation of stomatal immunity in Arabidopsis

The S-locus lectin receptor kinases (G-LecRKs) have been suggested as receptors for microbe/damage-associated molecular patterns (MAMPs/DAMPs) and to be involved in the pathogen defense responses, but the functions of most G-LecRKs in biotic stress response have not been characterized. Here, we identified a member of this family, G-LecRK-I.2, that positively regulates flg22- and Pseudomonas syringae pv. tomato (Pst) DC3000-induced stomatal closure. G-LecRK-I.2 was rapidly phosphorylated under flg22 treatment and could interact with the FLS2/BAK1 complex. Two T-DNA insertion lines, glecrk-i.2–1 and glecrk-i.2–2, had lower levels of reactive oxygen species (ROS) and nitric oxide (NO) production in guard cells, as compared with the wild-type Col-0, under Pst DC3000 infection. Also, the immunity marker genes CBP60g and PR1 were induced at lower levels under Pst DC3000 hrcC- infection in glecrk-i.2–1 and glecrk-i.2–2. The GUS reporter system also revealed that G-LecRK-I.2 was expressed only in guard cells. We also found that G-LecRK-I.2 could interact H+-ATPase AHA1 to regulate H+-ATPase activity in the guard cells. Taken together, our results show that G-LecRK-I.2 plays an important role in regulating stomatal closure under flg22 and Pst DC3000 treatments and in ROS and NO signaling specifically in guard cells.

Decomposition of dynamic transcriptomic responses during effector-triggered immunity reveals conserved responses in two distinct plant cell populations

Rapid plant immune responses in the appropriate cells are needed for effective defense against pathogens. Although transcriptome analysis is often used to describe overall immune responses, collection of transcriptome data with sufficient resolution in both space and time is challenging. We reanalyzed public Arabidopsis time-course transcriptome data obtained after low-dose inoculation with a Pseudomonas syringae strain expressing the effector AvrRpt2, which induces effector-triggered immunity in Arabidopsis. Double-peak time-course patterns are prevalent among thousands of upregulated genes. We implemented a multi-compartment modeling approach to decompose the double-peak pattern into two single-peak patterns for each gene. The decomposed peaks reveal an “echoing” pattern: the peak times of the first and second peaks correlate well across most upregulated genes. We demonstrated that the two peaks likely represent responses of two distinct cell populations that respond either cell autonomously or indirectly to AvrRpt2. Thus, the peak decomposition has extracted spatial information from the time-course data. The echoing pattern also indicates a conserved transcriptome response with different initiation times between the two cell populations despite different elicitor types. A gene set highly overlapping with the conserved gene set is also upregulated with similar kinetics during pattern-triggered immunity. Activation of a WRKY network via different entry-point WRKYs can explain the similar but not identical transcriptome responses elicited by different elicitor types. We discuss potential benefits of the properties of the WRKY activation network as an immune signaling network in light of pressure from rapidly evolving pathogens.

Cytokinin oxidase gene CKX5 is modulated in the immunity of Arabidopsis to Botrytis cinerea.

In our previous work, cytokinin (CK) signaling and biosynthesis were found to be modulated during Arabidopsis defense against infection by the necrotrophic pathogen Botrytis cinerea. Notably, the expression level of CYTOKININ OXIDASE/DEHYDROGENASE 5 (CKX5) was significantly induced in B. cinerea-infected leaves and later in distant B. cinerea-untreated leaves of the same plant. To confirm and determine how CKX5 is involved in the response to B. cinerea infection, transcript levels of CKX family genes were analyzed in B. cinerea-inoculated leaves, and only CKX5 was remarkably induced by B. cinerea infection. Furthermore, CKX5-overexpressing Arabidopsis plants were more resistant to B. cinerea than wild-type plants. Transcription factors (TFs) binding to the CKX5 promoter were then screened by yeast one-hybrid assays. Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) analysis further showed that genes encoding TFs, including WRKY40, WRKY33, ERF6, AHL15, AHL17, ANAC003, TCP13 and ANAC019, were also strongly induced in infected leaves, similar to CKX5. Analysis of ERF6-overexpressing plants and ERF6-and AHL15-knockout mutants indicated that ERF6 and AHL15 are involved in plant immunity to B. cinerea. Furthermore, CKX5 upregulation by B. cinerea infection was affected when ERF6 or AHL15 levels were altered. Our work suggests that CKX5 levels are controlled by the plant defense system to defend against attack by the pathogen B. cinerea.

IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss

Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

CRISPR-targeted mutagenesis of mitogen-activated protein kinase phosphatase 1 improves both immunity and yield in wheat.

Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over- or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plant Arabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock-down of TaMKP1 by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused by Puccinia striiformis f. sp. tritici (Pst) and powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), indicating that TaMKP1 negatively regulates disease resistance in wheat. Unexpectedly, while Tamkp1 mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild-type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK-mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield.

Marker and readout genes for defense priming in Pseudomonas cannabina pv. alisalensis interaction aid understanding systemic immunity in Arabidopsis

Following localized infection, the entire plant foliage becomes primed for enhanced defense. However, specific genes induced during defense priming (priming-marker genes) and those showing increased expression in defense-primed plants upon rechallenge (priming-readout genes) remain largely unknown. In our Arabidopsis thaliana study, genes AT1G76960 (function unknown), CAX3 (encoding a vacuolar Ca2+/H+ antiporter), and CRK4 (encoding a cysteine-rich receptor-like protein kinase) were strongly expressed during Pseudomonas cannabina pv. alisalensis-induced defense priming, uniquely marking the primed state for enhanced defense. Conversely, PR1 (encoding a pathogenesis-related protein), RLP23 and RLP41 (both encoding receptor-like proteins) were similarly activated in defense-primed plants before and after rechallenge, suggesting they are additional marker genes for defense priming. In contrast, CASPL4D1 (encoding Casparian strip domain-like protein 4D1), FRK1 (encoding flg22-induced receptor-like kinase), and AT3G28510 (encoding a P loop-containing nucleoside triphosphate hydrolases superfamily protein) showed minimal activation in uninfected, defense-primed, or rechallenged plants, but intensified in defense-primed plants after rechallenge. Notably, mutation in only priming-readout gene NHL25 (encoding NDR1/HIN1-like protein 25) impaired both defense priming and systemic acquired resistance, highlighting its previously undiscovered pivotal role in systemic plant immunity.

Exogenous application of pectin triggers stomatal closure and immunity in Arabidopsis.

Pectin has been extensively studied in animal immunity, and exogenous pectin as a food additive can provide protection against inflammatory bowel disease. However, the utility of pectin to improve immunity in plants is still unstudied. Here, we found exogenous application of pectin triggered stomatal closure in Arabidopsis in a dose- and time-dependent manner. Additionally, pectin activated peroxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to produce reactive oxygen species (ROS), which subsequently increased cytoplasmic Ca2+ concentration ([Ca2+ ]cyt ) and was followed by nitric oxide (NO) production, leading to stomatal closure in an abscisic acid (ABA) and salicylic acid (SA) signalling-dependent mechanism. Furthermore, pectin enhanced the disease resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) with mitogen-activated protein kinases (MPKs) MPK3/6 activated and upregulated expression of defence-responsive genes in Arabidopsis. These results suggested that exogenous pectin-induced stomatal closure was associated with ROS and NO production regulated by ABA and SA signalling, contributing to defence against Pst DC3000 in Arabidopsis.

A natural single‐nucleotide polymorphism in the CAMTA3 transcription factor regulates its function and transcription of its target genes

AbstractCAMTA3, a Ca2+/calmodulin‐binding transcription factor, is a key regulator of plant immunity in Arabidopsis. Here, we identified a novel naturally occurring single‐nucleotide polymorphism that results in a missense nonconservative mutation (CAMTA3H386D) in many Arabidopsis ecotypes. This region of CAMTA3 is not part of any previously characterized regulatory domains. To study the consequence of this change on the function of CAMTA3, we introduced the CAMTA3H386D into camta3, a loss‐of‐function mutant that exhibits a constitutive cell death phenotype, chlorotic lesions on leaves, and reduced plant size. Phenotypic and molecular analysis of these lines indicated that the expression of CAMTA3H386D in the camta3 mutant did not complement the mutant phenotypes. Also, the ecotypes containing the CAMTA3H386D exhibited camta3 phenotypes. Marker genes associated with salicylic acid biosynthesis and pathogen response were upregulated in the CAMTA3H386D lines and the Arabidopsis accessions 7127 (Est‐1) and 9941 (Fei‐0), as in camta3, indicating that H386D mutation alters CAMTA3 activity in regulating the expression of known target genes. In Nicotiana benthamiana transient expression assays, CAMTA3H386D failed to induce the expression of a luciferase reporter gene driven by the rapid stress‐responsive elements (RSRE) that contain the known binding sites of CAMTA3, suggesting that CAMTA3H386D mutation impairs its ability to activate its target genes. Transgenic lines and tested natural accessions expressing CAMTA3H386D showed enhanced levels of H2O2 and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Collectively, our results indicate that the H386D mutation in a previously unknown regulatory region of CAMTA3 is essential for its function.

The tRNA thiolation-mediated translational control is essential for plant immunity.

Plants have evolved sophisticated mechanisms to regulate gene expression to activate immune responses against pathogen infections. However, how the translation system contributes to plant immunity is largely unknown. The evolutionarily conserved thiolation modification of transfer RNA (tRNA) ensures efficient decoding during translation. Here, we show that tRNA thiolation is required for plant immunity in Arabidopsis. We identify a cgb mutant that is hyper-susceptible to the pathogen Pseudomonas syringae. CGB encodes ROL5, a homolog of yeast NCS6 required for tRNA thiolation. ROL5 physically interacts with CTU2, a homolog of yeast NCS2. Mutations in either ROL5 or CTU2 result in loss of tRNA thiolation. Further analyses reveal that both transcriptome and proteome reprogramming during immune responses are compromised in cgb. Notably, the translation of salicylic acid receptor NPR1 is reduced in cgb, resulting in compromised salicylic acid signaling. Our study not only reveals a regulatory mechanism for plant immunity but also uncovers an additional biological function of tRNA thiolation.

Plant U-box E3 ligases PUB20 and PUB21 negatively regulate pattern-triggered immunity in Arabidopsis.

Plant U-box E3 ligases PUB20 and PUB21 are flg22-triggered signaling components and negatively regulate immune responses. Plant U-box proteins (PUBs) constitute a class of E3 ligases that are associated with various stress responses. Among the class IV PUBs featuring C-terminal Armadillo (ARM) repeats, PUB20 and PUB21 are closely related homologs. Here, we show that both PUB20 and PUB21 negatively regulate innate immunity in plants. Loss of PUB20 and PUB21 function leads to enhanced resistance to surface inoculation with the virulent bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the resistance levels remain unaffected after infiltration inoculation, suggesting that PUB20 and PUB21 primarily function during the early defense stages. The enhanced resistance to Pst DC3000 in PUB mutant plants (pub20-1, pub21-1, and pub20-1/pub21-1) correlates with extensive flg22-triggered reactive oxygen production, strong MPK3 activation, and enhanced transcriptional activation of early immune response genes. Additionally, PUB mutant plants (except pub21-1) exhibit constitutive stomatal closure after Pst DC3000 inoculation, implying the significant role of PUB20 in stomatal immunity. Comparative analyses of flg22 responses between PUB mutants and wild-type plants reveals that the robust activation of the pattern-induced immune responses may enhance resistance against Pst DC3000. Notably, the hypersensitivity responses triggered by RPM1/avrRpm1 and RPS2/avrRpt2 are independent of PUB20 and PUB21. These results suggest that PUB20 and PUB21 knockout mutations affect bacterial invasion, likely during the early stages, acting as negative regulators of plant immunity.