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Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Enabling and constraining immune activation is of fundamental importance in maintaining cellular homeostasis. Depleting BAK1 and SERK4, the co-receptors of multiple pattern recognition receptors (PRRs), abolishes pattern-triggered immunity but triggers intracellular NOD-like receptor (NLR)-mediated autoimmunity with an elusive mechanism. By deploying RNAi-based genetic screens in Arabidopsis, we identified BAK-TO-LIFE 2 (BTL2), an uncharacterized receptor kinase, sensing BAK1/SERK4 integrity. BTL2 induces autoimmunity through activating Ca 2+ channel CNGC20 in a kinase-dependent manner when BAK1/SERK4 are perturbed. To compensate for BAK1 deficiency, BTL2 complexes with multiple phytocytokine receptors, leading to potent phytocytokine responses mediated by helper NLR ADR1 family immune receptors, suggesting phytocytokine signaling as a molecular link connecting PRR- and NLR-mediated immunity. Remarkably, BAK1 constrains BTL2 activation via specific phosphorylation to maintain cellular integrity. Thus, BTL2 serves as a surveillance rheostat sensing the perturbation of BAK1/SERK4 immune co-receptors in promoting NLR-mediated phytocytokine signaling to ensure plant immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a Pseudomonas syringae strain chromosomally tagged with the Nanoluc luciferase (NL) from the deep-sea shrimp Oplophorus gracilirostris , a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was more than 100 times brighter than P. syringae tagged with the luxCDABE operon from Photorhabdus luminescens , one of the existing luciferase-based strains. In planta imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, more than 30 epigenetic mutants were analyzed for their resistance traits because the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent Pseudomonas syringae pv. tomato . Dicer-like and Argonaute proteins, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for effector-triggered immunity and basal resistance to P. syringae pv. tomato . In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge about complex defense signaling pathways.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tracking RNA transcription has been one of the most powerful tools to gain insight into the biological process. While a wide range of molecular methods such as northern blotting, RNA-seq, and quantitative RT-PCR are available, one of the barriers in transcript analysis is an inability to accommodate a sufficient number of samples to achieve high resolution in dynamic transcriptional changes. RASL-seq (RNA-mediated oligonucleotide Annealing, Selection, and Ligation with next-generation sequencing) is a sequencing-based transcription profiling tool that processes hundreds of samples assessing a set of over a hundred genes with a fraction of the cost of a conventional RNA-seq. We described a RASL-seq protocol for assessing 288 genes mostly including defense genes to capture their dynamic nature. We demonstrated that this transcriptional profiling method produced a highly reliable outcome comparable to a conventional RNA-seq and quantitative RT-PCR., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Seed germination is an important stage in the lifecycle of a plant because it determines subsequent vegetative growth and reproduction. Here, we show that the E3 SUMO ligase AtSIZ1 regulates seed dormancy and germination. The germination rates of the siz1 mutants were less than 50%, even after a short period of ripening. However, their germination rates increased to wild-type levels after cold stratification or long periods of ripening. In addition, exogenous gibberellin (GA) application improved the germination rates of the siz1 mutants to the wild-type level. In transgenic plants, suppression of AtSIZ1 caused rapid post-translational decay of SLEEPY1 (SLY1), a positive regulator of GA signaling, during germination, and inducible AtSIZ1 overexpression led to increased SLY1 levels. In addition, overexpressing wild-type SLY1 in transgenic sly1 mutants increased their germination ratios to wild-type levels, whereas the germination ratio of transgenic sly1 mutants overexpressing mSLY1 was similar to that of sly1. The germination ratios of siz1 mutant seeds in immature developing siliques were much lower than those of the wild-type. Moreover, SLY1 and DELAY OF GERMINATION 1 (DOG1) transcript levels were reduced in the siz1 mutants, whereas the transcript levels of DELLA and ABSCISIC ACID INSENSITIVE 3 (ABI3) were higher than those of the wild-type. Taken together, these results indicate that the reduced germination of the siz1 mutants results from impaired GA signaling due to low SLY1 levels and activity, as well as hyperdormancy due to high levels of expression of dormancy-related genes including DOG1., (© 2016 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

The activities of mammalian DNA and histone methyltransferases are regulated by post-translational modifications such as phosphorylation and sumoylation; however, it is unclear how the activities of these enzymes are regulated at the post-translational level in plants. Here, we demonstrate that the DNA methylation activity of Arabidopsis CHROMOMETHYLASE 3 (CMT3) is positively regulated by the E3 SUMO ligase AtSIZ1. The methylation level of the Arabidopsis genome, including transposons, was significantly lower in the siz1-2 mutant than in wild-type plants. CMT3 was sumoylated by the E3 ligase activity of AtSIZ1 through a direct interaction, and the DNA methyltransferase activity of CMT3 was enhanced by this modification. In addition, the methylation levels of a large number of genes, including the nitrate reductase gene NIA2, were lower in siz1-2 and cmt3 plants than in wild-type plants. Furthermore, the CHG methylation activity of CMT3 was specific for NIA2and not NIA1 (the other nitrate reductase gene in Arabidopsis), indicating that CMT3 selectively regulates the CHG methylation levels of target genes. Taken together, our results indicate that the sumoylation of CMT3 is critical for its role in the control of gene expression and that AtSIZ1 positively controls the epigenetic repression of CMT3-mediated gene expression., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation., (© 2015 Authors; published by Portland Press Limited.)

Plant mitochondria possess alternative respiratory pathways mediated by the type II NAD(P)H dehydrogenases and alternative oxidases. Here, E3 SUMO ligase was shown to regulate alternative respiratory pathways and to participate in the maintenance of carbon and nitrogen balance in Arabidopsis. The transcript abundance of the type II NAD(P)H dehydrogenases NDA2 and NDB2 and alternative oxidases AOX1a and AOX1d genes was low in siz1-2 mutants compared to that in wild-type. The addition of nitrate or ammonium resulted in a decrease or an increase in the expression of the same gene families, respectively, in both wild-type and siz1-2 mutants. The amount of free sugar (glucose, fructose and sucrose) was lower in siz1-2 mutants than that in wild-type. These results indicate that low nitrate reductase activity due to the AtSIZ1 mutation is correlated with an overall decrease in alternative respiration and with a low carbohydrate content to maintain the carbon to nitrogen ratio in siz1-2 mutants.

Small peptides and proteins play critical regulatory roles in plant development and environmental stress responses; however, only a few of these molecules have been identified and characterized to date because of their poor annotation and other experimental challenges. Here, we present that rice (Oryza sativa L.) OsS1Fa1, a small 76-amino acid protein, confers drought stress tolerance in Arabidopsis thaliana. OsS1Fa1 was highly expressed in leaf, culm, and root tissues of rice seedlings during vegetative growth and was significantly induced under drought stress. OsS1Fa1 overexpression in Arabidopsis induced the expression of selected drought-responsive genes and enhanced the survival rate of transgenic lines under drought. The proteasome inhibitor MG132 protected the OsS1Fa1 protein from degradation. Together, our data indicate that the small protein OsS1Fa1 is induced by drought and is post-translationally regulated, and the ectopic expression of OsS1Fa1 protects plants from drought stress. [ABSTRACT FROM AUTHOR]