Microbe-induced coordination of plant iron-sulfur metabolism enhances high light stress tolerance of Arabidopsis.

High light stress in subtropical and tropical regions strongly limits agricultural production due to photo-oxidative damage, decreased growth and yield. Here, we investigated whether beneficial microbes can protect plants under high light stress. We found that Enterobacter sp. SA187 (SA187) supports Arabidopsis thaliana growth under high light stress by reducing the accumulation of reactive oxygen species (ROS) and maintaining photosynthesis. When subjected to high light stress, SA187 triggers dynamic changes in Arabidopsis gene expression related to fortified iron metabolism and redox regulation thereby enhancing the plant anti-oxidative glutathione/glutaredoxin redox system. Genetic analysis shows that SA187-enhanced iron and sulfur metabolism are coordinated by ethylene signaling. In summary, beneficial microbes could be an effective and inexpensive means for enhancing high light stress tolerance in plants.

Integrated multi-omics and genetic analyses reveal molecular determinants underlying Arabidopsis snap33 mutant phenotype.

The secretory pathway is essential for plant immunity, delivering diverse antimicrobial molecules into the extracellular space. Arabidopsis thaliana soluble N-ethylmaleimide-sensitive-factor attachment protein receptor SNAP33 is a key actor of this process. The snap33 mutant displays dwarfism and necrotic lesions, however the molecular determinants of its macroscopic phenotypes remain elusive. Here, we isolated several new snap33 mutants that exhibited constitutive cell death and H2O2 accumulation, further defining snap33 as an autoimmune mutant. We then carried out quantitative transcriptomic and proteomic analyses showing that numerous defense transcripts and proteins were up-regulated in the snap33 mutant, among which genes/proteins involved in defense hormone, pattern-triggered immunity, and nucleotide-binding domain leucine-rich-repeat receptor signaling. qRT-PCR analyses and hormone dosages supported these results. Furthermore, genetic analyses elucidated the diverse contributions of the main defense hormones and some nucleotide-binding domain leucine-rich-repeat receptor signaling actors in the establishment of the snap33 phenotype, emphasizing the preponderant role of salicylic acid over other defense phytohormones. Moreover, the accumulation of pattern-triggered immunity and nucleotide-binding domain leucine-rich-repeat receptor signaling proteins in the snap33 mutant was confirmed by immunoblotting analyses and further shown to be salicylic acid-dependent. Collectively, this study unveiled molecular determinants underlying the Arabidopsis snap33 mutant phenotype and brought new insights into autoimmunity signaling.

Salinity stress-induced phosphorylation of INDETERMINATE-DOMAIN 4 (IDD4) by MPK6 regulates plant growth adaptation in Arabidopsis.

The INDETERMINATE DOMAIN (IDD) family belongs to a group of plant-specific transcription factors that coordinates plant growth/development and immunity. However, the function and mode of action of IDDs during abiotic stress, such as salt, are poorly understood. We used idd4 transgenic lines and screened them under salt stress to find the involvement of IDD4 in salinity stress tolerance The genetic disruption of IDD4 increases salt-tolerance, characterized by sustained plant growth, improved Na+/K+ ratio, and decreased stomatal density/aperture. Yet, IDD4 overexpressing plants were hypersensitive to salt-stress with an increase in stomatal density and pore size. Transcriptomic and ChIP-seq analyses revealed that IDD4 directly controls an important set of genes involved in abiotic stress/salinity responses. Interestingly, using anti-IDD4-pS73 antibody we discovered that IDD4 is specifically phosphorylated at serine-73 by MPK6 in vivo under salinity stress. Analysis of plants expressing the phospho-dead and phospho-mimicking IDD4 versions proved that phosphorylation of IDD4 plays a crucial role in plant transcriptional reprogramming of salt-stress genes. Altogether, we show that salt stress adaption involves MPK6 phosphorylation of IDD4 thereby regulating IDD4 DNA-binding and expression of target genes.

Dynamics of ribosome composition and ribosomal protein phosphorylation in immune signaling in Arabidopsis thaliana.

In plants, the detection of microbe-associated molecular patterns (MAMPs) induces primary innate immunity by the activation of mitogen-activated protein kinases (MAPKs). We show here that the MAMP-activated MAPK MPK6 not only modulates defense through transcriptional regulation but also via the ribosomal protein translation machinery. To understand the effects of MPK6 on ribosomes and their constituent ribosomal proteins (RPs), polysomes, monosomes and the phosphorylation status of the RPs, MAMP-treated WT and mpk6 mutant plants were analysed. MAMP-activation induced rapid changes in RP composition of monosomes, polysomes and in the 60S ribosomal subunit in an MPK6-specific manner. Phosphoproteome analysis showed that MAMP-activation of MPK6 regulates the phosphorylation status of the P-stalk ribosomal proteins by phosphorylation of RPP0 and the concomitant dephosphorylation of RPP1 and RPP2. These events coincide with a significant decrease in the abundance of ribosome-bound RPP0s, RPP1s and RPP3s in polysomes. The P-stalk is essential in regulating protein translation by recruiting elongation factors. Accordingly, we found that RPP0C mutant plants are compromised in basal resistance to Pseudomonas syringae infection. These data suggest that MAMP-induced defense also involves MPK6-induced regulation of P-stalk proteins, highlighting a new role of ribosomal regulation in plant innate immunity.

Essential role of the CD docking motif of MPK4 in plant immunity, growth, and development.

MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators. We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand-bound MPK4 crystal structure. We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild-type (WT) MPK4-C181, nonsulfenylatable MPK4-C181S, and potentially sulfenylation mimicking MPK4-C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4-C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4-C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4. Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase.

The wheat stem rust resistance gene Sr43 encodes an unusual protein kinase.

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool1. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43, which was crossed into bread wheat from the wild grass Thinopyrum elongatum2,3. Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.

Linker histone H1 modulates defense priming and immunity in plants.

Linker H1 histones play an important role in animal and human pathogenesis, but their function in plant immunity is poorly understood. Here, we analyzed mutants of the three canonical variants of Arabidopsis H1 histones, namely H1.1, H1.2 and H1.3. We observed that double h1.1h1.2 and triple h1.1h1.2h1.3 (3h1) mutants were resistant to Pseudomonas syringae and Botrytis cinerea infections. Transcriptome analysis of 3h1 mutant plants showed H1s play a key role in regulating the expression of early and late defense genes upon pathogen challenge. Moreover, 3h1 mutant plants showed enhanced production of reactive oxygen species and activation of mitogen activated protein kinases upon pathogen-associated molecular pattern (PAMP) treatment. However, 3h1 mutant plants were insensitive to priming with flg22, a well-known bacterial PAMP which induces enhanced resistance in WT plants. The defective defense response in 3h1 upon priming was correlated with altered DNA methylation and reduced global H3K56ac levels. Our data place H1 as a molecular gatekeeper in governing dynamic changes in the chromatin landscape of defense genes during plant pathogen interaction.

OXIDATIVE SIGNAL-INDUCIBLE1 induces immunity by coordinating N-hydroxypipecolic acid, salicylic acid, and camalexin synthesis.

Expression of OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1) is induced by a number of stress conditions and regulates the interaction of plants with pathogenic and beneficial microbes. In this work, we generated Arabidopsis OXI1 knockout and genomic OXI1 overexpression lines and show by transcriptome, proteome, and metabolome analysis that OXI1 triggers ALD1, SARD4, and FMO1 expressions to promote the biosynthesis of pipecolic acid (Pip) and N-hydroxypipecolic acid (NHP). OXI1 contributes to enhanced immunity by induced SA biosynthesis via CBP60g-induced expression of SID2 and camalexin accumulation via WRKY33-targeted transcription of PAD3. OXI1 regulates genes involved in reactive oxygen species (ROS) generation such as RbohD and RbohF. OXI1 knock out plants show enhanced expression of nuclear and chloroplast genes of photosynthesis and enhanced growth under ambient conditions, while OXI1 overexpressing plants accumulate NHP, SA, camalexin, and ROS and show a gain-of-function (GOF) cell death phenotype and enhanced pathogen resistance. The OXI1 GOF phenotypes are completely suppressed when compromising N-hydroxypipecolic acid (NHP) synthesis in the fmo1 or ald1 background, showing that OXI1 regulation of immunity is mediated via the NHP pathway. Overall, these results show that OXI1 plays a key role in basal and effector-triggered plant immunity by regulating defense and programmed cell death via biosynthesis of salicylic acid, N-hydroxypipecolic acid, and camalexin.

Analysis of the Arabidopsis coilin mutant reveals a positive role of AtCOILIN in plant immunity.

Biogenesis of ribonucleoproteins occurs in dynamic subnuclear compartments called Cajal bodies (CBs). COILIN is a critical scaffolding component essential for CB formation, composition, and activity. We recently showed that Arabidopsis (Arabidopsis thaliana) AtCOILIN is phosphorylated in response to bacterial elicitor treatment. Here, we further investigated the role of AtCOILIN in plant innate immunity. Atcoilin mutants are compromised in defense responses to bacterial pathogens. Besides confirming a role of AtCOILIN in alternative splicing (AS), Atcoilin showed differential expression of genes that are distinct from those of AS, including factors involved in RNA biogenesis, metabolism, plant immunity, and phytohormones. Atcoilin mutant plants have reduced levels of defense phytohormones. As expected, the mutant plants were more sensitive to the necrotrophic fungal pathogen Botrytis cinerea. Our findings reveal an important role for AtCOILIN in innate plant immunity.

Comprehensive evolutionary analysis and nomenclature of plant G3BPs.

Stress induces extensive reprogramming of mRNA metabolism, which includes the transcription and translation of stress-related genes and the formation of stress granules. RasGAP SH3 domain-binding proteins (G3BPs, also called Rasputins) form a highly conserved family of proteins found throughout eukaryotic evolution, which coordinate signal transduction and posttranscriptional gene regulation and play a key role in the formation of stress granules. G3BPs play a role in osmotic, oxidative, and biotic stress in mammals, and recent results revealed that they play similar functions in higher plants. Although simple eukaryotes such as yeast have only one G3BP gene, higher plants show a massive expansion of their G3BP genes into distinct subfamilies. However, because this family of genes has not been well-characterized in plants, functions that have evolved during this expansion remain unidentified. Therefore, we carried out a phylogenetic analysis of G3BPs in different eukaryotes, particularly focusing on the green lineage. On the basis of this evolutionary analysis of G3BPs in eukaryotes, we propose a uniform nomenclature for plant G3BPs that should help predict the evolutionary and functional diversification in this family.

The Lys-motif receptor LYK4 mediates Enterobacter sp. SA187 triggered salt tolerance in Arabidopsis thaliana.

Root endophytes establish beneficial interactions with plants, improving holobiont resilience and fitness, but how plant immunity accommodates beneficial microbes is poorly understood. The multi-stress tolerance-inducing endophyte Enterobacter sp. SA187 triggers a canonical immune response in Arabidopsis only at high bacterial dosage (>108 CFUs ml-1 ), suggesting that SA187 is able to evade or suppress the plant defence system at lower titres. Although SA187 flagellin epitopes are recognized by the FLS2 receptor, SA187-triggered salt tolerance functions independently of the FLS2 system. In contrast, overexpression of the chitin receptor components LYK4 and LYK5 compromised the beneficial effect of SA187 on Arabidopsis, while it was enhanced in lyk4 mutant plants. Transcriptome analysis revealed that the role of LYK4 is intertwined with a function in remodelling defence responses with growth and root developmental processes. LYK4 interferes with modification of plant ethylene homeostasis by Enterobacter SA187 to boost salt stress resistance. Collectively, these results contribute to unlock the crosstalk between components of the plant immune system and beneficial microbes and point to a new role for the Lys-motif receptor LYK4 in beneficial plant-microbe interaction.

In vivo identification of putative CPK5 substrates in Arabidopsis thaliana.

Calcium signaling mediates most developmental processes and stress responses in plants. Among plant calcium sensors, the calcium-dependent protein kinases display a unique structure harboring both calcium sensing and kinase responding activities. AtCPK5 is an essential member of this family in Arabidopsis that regulates immunity and abiotic stress tolerance. To understand the underlying molecular mechanisms, we implemented a biochemical approach to identify in vivo substrates of AtCPK5. We generated transgenic lines expressing a constitutively active form of AtCPK5 under the control of a dexamethasone-inducible promoter. Lines expressing a kinase-dead version were used as a negative control. By comparing the phosphoproteome of the kinase-active and kinase-dead lines upon dexamethasone treatment, we identified 5 phosphopeptides whose abundance increased specifically in the kinase-active lines. Importantly, we showed that all 5 proteins were phosphorylated in vitro by AtCPK5 in a calcium-dependent manner, suggesting that they are direct targets of AtCPK5. We also detected several interaction patterns between the kinase and the candidates in the cytosol, membranes or nucleus, consistent with the ubiquitous localization of AtCPK5. Finally, we further validated the two phosphosites S245 and S280 targeted by AtCPK5 in the E3 ubiquitin ligase ATL31. Altogether, those results open new perspectives to decipher AtCPK5 biological functions.

A Semi-In Vivo Transcriptional Assay to Dissect Plant Defense Regulatory Modules.

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.

G3BPs in Plant Stress.

The sessile nature of plants enforces highly adaptable strategies to adapt to different environmental stresses. Plants respond to these stresses by a massive reprogramming of mRNA metabolism. Balancing of mRNA fates, including translation, sequestration, and decay is essential for plants to not only coordinate growth and development but also to combat biotic and abiotic environmental stresses. RNA stress granules (SGs) and processing bodies (P bodies) synchronize mRNA metabolism for optimum functioning of an organism. SGs are evolutionarily conserved cytoplasmic localized RNA-protein storage sites that are formed in response to adverse conditions, harboring mostly but not always translationally inactive mRNAs. SGs disassemble and release mRNAs into a translationally active form upon stress relief. RasGAP SH3 domain binding proteins (G3BPs or Rasputins) are "scaffolds" for the assembly and stability of SGs, which coordinate receptor mediated signal transduction with RNA metabolism. The role of G3BPs in the formation of SGs is well established in mammals, but G3BPs in plants are poorly characterized. In this review, we discuss recent findings of the dynamics and functions of plant G3BPs in response to environmental stresses and speculate on possible mechanisms such as transcription and post-translational modifications that might regulate the function of this important family of proteins.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity.

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Nanofabrication of Isoporous Membranes for Cell Fractionation.

Cell fractionations and other biological separations frequently require several steps. They could be much more effectively done by filtration, if isoporous membranes would be available with high pore density, and sharp pore size distribution in the micro- and nanoscale. We propose a combination of two scalable methods, photolithography and dry reactive ion etching, to fabricate a series of polyester membranes with isopores of size 0.7 to 50 µm and high pore density with a demonstrated total area of 38.5 cm2. The membranes have pore sizes in the micro- and submicro-range, and pore density 10-fold higher than track-etched analogues, which are the only commercially available isoporous polymeric films. Permeances of 220,000 L m-2 h-1bar-1 were measured with pore size 787 nm. The method does not require organic solvents and can be applied to many homopolymeric materials. The pore reduction from 2 to 0.7 µm was obtained by adding a step of chemical vapor deposition. The isoporous system was successfully demonstrated for the organelle fractionation of Arabidopsis homogenates and could be potentially extended to other biological fractionations.

Phosphorylation regulates the activity of INDETERMINATE-DOMAIN (IDD/BIRD) proteins in response to diverse environmental conditions.

INDETERMINATE-DOMAIN proteins (IDDs) belong to a diverse plant-specific family of transcriptional regulators that coordinate distinct functions during plant growth and development. The functions of several of these IDD members are transcriptionally regulated, but so far nothing is known about the regulation at the post-translational level in spite of the fact that post-translational modifications of these proteins have been reported in several large-scale proteomics studies. Recently, we showed that IDD4 is a repressor of basal immunity and its characteristic traits are predominantly determined by the phosphorylation at two distinct phosphorylation sites. This finding prompted us to comprehensively review phosphorylation of the various IDD members from the plethora of phosphoproteomics studies demonstrating the post-translational modification of IDDs at highly conserved sites under various experimental conditions. We reckon that the phosphorylation of IDDs is an underrated mechanistic aspect in their regulation and we postulate their importance in IDD/BIRD functioning.

INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana.

INDETERMINATE DOMAIN (IDD)/ BIRD proteins are a highly conserved plant-specific family of transcription factors which play multiple roles in plant development and physiology. Here, we show that mutation in IDD4/IMPERIAL EAGLE increases resistance to the hemi-biotrophic pathogen Pseudomonas syringae, indicating that IDD4 may act as a repressor of basal immune response and PAMP-triggered immunity. Furthermore, the idd4 mutant exhibits enhanced plant-growth indicating IDD4 as suppressor of growth and development. Transcriptome comparison of idd4 mutants and IDD4ox lines aligned to genome-wide IDD4 DNA-binding studies revealed major target genes related to defense and developmental-biological processes. IDD4 is a phospho-protein that interacts and becomes phosphorylated on two conserved sites by the MAP kinase MPK6. DNA-binding studies of IDD4 after flg22 treatment and with IDD4 phosphosite mutants show enhanced binding affinity to ID1 motif-containing promoters and its function as a transcriptional regulator. In contrast to the IDD4-phospho-dead mutant, the IDD4 phospho-mimicking mutant shows altered susceptibility to PstDC3000, salicylic acid levels and transcriptome reprogramming. In summary, we found that IDD4 regulates various hormonal pathways thereby coordinating growth and development with basal immunity.

The Lamin-Like LITTLE NUCLEI 1 (LINC1) Regulates Pattern-Triggered Immunity and Jasmonic Acid Signaling.

Pathogen-associated molecular pattern (PAMP) recognition occurs by plasma membrane located receptors that induce among other processes nuclear gene expression. However, signaling to the nuclear compartment is restricted by the nuclear envelope and nuclear pore complexes. We show here that among the four Arabidopsis lamin homologs LITTLE NUCLEI/CROWDED NUCLEI (LINC/CRWN), LINC1 plays an important role in PTI and jasmonic acid (JA) signaling. We show that linc1 knock out mutants affect PAMP-triggered MAPK activation and growth inhibition, but not reactive oxygen species or callose accumulation. We also demonstrate that linc1 mutants are compromised in regulating PAMP-triggered pathogen-related genes, in particular encoding factors involved in JA signaling and responses. Expression of a number of JAZ domain proteins, the key JA-related transcription factor MYC2 as well as key MYB transcription factors and biosynthesis genes of both the indole and aliphatic glucosinolate pathways are changed in linc1 mutants. Moreover, PAMP triggers JA and JA-Ile accumulation in linc1 mutants, whereas salicylic acid levels are unchanged. Despite impairment in PAMP-triggered immunity, linc1 mutants still show basal immunity towards Pseudomonas syringae DC3000 strains. High JA levels usually render plants resistant to necrotrophic pathogen. Thus, linc1 mutants show enhanced resistance to Botrytis cinerea infection. In accordance with a general role of LINC1 in JA signaling, linc1 mutants are hypersensitive to growth inhibition to external JA. In summary, our findings show that the lamin-like LINC1 protein plays a key role in JA signaling and regulation of PTI responses in Arabidopsis.