14-3-3 proteins as a major hub for plant immunity.

14-3-3 proteins, ubiquitously present in eukaryotic cells, are regulatory proteins involved in a plethora of cellular processes. In plants, they have been studied in the context of metabolism, development, and stress responses. Recent studies have highlighted the pivotal role of 14-3-3 proteins in regulating plant immunity. The ability of 14-3-3 proteins to modulate immune responses is primarily attributed to their function as interaction hubs, mediating protein-protein interactions and thereby regulating the activity and overall function of their binding partners. Here, we shed light on how 14-3-3 proteins contribute to plant defense mechanisms, the implications of their interactions with components of plant immunity cascades, and the potential for leveraging this knowledge for crop improvement strategies.

Host genotype, soil composition, and geo-climatic factors shape the fonio seed microbiome.

BACKGROUND: Fonio (Digitaria exilis), an orphan millet crop, is the oldest indigenous crop in West Africa. Although the yield is low due to pre-domestication characteristics, the quick maturation time, drought tolerance, and the ability to thrive on poor soils make fonio a climate-smart crop. Being holobionts, plants evolve in close interaction with microbial partners, which is crucial for plant phenology and fitness. As seeds are the bottleneck of vertically transmitting plant microbiota, we proposed to unravel the seed microbiome of the under-domesticated and resilient crop fonio. Our study investigated the bacterial seed endophyte diversity across 126 sequenced fonio accessions from distinct locations in West Africa. We conducted a correlation study of the structures and functions of the seed-associated microbiomes with the native geo-climate and soil structure data. We also performed Genome-wide association studies (GWAS) to identify genetic loci associated with seed endophyte diversity. RESULT: We report that fonio millet has diverse heritable seed endophytic taxa. We analyzed the seed microbiomes of 126 fonio accessions and showed that despite the diversity of microbiomes from distinct geographical locations, all fonio genetic groups share a core microbiome. In addition, we observed that native soil composition, geo-climatic factors, and host genotype correlate with the seed microbiomes. GWAS analysis of genetic loci associated with endophyte seed bacterial diversity identified fonio SNPs associated with genes functioning in embryo development and stress/defense response. CONCLUSION: Analysis of the seed endophyte of the climate-smart crop fonio indicated that despite possessing a heritable core microbiome, native conditions may shape the overall fonio seed microbiomes in different populations. These distinct microbiomes could play important roles in the adaptation of fonio to different environmental conditions. Our study identified the seed microbiome as a potential target for enhancing crop resilience to climate stress in a sustainable way. Video Abstract.

m6A RNA methylation counteracts dark-induced leaf senescence in Arabidopsis.

Senescence is an important physiological process which directly affects many agronomic traits in plants. Senescence induces chlorophyll degradation, phytohormone changes, cellular structure damage, and altered gene regulation. Although these physiological outputs are well defined, the molecular mechanisms employed are not known. Using dark-induced leaf senescence (DILS) as the experimental system, we investigated the role of N6-methyladenosine (m6A) mRNA methylation during senescence in Arabidopsis (Arabidopsis thaliana). Plants compromised in m6A machinery components like METHYLTRANSFERASE A (mta mutant) and VIRILIZER1 (vir-1 mutant) showed an enhanced DILS phenotype. This was accompanied by compromised chloroplast and photosynthesis performance in mta as well as accumulation of senescence-promoting camalexin and phytohormone jasmonic acid after dark treatment. m6A levels increased during DILS and destabilized senescence-related transcripts thereby preventing premature aging. Due to inefficient decay, senescence-related transcripts like ORESARA1 (ORE1), SENESCENCE-ASSOCIATED GENE 21 (SAG21), NAC-like, activated by AP3/PI (NAP), and NONYELLOWING 1 (NYE1) over-accumulated in mta thereby causing accelerated senescence during DILS. Overall, our data propose that m6A modification is involved in regulating the biological response to senescence in plants, providing targets for engineering stress tolerance of crops.

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.

Pathogen-induced m6A dynamics affect plant immunity.

Posttranscriptional regulation of mRNA mediated by methylation at the N6 position of adenine (N6-methyladenosine [m6A]) has profound effects on transcriptome regulation in plants. Focused studies across eukaryotes offer glimpses into the processes governed by m6A throughout developmental and disease states. However, we lack an understanding of the dynamics and the regulatory potential of m6A during biotic stress in plants. Here, we provide a comprehensive look into the effects of m6A on both the short-term and long-term responses to pathogen signaling in Arabidopsis (Arabidopsis thaliana). We demonstrate that m6A-deficient plants are more resistant to bacterial and fungal pathogen infections and have altered immune responses. Furthermore, m6A deposition is specifically coordinated on transcripts involved in defense and immunity prior to and proceeding the pathogen signal flagellin. Consequently, the dynamic modulation of m6A on specific stress-responsive transcripts is correlated with changes in abundance and cleavage of these transcripts. Overall, we show that the m6A methylome is regulated prior to and during simulated and active pathogen stress and functions in the coordination and balancing of normal growth and pathogen responses.

Microbe-induced drought tolerance by ABA-mediated root architecture and epigenetic reprogramming.

The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis strain SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic ABA-dependent manner. Application of SA190 priming on crops is demonstrated for alfalfa, showing enhanced performance under drought conditions. In summary, a single beneficial root bacterial strain can help plants to resist drought conditions.

Dynamic changes of the Prf/Pto tomato resistance complex following effector recognition.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.

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.

Development, validation, and application of an HPLC-MS/MS method for quantification of oxidized fatty acids in plants.

Oxylipins constitute a huge class of compounds produced by oxidation of long-chain unsaturated fatty acids either chemically (by radicals such as reactive oxygen species, ROS) or enzymatically (by lipoxygenases, LOX; cyclooxygenases, COX; or cytochrome P450 pathways). This process generates fatty acids peroxides, which can then be further modified in a broad range to epoxy, hydroxy, keto, ether fatty acids, and also hydrolyzed to generate small aldehydes and alcohols. In general, oxylipins are present in almost all living organisms and have a wide range of signaling, metabolic, physiological, and ecological roles depending on the particular organism and on their structure. In plants, oxylipins have been extensively studied over the past 35 years. However, these studies have focused mainly on the jasmonates and so-called green leaves volatiles. The function of early LOX products (like keto and hydroxy fatty acids) is yet not well understood in plants, where they are mainly analyzed by indirect methods or by GC-MS what requires a laborious sample preparation. Here, we developed and validated a straightforward, precise, accurate, and sensitive method for quantifying oxylipins in plant tissues using HPLC-MS/MS, with a one-step extraction procedure using low amount of plant tissues. We successfully applied this method to quantify the oxylipins in different plant species and Arabidopsis thaliana plants treated with various biotic and abiotic stress conditions.

Root endophyte induced plant thermotolerance by constitutive chromatin modification at heat stress memory gene loci.

Global warming has become a critical challenge to food security, causing severe yield losses of major crops worldwide. Conventional and transgenic breeding strategies to enhance plant thermotolerance are laborious and expensive. Therefore, the use of beneficial microbes could be an alternative approach. Here, we report that the root endophyte Enterobacter sp. SA187 induces thermotolerance in wheat in the laboratory as well as in open-field agriculture. To unravel the molecular mechanisms, we used Arabidopsis thaliana as model plant. SA187 reprogramed the Arabidopsis transcriptome via HSFA2-dependent enhancement of H3K4me3 levels at heat stress memory gene loci. Unlike thermopriming, SA187-induced thermotolerance is mediated by ethylene signaling via the transcription factor EIN3. In contrast to the transient chromatin modification by thermopriming, SA187 induces constitutive H3K4me3 modification of heat stress memory genes, generating robust thermotolerance in plants. Importantly, microbial community composition of wheat plants in open-field agriculture is not influenced by SA187, indicating that beneficial microbes can be a powerful tool to enhance thermotolerance of crops in a sustainable manner.

Arabidopsis WRKY50 and TGA Transcription Factors Synergistically Activate Expression of PR1.

Arabidopsis PR1 is a salicylic acid (SA) inducible marker gene for systemic acquired resistance (SAR). However, the regulation of PR1 in plants is poorly understood. In this study, we showed that AtWRKY50 transcription factor binds to two promoter elements of PR1 via its DNA binding domain. Interestingly, the DNA-binding sites for AtWRKY50 deviate significantly from the consensus WRKY binding W-box. The binding sites are located in close proximity to the binding sites for TGA transcription factors. Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1 and tga256 mutant plants indicated that AtWRKY50 alone was able to induce expression of a PR1::ß-glucuronidase (GUS) reporter gene, independent of TGAs or NPR1. However, co-expression of TGA2 or TGA5 with AtWRKY50 synergistically enhanced expression to high levels. Yeast-2-hybrid assays and bimolecular fluorescence complementation (BiFC) experiments revealed that AtWRKY50 could interact with TGA2 and TGA5. Using electrophoretic mobility shift assays (EMSA) it was established that AtWRKY50 and TGA2 or TGA5 simultaneously bind to the PR1 promoter. Taken together, these results support a role of AtWRKY50 in SA-induced expression of PR1. Highlights: AtWRKY50 specifically binds to LS10 region of PR1 promoter and interacts with TGAs to synergistically activate PR1 expression.

Possible role of plant MAP kinases in the biogenesis and transcription regulation of rice microRNA pathway factors.

Signalling pathways play vital roles as determinants of almost all the molecular processes inside a eukaryotic cell. They are more often considered to be the link between extracellular and intracellular environmental cues. Gene silencing pathways have emerged to be involved in regulation of stress responses and developmental processes. However, very little is known about the crosstalk between signalling and silencing pathways and their influence on each other. The present work describes the effects of general protein kinase inhibitors and specific mitogen activated protein kinase (MAPK) pathway inhibitors on the components of microRNA pathway in rice. The kinase inhibitors significantly reduced the activities of miRNA biogenesis complex and changed the transcript expression of miRNA pathway factors. This study suggests a possible regulation of microRNA machinery by plant kinases and MAP kinases in particular.

The Proteasome Acts as a Hub for Plant Immunity and Is Targeted by Pseudomonas Type III Effectors.

Recent evidence suggests that the ubiquitin-proteasome system is involved in several aspects of plant immunity and that a range of plant pathogens subvert the ubiquitin-proteasome system to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis (Arabidopsis thaliana). Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv tomato DC3000 (Pst) and Pseudomonas syringae pv maculicola ES4326. Both proteasome subunits are required for pathogen-associated molecular pattern-triggered immunity responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR In addition, we show that Pst inhibits proteasome activity in a type III secretion-dependent manner. A screen for type III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1, and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome, leading to its inhibition. Thus, the proteasome is an essential component of pathogen-associated molecular pattern-triggered immunity and SAR, which is targeted by multiple bacterial effectors.

Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense.

MicroRNA biogenesis factor DRB1 is a phosphorylation target of mitogen activated protein kinase MPK3 in both rice and Arabidopsis.

MicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein-protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant.

Regulation of MAP kinase signaling cascade by microRNAs in Oryza sativa.

Mitogen activated protein kinase (MAPK) pathway is one of the most conserved signaling cascade in plants regulating a plethora of cellular processes including normal growth and development, abiotic and biotic stress responses. The perception of external cues triggers the phosphorylation of three tier MAPKKK-MAPKK-MAPK cascade which finally modifies a downstream substrate thereby regulating the cellular processes. Whereas, the transcription regulation by MAPKs, mediated through their substrates is well studied in plants, the transcription and post-transcriptional regulation of the MAPK genes are poorly understood. Previous studies from the animals systems suggested the miRNAs regulate the post-transcriptional regulation of MAPK transcripts. Here we attempt to unravel the post-transcriptional regulation of MAPKs by miRNAs in model crop plant Oryza sativa. Using in silico tools, we predict the miRNAs for 98 out of 99 MAPK transcripts. The predicted miRNAs were validated for the biological relevance of their function. The inverse correlation between relative transcript levels between the MAPKs and their predicted miRNAs validated the in silico prediction. Taken together, this report demonstrates the significance of miRNAs in regulation of the MAPK pathway in plants with a new direction to study the plant signaling molecules.

Agroinfiltration by cytokinin-producing Agrobacterium sp. strain GV3101 primes defense responses in Nicotiana tabacum.

Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies.

Interaction between two rice mitogen activated protein kinases and its possible role in plant defense.

BACKGROUND: The canonical mitogen activated protein kinase (MAPK) signaling pathway plays a vital role in carrying out the normal growth and development of the plant. The pathway, connecting the upstreams signal with the downstream target is considered to be linear, mostly starting with a MAPKKK and ending in a MAPK. RESULTS: Here we report a novel interaction between two rice MAPKs, OsMPK20-4 and OsMPK3 suggesting the complex nature of the pathway rather than a linear one at individual steps. The interaction between OsMPK20-4 and OsMPK3 found by yeast two-hybrid analysis was confirmed in planta by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. The interaction is specific and is phosphorylation independent. The results suggest a role of the interaction between OsMPK20-4 and OsMPK3 in basic plant defense. CONCLUSIONS: The current novel work showing the physical interaction between two plant MAPKs, OsMPK20-4 and OsMPK3 is the diversion from the dogma of a typical MAPK cascade thereby opening a new dimension to the MAPK signal transduction.

CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids.

BACKGROUND: Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress. RESULTS: CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA) activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs. CONCLUSION: Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs.