Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Mitogen-activated protein kinase signaling in plant-interacting fungi: distinct messages from conserved messengers

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved proteins that function as key signal transduction components in fungi, plants, and mammals. During interaction between phytopathogenic fungi and plants, fungal MAPKs help to promote mechanical and/or enzymatic penetration of host tissues, while plant MAPKs are required for activation of plant immunity. However, new insights suggest that MAPK cascades in both organisms do not operate independently but that they mutually contribute to a highly interconnected molecular dialogue between the plant and the fungus. As a result, some pathogenesis-related processes controlled by fungal MAPKs lead to the activation of plant signaling, including the recruitment of plant MAPK cascades. Conversely, plant MAPKs promote defense mechanisms that threaten the survival of fungal cells, leading to a stress response mediated in part by fungal MAPK cascades. In this review, we make use of the genomic data available following completion of whole-genome sequencing projects to analyze the structure of MAPK protein families in 24 fungal taxa, including both plant pathogens and mycorrhizal symbionts. Based on conserved patterns of sequence diversification, we also propose the adoption of a unified fungal MAPK nomenclature derived from that established for the model species Saccharomyces cerevisiae. Finally, we summarize current knowledge of the functions of MAPK cascades in phytopathogenic fungi and highlight the central role played by MAPK signaling during the molecular dialogue between plants and invading fungal pathogens.     

Modeling of the MAPK machinery activation in response to various abiotic and biotic stresses in plants by a system biology approach

Mitogen-Activated Protein Kinases (MAPKs) cascade plays an important role in regulating plant growth and development, generating cellular responses to the extracellular stimuli. MAPKs cascade mainly consist of three sub-families i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK), several cascades of which are activated by various abiotic and biotic stresses. In this work we have modeled the holistic molecular mechanisms essential to MAPKs activation in response to several abiotic and biotic stresses through a system biology approach and performed its simulation studies. As extent of abiotic and biotic stresses goes on increasing, the process of cell division, cell growth and cell differentiation slow down in time dependent manner. The models developed depict the combinatorial and multicomponent signaling triggered in response to several abiotic and biotic factors. These models can be used to predict behavior of cells in event of various stresses depending on their time and exposure through activation of complex signaling cascades.     

Soybean homologs of MPK4 negatively regulate defense responses and positively regulate growth and development

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). However, the importance of MAPK signaling pathways in the disease resistance of crops is still largely uninvestigated. To better understand the role of MAPK signaling pathways in disease resistance in soybean (Glycine max), 13, nine, and 10 genes encoding distinct MAPKs, MAPKKs, and MAPKKKs, respectively, were silenced using virus-induced gene silencing mediated by Bean pod mottle virus. Among the plants silenced for various MAPKs, MAPKKs, and MAPKKKs, those in which GmMAPK4 homologs (GmMPK4s) were silenced displayed strong phenotypes including stunted stature and spontaneous cell death on the leaves and stems, the characteristic hallmarks of activated defense responses. Microarray analysis showed that genes involved in defense responses, such as those in salicylic acid (SA) signaling pathways, were significantly up-regulated in GmMPK4-silenced plants, whereas genes involved in growth and development, such as those in auxin signaling pathways and in cell cycle and proliferation, were significantly down-regulated. As expected, SA and hydrogen peroxide accumulation was significantly increased in GmMPK4-silenced plants. Accordingly, GmMPK4-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants. Using bimolecular fluorescence complementation analysis and in vitro kinase assays, we determined that GmMKK1 and GmMKK2 might function upstream of GmMPK4. Taken together, our results indicate that GmMPK4s negatively regulate SA accumulation and defense response but positively regulate plant growth and development, and their functions are conserved across plant species.     

An Extracellular Subtilase Switch for Immune Priming in Arabidopsis

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.     

Identification of MAPKs and their possible MAPK kinase activators involved in the Pto-mediated defense response of tomato

The Pto kinase mediates resistance to bacterial speck disease in tomato by activating host defenses upon recognition of Pseudomonas syringae pv. tomato strains expressing the AvrPto or AvrPtoB proteins. Previous gene-silencing experiments have indicated that mitogen activated protein kinase (MAPK) cascades play a key role downstream of the Pto kinase to activate host defense responses. Here we use biochemical methods to demonstrate that two tomato MAPKs, LeMPK2 and LeMPK3, are activated in leaves in a Pto-specific manner upon expression of AvrPto and AvrPtoB. We show that these same MAPKs are activated upon overexpression of LeMAPKKKalpha, a protein previously demonstrated to be involved in Pto-mediated immunity. We identified two phylogenetically unrelated MAPK kinases (LeMKK2 and LeMKK4) that when overexpressed in leaves elicit cell death and activate LeMPK2 and LeMPK3. In vitro analysis demonstrated that LeMKK2 and LeMKK4 each phosphorylate the same subset of three MAPKs. Together these data provide biochemical evidence for the involvement of MAPK cascades in Pto-mediated resistance.     

Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in

ABSTRACT: BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKKalpha of Nicotiana benthamiana (NbMAPKKKalpha), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKepsilon2. Transient overexpression of full-length NbMAPKKKbeta or NbMAPKKKgamma or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKKbeta or NbMAPKKKgamma expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKalpha (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKalpha suppressed cell death induced by the overexpression of the NbMAPKKKbeta kinase domain or of NbMAPKKKgamma, but silencing of NbMAPKKKbeta failed to suppress cell death induced by the overexpression of NbMAPKKKalpha or NbMAPKKKgamma. Silencing of NbMAPKKKgamma suppressed cell death induced by the NbMAPKKKbeta kinase domain but not that induced by NbMAPKKKalpha. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKKalpha, NbMAPKKKbeta and NbMAPKKKgamma also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKbeta to NbMAPKKKgamma to NbMAPKKKalpha.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.     

Mitogen-activated protein kinases: A new therapeutic target in cardiac pathology

Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene targeting or expression of constitutively active and dominant-negative forms of enzymes (adenovirus-mediated gene transfer).This review is focused on the regulatory role of MAPKs in the myocardium, with particular regard to their involvement in pathophysiological processes, such as myocardial hypertrophy and heart failure, ischemia/reperfusion injury, as well as in the mechanisms of cardioprotection. In addition, it summarizes current information on pharmacological modulations of MAPKs activity and their impact on the cardiac response to pathophysiological processes.     

A MAPK pathway mediates ethylene signaling in plants

Ethylene signal transduction involves ETR1, a two-component histidine protein kinase receptor. ETR1 functions upstream of the negative regulator CTR1. The similarity of CTR1 to members of the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs) suggested that ethylene signaling in plants involves a MAPK pathway, but no direct evidence for this has been provided. Here we show that distinct MAPKs are activated by the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) in Medicago and ARABIDOPSIS: In Medicago, the ACC-activated MAPKs were SIMK and MMK3, while in Arabidopsis MPK6 and another MAPK were identified. Medicago SIMKK specifically mediated ACC-induced activation of SIMK and MMK3. Transgenic Arabidopsis plants overexpressing SIMKK have constitutive MPK6 activation and ethylene-induced target gene expression. SIMKK overexpressor lines resemble ctr1 mutants in showing a triple response phenotype in the absence of ACC. Whereas MPK6 was not activated by ACC in etr1 mutants, ein2 and ein3 mutants showed normal activation profiles. In contrast, ctr1 mutants showed constitutive activation of MPK6. These data indicate that a MAPK cascade is part of the ethylene signal transduction pathway in plants.     

Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.