MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.     

The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.     

Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.     

The MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates immunity mediated by a mitogen-activated protein kinase kinase kinase in Arabidopsis

In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding-leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses.     

Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22

Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK.     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1's full kinase activity.     

EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling

Mitogen‐activated protein kinase (MAPK) signaling plays important roles in diverse biological processes. In Arabidopsis, MPK3/MPK6, MKK4/MKK5, and the MAPKKK YODA (YDA) form a MAPK pathway that negatively regulates stomatal development. Brassinosteroid (BR) stimulates this pathway to inhibit stomata production. In addition, MPK3/MPK6 and MKK4/MKK5 also serve as critical signaling components in plant immunity. Here, we report that MAPKKK3/MAPKKK5 form a kinase cascade with MKK4/MKK5 and MPK3/MPK6 to transduce defense signals downstream of multiple plant receptor kinases. Loss of MAPKKK3/MAPKKK5 leads to reduced activation of MPK3/MPK6 in response to different pathogen‐associated molecular patterns (PAMPs) and increased susceptibility to pathogens. Surprisingly, developmental defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. On the other hand, loss of YDA or blocking BR signaling leads to increased PAMP‐induced activation of MPK3/MPK6. These results reveal antagonistic interactions between a developmental MAPK pathway and an immune signaling MAPK pathway.     

Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) serves as a primary plant defense response against microbial pathogens, with MEKK1, MKK1/MKK2, and MPK4 functioning as a MAP kinase cascade downstream of PAMP receptors. Plant Resistance (R) proteins sense specific pathogen effectors to initiate a second defense mechanism, termed effector-triggered immunity (ETI). In a screen for suppressors of the mkk1 mkk2 autoimmune phenotype, we identify the nucleotide-binding leucine-rich repeat (NB-LRR) protein SUMM2 and find that the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulates SUMM2-mediated immunity. Further, the MEKK1-MKK1/MKK2-MPK4 cascade positively regulates basal defense targeted by the Pseudomonas syringae pathogenic effector HopAI1, which inhibits MPK4 kinase activity. Inactivation of MPK4 by HopAI1 results in activation of SUMM2-mediated defense responses. Our data suggest that SUMM2 is an R protein that becomes active when the MEKK1-MKK1/MKK2-MPK4 cascade is disrupted by pathogens, supporting the hypothesis that R proteins evolved to protect plants when microbial effectors suppress basal resistance.     

MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. An Arabidopsis MAPK cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) has been proposed to function downstream of the flagellin receptor FLS2 based on biochemical assays using transient overexpression of candidate components. To genetically test this model, we characterized two mekk1 mutants. We show here that MEKK1 is not required for flagellin-triggered activation of MPK3 and MPK6. Instead, MEKK1 is essential for activation of MPK4, a MAPK that negatively regulates systemic acquired resistance. We also showed that MEKK1 negatively regulates temperature-sensitive and tissue-specific cell death and H(2)O(2) accumulation that are partly dependent on both RAR1, a key component in resistance protein function, and SID2, an isochorismate synthase required for salicylic acid production upon pathogen infection.     

The NLR protein SUMM2 senses the disruption of an immune signaling MAP kinase cascade via CRCK3

MAP kinase signaling is an integral part of plant immunity. Disruption of the MEKK1‐MKK1/2‐MPK4 kinase cascade results in constitutive immune responses mediated by the NLR protein SUMM2, but the molecular mechanism is so far poorly characterized. Here, we report that SUMM2 monitors a substrate protein of MPK4, CALMODULIN‐BINDING RECEPTOR‐LIKE CYTOPLASMIC KINASE 3 (CRCK3). Similar to SUMM2, CRCK3 was isolated from a suppressor screen of mkk1 mkk2 and is required for the autoimmunity phenotypes in mekk1, mkk1 mkk2, and mpk4 mutants. In wild‐type plants, CRCK3 is mostly phosphorylated. MPK4 interacts with CRCK3 and can phosphorylate CRCK3 in vitro. In mpk4 mutant plants, phosphorylation of CRCK3 is substantially reduced, suggesting that MPK4 phosphorylates CRCK3 in vivo. Further, CRCK3 associates with SUMM2 in planta, suggesting SUMM2 senses the disruption of the MEKK1‐MKK1/2‐MPK4 kinase cascade through CRCK3. Our study suggests that a MAP kinase substrate is used as a guardee or decoy for monitoring the integrity of MAP kinase signaling.     

Arabidopsis MPK3 and MPK6 play different roles in basal and oligogalacturonide- or flagellin-induced resistance against Botrytis cinerea

Mitogen-activated protein kinases (MAPKs) are fundamental components of the plant innate immune system. MPK3 and MPK6 are Arabidopsis (Arabidopsis thaliana) MAPKs activated by pathogens and elicitors such as oligogalacturonides (OGs), which function as damage-associated molecular patterns, and flg22, a well-known microbe-associated molecular pattern. However, the specific contribution of MPK3 and MPK6 to the regulation of elicitor-induced defense responses is not completely defined. In this work we have investigated the roles played by these MAPKs in elicitor-induced resistance against the fungal pathogen Botrytis cinerea. Analysis of single mapk mutants revealed that lack of MPK3 increases basal susceptibility to the fungus, as previously reported, but does not significantly affect elicitor-induced resistance. Instead, lack of MPK6 has no effect on basal resistance but suppresses OG- and flg22-induced resistance to B. cinerea. Overexpression of the AP2C1 phosphatase leads to impaired OG- and flg22-induced phosphorylation of both MPK3 and MPK6, and to phenotypes that recapitulate those of the single mapk mutants. These data indicate that OG- and flg22-induced defense responses effective against B. cinerea are mainly dependent on MAPKs, with a greater contribution of MPK6.