Tobacco Mitogen-activated Protein Kinase Research Articles

Mitogen-Activated Protein Kinases Associated Sites of Tobacco Repression of Shoot Growth Regulates Its Localization in Plant Cells.

Plant defense and growth rely on multiple transcriptional factors (TFs). Repression of shoot growth (RSG) is a TF belonging to a bZIP family in tobacco, known to be involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG previously. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as an RSG-interacting protein kinase. Moreover, the mutation of the predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74, and Thr-135) significantly altered the intracellular localization of the NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acid mutants (T30A and S74A) were observed after being treated with plant defense elicitor peptide flg22 within 5 min, and the two mutated RSG swiftly re-localized in tobacco cytoplasm within 30 min. In addition, triple-point mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and is predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no re-localization effect under the treatments of flg22, B. cereus AR156, or GA3, and over-expression of this mutant in tobacco resulted in lower expression levels of downstream gene GA20ox1. Our results suggest that MAPK-associated phosphorylation sites of RSG regulate its localization in tobacco, and that constant unphosphorylation of RSG in Thr-30, Ser-74, and Thr-135 keeps RSG predominantly localized in cytoplasm.

Protein phosphatase NtPP2C2b and MAP kinase NtMPK4 act in concert to modulate nicotine biosynthesis.

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.

Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae.

The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed.

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.

Tobacco MAP Kinase Phosphatase (NtMKP1) Negatively Regulates Wound Response and Induced Resistance Against Necrotrophic Pathogens and Lepidopteran Herbivores

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction pathways in eukaryotic cells. In tobacco, two MAPK, wound-induced protein kinase (WIPK) and salicylic acid (SA)-induced protein kinase (SIPK), are activated by biotic and abiotic stresses. Both WIPK and SIPK positively regulate the biosynthesis of jasmonic acid (JA) or ethylene (ET) while negatively regulating SA accumulation. We showed previously that recombinant tobacco MAPK phosphatase (NtMKP1) protein dephosphorylates and inactivates SIPK in vitro, and overexpression of NtMKP1 repressed wound-induced activation of both SIPK and WIPK. To elucidate the role of NtMKP1 in response to biotic and abiotic stresses, we generated transgenic tobacco plants in which NtMKP1 expression was suppressed. Suppression of NtMKP1 expression resulted in enhanced activation of WIPK and SIPK and production of both JA and ET upon wounding. Wound-induced expression of JA- or ET-inducible genes, basic PR-1 and PI-II, was also significantly enhanced in these plants. Furthermore, NtMKP1-suppressed plants exhibited enhanced resistance against a necrotrophic pathogen, Botrytis cinerea, and lepidopteran herbivores, Mamestra brassicae and Spodoptera litura. These results suggest that NtMKP1 negatively regulates wound response and resistance against both necrotrophic pathogens and herbivorous insects through suppression of JA or ET pathways via inactivation of MAPK.

Rice MAPK phosphatase IBR5 negatively regulates drought stress tolerance in transgenic Nicotiana tabacum

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, regulation of development and response to environment stresses. Several MAPKs have been reported to be involved in the drought stress response, however, there is no evidence for the specific function of MKPs in drought stress. Here, a putative MKP in rice (Oryza sativa), OsIBR5, was characterized. Expression of OsIBR5 was induced by PEG6000, abscisic acid (ABA) and hydrogen peroxide (H2O2). Overexpression of OsIBR5 in tobacco plants resulted in hypersensitivity to drought and H2O2 treatments. Drought and ABA-induced stomatal closure was significantly reduced in OsIBR5-overexpressing tobacco plants compared with controls. Moreover, OsIBR5 was found to interact with tobacco MAPKs SIPK and WIPK, and drought-induced WIPK activity was impaired in OsIBR5-overexpressing tobacco plants. These results indicated that OsIBR5 is a MKP which was induced by abiotic stresses and decreased tolerance to drought stress in transgenic tobacco plants.

Phytophthora sojae effector Avh331 suppresses the plant defence response by disturbing the MAPK signalling pathway

Oomycete plant pathogens translocate hundreds of RXLR effector proteins into host cells to establish an infection; however, the mechanism by which the effector manipulates host defence and promotes pathogenicity remains largely unknown. Here, we generated two independent transgenic Arabidopsis lines of Avh331, an RXLR effector of Phytophthora sojae, and showed that Avh331 suppresses the Arabidopsis mitogen-activated protein kinase (MAPK)-based plant defence activated by the Phytophthora elicitor to promote colonisation of the Phytophthora pathogen. Furthermore, Avh331 suppressed transcriptional activation of resistance marker genes downstream of the MAPK signalling pathway. Similar to the phenotype in Arabidopsis, Avh331 suppressed the MAPK signalling pathway to inhibit the basal defence induced by Phytophthora elicitor INF1 or tobacco MAPK proteins in Nicotiana benthamiana. Taken together, these results indicate that the RXLR effector Avh331 promoted the colonisation of plant pathogens by manipulating the MAPK signalling pathway in plant cells.

Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode

The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca(2+)-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca(2+)-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel "double anchor" motif. Moreover, the orientation of the helical peptide on the surface of Ca(2+)-SCaM4 is distinct from other known complexes. The N-lobe of Ca(2+)-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca(2+) are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.

Transgenic Rice Plants Expressing an Active Tobacco Mitogen-activated Protein Kinase Kinase Induce Multiple Defense Responses

It is well known that NtMEK2, a tobacco MAPK kinase, is the upstream kinase of both salicylic acid-induced protein kinase and wound-induced protein kinase. In addition, expression of <TEX>$NtMEK2^{DD}$</TEX>, a constitutively active mutant of NtMEK2, is known to induce multiple defense responses in tobacco. In this study, transgenic rice plants that contained an active or inactive mutant of NtMEK2 under the control of a steroid inducible promoter were generated and used to determine if a similar MAPK cascade is involved in disease resistance in rice. The expression of <TEX>$NtMEK2^{DD}$</TEX> in transgenic rice plants resulted in HR-like cell death. The observed cell death was preceded by the activation of endogenous rice 48-kDa MBP kinase, which is also activated by Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice. In addition, prolonged activation of the MAPK induced the generation of hydrogen peroxide and up-regulated the expression of defense-related genes including the pathogenesis-related genes, peroxidases and glutathione S-transferases. These results demonstrate that NtMEK2 is functionally replaceable with rice MAPK kinase in inducing the activation of the downstream MAPK, which in turn induces multiple defense responses in rice.

Chloroplast‐generated reactive oxygen species are involved in hypersensitive response‐like cell death mediated by a mitogen‐activated protein kinase cascade

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.

Calcium-dependent and -independent Binding of Soybean Calmodulin Isoforms to the Calmodulin Binding Domain of Tobacco MAPK Phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.

The mitogen‐activated protein kinases WIPK and SIPK regulate the levels of jasmonic and salicylic acids in wounded tobacco plants

In tobacco (Nicotiana tabacum), wounding causes rapid activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid (SA)-induced protein kinase (SIPK), and the subsequent accumulation of jasmonic acid (JA). Our previous studies suggested that activation of WIPK is required for the production of wound-induced JA. However, the exact role of WIPK remains unresolved. We generated transgenic tobacco plants in which either WIPK or SIPK were silenced using RNA interference to define the roles of WIPK and SIPK in the wound response. In addition, transgenic tobacco plants were generated in which both WIPK and SIPK were silenced to examine the possibility that they have redundant roles. Wound-induced JA production was reduced compared with non-silenced plants in all of the WIPK-, SIPK- and WIPK/SIPK-silenced plants. Transgenic plants over-expressing NtMKP1, a gene encoding tobacco MAPK phosphatase, which inactivates WIPK and SIPK, also exhibited reduced JA production in response to wounding. In both WIPK/SIPK-silenced and NtMKP1-over-expressing plants, wounding resulted in an abnormal accumulation of both SA and transcripts for SA-responsive genes. These results suggest that WIPK and SIPK play an important role in JA production in response to wounding, and that they function cooperatively to control SA biosynthesis.

Activation of Ntf4, a Tobacco Mitogen-Activated Protein Kinase, during Plant Defense Response and Its Involvement in Hypersensitive Response-Like Cell Death

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells. They function downstream of sensors/receptors and regulate cellular responses to external and endogenous stimuli. Recent studies demonstrated that SIPK and WIPK, two tobacco (Nicotiana spp.) MAPKs, are involved in signaling plant defense responses to various pathogens. Ntf4, another tobacco MAPK that shares 93.6% and 72.3% identity with SIPK and WIPK, respectively, was reported to be developmentally regulated and function in pollen germination. We found that Ntf4 is also expressed in leaves and suspension-cultured cells. Genomic analysis excluded the possibility that Ntf4 and SIPK are orthologs from the two parental lines of the amphidiploid common tobacco. In vitro and in vivo phosphorylation and activation assays revealed that Ntf4 shares the same upstream MAPK kinase, NtMEK2, with SIPK and WIPK. Similar to SIPK and WIPK, Ntf4 is also stress responsive and can be activated by cryptogein, a proteinaceous elicitin from oomycetic pathogen Phytophthora cryptogea. Tobacco recognition of cryptogein induces rapid hypersensitive response (HR) cell death in tobacco. Transgenic Ntf4 plants with elevated levels of Ntf4 protein showed accelerated HR cell death when treated with cryptogein. In addition, conditional overexpression of Ntf4, which results in high cellular Ntf4 activity, is sufficient to induce HR-like cell death. Based on these results, we concluded that Ntf4 is multifunctional. In addition to its role in pollen germination, Ntf4 is also a component downstream of NtMEK2 in the MAPK cascade that regulates pathogen-induced HR cell death in tobacco.

Phosphorylation of NtMAP65-1 by a MAP kinase down-regulates its activity of microtubule bundling and stimulates progression of cytokinesis of tobacco cells

The tobacco mitogen-activated protein kinase (MAPK) cascade, which includes MAPK NRK1/NTF6, positively regulates expansion of the cytokinetic machinery known as the phragmoplast, which is followed by the synthesis of cell plates for completion of cell division. However, molecular events lying between the MAPK and phragmoplast expansion were not known. Here, we show that NRK1/NTF6 phosphorylates the threonine residue at position 579 in NtMAP65-1a, a microtubule-associated (MT-associated) protein. Levels of phosphorylated NtMAP65-1 increase during late M phase of the cell cycle, when NRK1/NTF6 is activated. Phosphorylated NtMAP65-1 is concentrated at the equator of phragmoplast, as is NRK1/NTF6. Overexpression of mutant forms of NtMAP65-1a that cannot be phosphorylated by NRK1 delays progression of the M phase and phragmoplast expansion, also rendering phragmoplast structures resistant to an MT-depolymerizing drug. Phosphorylation of NtMAP65-1 by NRK1/NTF6 down-regulates its MT-bundling activity in vitro. These results suggest that phosphorylation of NtMAP65-1 by NRK1/NTF6 also reduces its MT-bundling activity in vivo, which enhances destabilization and turnover of MTs at the phragmoplast equator, perhaps facilitating phragmoplast expansion.

A Mitogen-activated Protein Kinase NtMPK4 Activated by SIPKK is Required for Jasmonic Acid Signaling and Involved in Ozone Tolerance via Stomatal Movement in Tobacco

The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.

Activation of a mitogen-activated protein kinase cascade induces WRKY family of transcription factors and defense genes in tobacco.

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells that convert signals generated from the receptors/sensors to cellular responses. Upon activation, MAPKs can be translocated into nuclei where they phosphorylate transcription factors, which in turn activate gene expression. We recently identified NtMEK2, a tobacco MAPK kinase, as the upstream kinase of SIPK and WIPK, two well-characterized tobacco stress-responsive MAPKs. In the conditional gain-of-function NtMEK2DD transgenic tobacco plants, the activation of endogenous SIPK and WIPK by NtMEK2DD induces several groups of defense genes, including 3-hydroxy-3-methlyglutaryl CoA reductase (HMGR), basic pathogenesis related (PR) genes, systemic acquired resistance gene 8.2 (Sar 8.2), and harpin-induced gene1 (Hin1). To identify the transcription factor(s) involved in the activation of these defense genes, we performed gel-mobility shift assays using nuclear extracts from NtMEK2DD plants. Among the common cis-acting elements present in the promoters of defense-related genes, we observed a strong increase in the binding activity to the W box in nuclear extracts from the NtMEK2DD plants but not the control NtMEK2KR plants. The elevated W-box-binding activity in the nuclear extracts cannot be reversed by phosphatase treatment, excluding the possibility of a direct phosphorylation regulation of WRKY transcription factors by SIPK/WIPK. Instead, we observed a rapid increase in the expression of several WRKY genes in the NtMEK2DD plants. These results suggest that the increase in W-box-binding activity after SIPK/WIPK activation is a result of WRKY gene activation, and the NtMEK2-SIPK/WIPK cascade is involved in regulating the expression of genes ranging from transcription factors to defense genes further downstream during plant defense responses.

Expression of an active tobacco mitogen-activated protein kinase kinase kinase enhances freezing tolerance in transgenic maize.

Cold acclimation is the major process that prepares plants for freezing tolerance. In addition to extensive transcription regulation by cold-inducible master transcription factors, oxidative stress signaling has been postulated to play a role in freezing tolerance. Activation of oxidative signaling through the expression of an active mitogen-activated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing temperature bypassing cold acclimation. Because involvement of the mitogen-activated protein kinase cascade in oxidative stress signaling is evolutionarily conserved in eukaryotes from yeast to mammals, we tested the effect of expressing a heterologous tobacco mitogen-activated protein kinase kinase kinase (Nicotiana PK1), which can mimic H(2)O(2) signaling, in a major cereal crop. We demonstrate that low-level but constitutive expression of the Nicotiana PK1 gene enhances freezing tolerance in transgenic maize plants that are normally frost sensitive. Our results suggest that a new molecular approach can be designed to genetically enhance freezing tolerance in important crops.

Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants.

Plants under stress from both biotic and abiotic sources produce increased levels of ethylene, which is perceived by ethylene receptors and triggers cellular responses further downstream. Protein phosphorylation and dephosphorylation were implicated in the regulation of ethylene induction by stresses based on studies using protein kinase and phosphatase inhibitors. However, the kinase(s) involved remains to be determined. Using a conditional gain-of-function transgenic system, we demonstrate that the activation of SIPK, a tobacco mitogen-activated protein kinase (MAPK), by NtMEK2DD, an active mutant of the upstream kinase of SIPK, resulted in a dramatic increase in ethylene production. The increase in ethylene after the activation of SIPK coincided with a dramatic increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, which was followed by the activation of a subgroup of ACS and ACC oxidase (ACO) genes, suggesting that either the activation of unidentified ACS(s) or post-transcriptional regulation is involved. Infection with Tobacco mosaic virus (TMV), which is known to activate the SIPK cascade and induce ethylene biosynthesis, also induced the same ACSs and ACOs. After ethylene production in NtMEK2DD plants, strong activation of ETHYLENE-RESPONSE FACTOR (ERF) genes was observed, similar to the effect in NN tobacco plants infected with TMV. In contrast to previous reports, no major increase in jasmonic acid (JA) and methyl jasmonate (MJ) was detected after the activation of SIPK/WIPK in NtMEK2DD transgenic plants. These results suggest that the induction of ethylene but not JA/MJ is involved in plant defense responses mediated by the NtMEK2-SIPK/WIPK pathway.

Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco.

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein.

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.