Activation of Salicylic Acid–Induced Protein Kinase, a Mitogen-Activated Protein Kinase, Induces Multiple Defense Responses in Tobacco

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2^DD, in tobacco induces the expression of defense genes and hypersensitive response–like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid–induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response–like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.     

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.     

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.     

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen,but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs ( NbWIPK and NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen ( Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.Communicated by R. G. Herrmann     

MAP kinase cascades in elicitor signal transduction

 Protein kinases play important roles in elicitor signal transduction. In this article, I describe the current view of the role of mitogen-activated protein kinase (MAPK) cascades in elicitor signal transduction of plant cells based on our own research and recent developments in this field. In the past several years, it has become apparent that MAPK cascades play important roles in elicitor signal transduction in plants. Our early studies demonstrated the identification of p47 MAPK in tobacco as an elicitor-responsive protein kinase and possible involvement of p47 MAPK in elicitor signal transduction to induce defense responses, including defense gene expression and hypersensitive cell death. However, the molecular identity of p47 MAPK is still unclear. Recent important studies suggest that tobacco MAPK cascades that include SIPK, and/or WIPK, and NtMEK2, an upstream kinase for both SIPK and WIPK, have a crucial function in induction of defense responses and hypersensitive cell death. The orthologs of these protein kinases in Arabidopsis and alfalfa are also suggested to have similar functions. Furthermore, the identification of loss-of-function mutation in Arabidopsis reveals a negative regulatory role for putative MAPK cascades in plant defense mechanisms. Received: February 7, 2002 / Accepted: February 25, 2002     

Involvement of PPS3 phosphorylated by elicitor-responsive mitogen-activated protein kinases in the regulation of plant cell death

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant innate immunity. Overexpression of StMEK1(DD), a constitutively active MAPK kinase that activates salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), provokes hypersensitive response-like cell death in Nicotiana benthamiana. Here we purified a 51-kD MAPK, which was activated in potato (Solanum tuberosum) tubers treated with hyphal wall elicitor of a plant pathogen, and isolated the cDNA designated StMPK1. The deduced amino acid sequence of the StMPK1 showed strong similarity to stress-responsive MAPKs, such as tobacco (Nicotiana tabacum) SIPK and Arabidopsis (Arabidopsis thaliana) AtMPK6. To investigate the downstream signaling of StMPK1, we identified several proteins phosphorylated by StMPK1 (PPSs) using an in vitro expression cloning method. To dissect the biological function of PPSs in the plant defense, we employed virus-induced gene silencing (VIGS) in N. benthamiana. VIGS of NbPPS3 significantly delayed cell death induced by the transient expression of StMEK1(DD) and treatment with hyphal wall elicitor. Furthermore, the mobility shift of NbPPS3 on SDS-polyacrylamide gel was induced by transient expression of StMEK1(DD). The mobility shift of NbPPS3 induced by StMEK1(DD) was not compromised by VIGS of WIPK or SIPK alone, but drastically reduced by the silencing of both WIPK and SIPK. This work strongly supports the idea that PPS3 is a physiological substrate of StMPK1 and is involved in cell death activated by a MAPK cascade.     

A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco

A cellular signal transduction pathway induced by the polyamine, spermine (Spm), and transmitted by mitochondrial dysfunction is proposed in tobacco. In this investigation, we further resolve the pathway by identifying a subset of hypersensitive response (HR) marker genes as downstream components. In a previous report, we identified harpin-induced 1 (HIN1) and two closely related genes as responsive to Spm. Other HR marker genes, HSR203J, HMGR, HSR201, and HSR515, are also Spm-responsive. Induction of these HR marker genes, including HIN1, by Spm was suppressed by pre-treatment with antioxidants, calcium channel blockers, inhibitor of mitochondrial permeability transition pore openings, and blockers of amine oxidase/polyamine oxidase. Such quenching is also observed for Spm-induced activation of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK), and upregulation of the WIPK gene, suggesting that all these components are part of the same signaling pathway. Furthermore, gain-of-function and loss-of-function studies on MAPK cascade members reveal that the expression of Spm-induced HR marker genes varies with respect to involvement of SIPK/WIPK activation.     

The Colletotrichum orbiculare ssd1 Mutant Enhances Nicotiana benthamiana Basal Resistance by Activating a Mitogen-Activated Protein Kinase Pathway

Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance.     

Cloning and evolutionary analysis of tobacco MAPK gene family

The mitogen-activated protein (MAP) kinase cascade is an important signaling module which is involved in biotic and abiotic stress responses as well as plant growth and development. In this study, we identified 17 tobacco MAPKs including 11 novel tobacco MAPK genes that have not been identified before. Comparative analysis with MAPK gene families from other plants, such as Athaliana thaliana, rice and poplar, suggested that tobacco MAPKs (such as NtMPK1, NtMPK3 and NtMPK8) might play similar functions in response to abiotic and biotic stresses. QRT-PCR analysis revealed that a total of 14 NtMPKs were regulated by SA and/or MeJA, suggesting their potential roles involved in plant defense response. In addition, 6 NtMPKs were induced by drought treatment, implying their roles in response to drought stress. Our results indicated that most of tobacco MAPK might be involved in plant defense response, which provides the basis for further analysis on physiological functions of tobacco MAPKs.     

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.     

MAP kinases function downstream of HSP90 and upstream of mitochondria in TMV resistance gene N-mediated hypersensitive cell death

Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2(DD), a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2(DD)- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2(DD)- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.     

Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2(DD), in tobacco induces the expression of defense genes and hypersensitive response-like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response-like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.     

An Arabidopsis kinase cascade influences auxin‐responsive cell expansion

Mitogen‐activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin‐related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1‐1 as a mutant that displays hypersensitivity in auxin‐responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin‐responsive cell expansion assays, suggesting that this MPK cascade affects auxin‐influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho‐like GTPases from Plants (ROP) small GTPase family. Similar to mpk1‐1 and mkk3‐1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin‐responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin‐responsive cell expansion are mediated through phosphorylation‐dependent modulation of ROP activity. Our data suggest a MKK3 ? MPK1 ? RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion.     

Double jeopardy: both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive

In plants, the role of mitogen-activated protein kinase (MAPK) in reactive oxygen species (ROS)-based signal transduction processes is elusive. Despite the fact that ROS can induce MAPK activation, no direct genetic evidence has linked ROS-induced MAPK activation with the hypersensitive response, a form of programmed cell death. In tobacco, the major ROS-induced MAPK is salicylate-induced protein kinase (SIPK). We found through gain-of-function and loss-of-function approaches that both overexpression and RNA interference-based suppression of SIPK render the plant sensitive to ROS stress. Transgenic lines overexpressing a nonphosphorylatable version of SIPK were not ROS sensitive. Analysis of the MAPK activation profiles in ROS-stressed transgenic and wild-type plants revealed a striking interplay between SIPK and another MAPK (wound-induced protein kinase [WIPK]) in the different kinotypes. During continuous ozone exposure, abnormally prolonged activation of SIPK was seen in the SIPK-overexpression genotype, without WIPK activation, whereas strong and stable activation of WIPK was observed in the SIPK-suppressed lines. Thus, one role of activated SIPK in tobacco cells upon ROS stimulation appears to be control of the inactivation of WIPK.