The Peroxisomal ß-Oxidative Pathway and Benzyl Alcohol O-Benzoyltransferase HSR201 Cooperatively Contribute to the Biosynthesis of Salicylic Acid.

The phytohormone salicylic acid (SA) regulates plant defense responses against pathogens. Previous studies have suggested that SA is mainly produced from trans-cinnamic acid (CA) in tobacco, but the underlying mechanisms remain largely unknown. SA synthesis is activated by wounding in tobacco plants in which the expression of WIPK and SIPK, two stress-related mitogen-activated protein kinases, is suppressed. Using this phenomenon, we previously revealed that HSR201 encoding benzyl alcohol O-benzoyltransferase is required for pathogen signal-induced SA synthesis. In this study, we further analyzed the transcriptomes of wounded WIPK-/SIPK-suppressed plants and found that the expression of NtCNL, NtCHD and NtKAT1, homologous to cinnamate-coenzyme A (CoA) ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD) and 3-ketoacyl-CoA thiolase (KAT), respectively, is associated with SA biosynthesis. CNL, CHD and KAT constitute a ß-oxidative pathway in the peroxisomes and produce benzoyl-CoA, a precursor of benzenoid compounds in petunia flowers. Subcellular localization analysis showed that NtCNL, NtCHD and NtKAT1 localize in the peroxisomes. Recombinant NtCNL catalyzed the formation of CoA esters of CA, whereas recombinant NtCHD and NtKAT1 proteins converted cinnamoyl-CoA to benzoyl-CoA, a substrate of HSR201. Virus-induced gene silencing of any one of NtCNL, NtCHD and NtKAT1 homologs compromised SA accumulation induced by a pathogen-derived elicitor in Nicotiana benthamiana leaves. Transient overexpression of NtCNL in N. benthamiana leaves resulted in SA accumulation, which was enhanced by co-expression of HSR201, although overexpression of HSR201 alone did not cause SA accumulation. These results suggested that the peroxisomal ß-oxidative pathway and HSR201 cooperatively contribute to SA biosynthesis in tobacco and N. benthamiana.

Hypersensitivity-Related Genes HSR201 and HSR203J Are Regulated by Calmodulin-Binding Protein 60-Type Transcription Factors and Required for Pathogen Signal-Induced Salicylic Acid Synthesis.

Salicylic acid (SA) plays a key role in plant resistance to pathogens. In Arabidopsis, the isochorismate synthase pathway mainly contributes to pathogen-induced SA synthesis, and the expression of SA synthesis genes is activated by two calmodulin (CaM)-binding protein 60 (CBP60)-type transcription factors, CBP60g and SARD1. In tobacco, the mechanisms underlying SA synthesis remain largely unknown. SA production is induced by wounding in tobacco plants in which the expression of two stress-related mitogen-activated protein kinases is suppressed. Using this phenomenon, we identified genes whose expression is associated with SA synthesis. One of the genes, NtCBP60g, showed 23% amino acid sequence identity with CBP60g. Transient overexpression of NtCBP60g as well as NtSARD1, a tobacco homolog of SARD1, induced SA accumulation in Nicotiana benthamiana leaves. NtCBP60g and NtSARD1 bound CaM, and CaM enhanced SA accumulation induced by NtCBP60g and NtSARD1. Conversely, mutations in NtCBP60g and NtSARD1 that abolished CaM binding reduced their ability to induce SA. Expression profiling and promoter analysis identified two hypersensitivity-related genes, HSR201 and HSR203J as the targets of NtCBP60g and NtSARD1. Virus-induced gene silencing of both NtCBP60g and NtSARD1 homologs compromised SA accumulation and the expression of HSR201 and HSR203J homologs, which were induced by a pathogen-derived elicitor in N. benthamiana leaves. Moreover, elicitor-induced SA accumulation was compromised by silencing of the HSR201 homolog and the HSR203J homolog. These results suggested that HSR201 and HSR203J are regulated by NtCBP60g and NtSARD1 and are required for elicitor-induced SA synthesis.

Transcriptional induction of capsidiol synthesis genes by wounding can promote pathogen signal-induced capsidiol synthesis.

BACKGROUND: Plants are exposed to various forms of environmental stress. Penetration by pathogens is one of the most serious environmental insults. Wounding caused by tissue damage or herbivory also affects the growth and reproduction of plants. Moreover, wounding disrupts physical barriers present at the plant surface and increases the risk of pathogen invasion. Plants cope with environmental stress by inducing a variety of responses. These stress responses must be tightly controlled, because their unnecessary induction is detrimental to plant growth. In tobacco, WIPK and SIPK, two wound-responsive mitogen-activated protein kinases, have been shown to play important roles in regulating wound responses. However, their contribution to downstream wound responses such as gene expression is not well understood. RESULTS: To identify genes regulated by WIPK and SIPK, the transcriptome of wounded WIPK/SIPK-suppressed plants was analyzed. Among the genes down-regulated in WIPK/SIPK-suppressed plants, the largest group consisted of those involved in the production of antimicrobial phytoalexins. Almost all genes involved in the biosynthesis of capsidiol, a major phytoalexin in tobacco, were transcriptionally induced by wounding in WIPK/SIPK-dependent and -independent manners. 5-epi-aristolochene synthase (EAS) is the committing enzyme for capsidiol synthesis, and the promoter of EAS4, a member of the EAS family, was analyzed. Reporter gene analysis revealed that at least two regions each 40-50 bp length were involved in activation of the EAS4 promoter by wounding, as well as by artificial activation of WIPK and SIPK. Unlike transcripts of the capsidiol synthesis genes, accumulation of EAS protein and capsidiol itself were not induced by wounding; however, wounding significantly enhanced their subsequent induction by a pathogen-derived elicitor. CONCLUSIONS: Our results suggest a so-called priming phenomenon since the induction of EAS by wounding is only visible at the transcript level. By inducing transcripts, not the proteins, of EAS and possibly other capsidiol synthesis genes at wound sites, plants can produce large quantities of capsidiol quickly if pathogens invade the wound site, whereas plants can minimize energy loss and avoid the cytotoxic effects of capsidiol where pathogens do not gain entry during wound healing.

Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties.

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from Nicotiana benthamiana, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in N. benthamiana leaves. Chimeric and point-mutation analyses revealed that Thr531, Ser537, and Ile550 of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.

EIN2-mediated signaling is involved in pre-invasion defense in Nicotiana benthamiana against potato late blight pathogen, Phytophthora infestans.

Nicotiana benthamiana ABCG1 and ABCG2 are ABC transporters which are probably involved in the export of capsidiol, the major phytoalexin of Nicotiana species. While capsidiol export by these transporters plays an essential role in post-invasion defense against Phytophthora infestans, they also export unidentified antimicrobial compound(s) involved in pre-invasion defense. In this study, promoter activity of NbABCG2 (Pabcg2a) was analyzed using a GFP marker. Expression of GFP under the control of Pabcg2a was significantly increased by co-expression with the INF1 elicitor from P. infestans. Disruption of the ethylene-responsive GCC box in Pabcg2a compromised INF1-induced activation of Pabcg2a. Consistently, penetration by P. infestans was increased by gene-silencing of NbEIN2, the key ethylene-signaling component, suggesting the involvement of ethylene for pre-invasion defense of N. benthamiana.

Transcriptome analysis of WIPK/SIPK-suppressed plants reveals induction by wounding of disease resistance-related genes prior to the accumulation of salicylic acid.

Salicylic acid (SA) plays a key role in plant resistance to pathogens. Accumulation of SA is induced by wounding in tobacco plants in which the expression of WIPK and SIPK, two mitogen-activated protein kinases, is suppressed. Here, the mechanisms underlying the abnormal accumulation of SA in WIPK/SIPK-suppressed plants have been characterized. SA accumulation started around 12 h after wounding and was inhibited by cycloheximide (CHX), a protein synthesis inhibitor. SA accumulation, however, was enhanced several fold when leaf discs were transferred onto CHX after floating on water for ≥6 h. Temporal and spatial analyses of wound-induced and CHX-enhanced SA accumulation suggested that wounding induces activators for SA accumulation followed by the generation of repressors, and late CHX treatment inhibits the production of repressors more efficiently than that of activators. Microarray analysis revealed that the expression of many disease resistance-related genes, including N, a Resistance (R) gene for Tobacco mosaic virus and R gene-like genes, was up-regulated in wounded WIPK/SIPK-suppressed plants. Expression of the N gene and R gene-like genes peaked earlier than that of most other genes as well as SA accumulation, and was mainly induced in those parts of leaf discs where SA was highly accumulated. Moreover, wound-induced SA accumulation was decreased by the treatments which compromise the function of R proteins. These results indicate that signaling leading to the expression of disease resistance-related genes is activated by wounding in WIPK/SIPK-suppressed plants, and induction of R gene and R gene-like genes might lead to the biosynthesis of SA.

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.

Phosphorylation of the Nicotiana benthamiana WRKY8 transcription factor by MAPK functions in the defense response.

Mitogen-activated protein kinase (MAPK) cascades have pivotal roles in plant innate immunity. However, downstream signaling of plant defense-related MAPKs is not well understood. Here, we provide evidence that the Nicotiana benthamiana WRKY8 transcription factor is a physiological substrate of SIPK, NTF4, and WIPK. Clustered Pro-directed Ser residues (SP cluster), which are conserved in group I WRKY proteins, in the N-terminal region of WRKY8 were phosphorylated by these MAPKs in vitro. Antiphosphopeptide antibodies indicated that Ser residues in the SP cluster of WRKY8 are phosphorylated by SIPK, NTF4, and WIPK in vivo. The interaction of WRKY8 with MAPKs depended on its D domain, which is a MAPK-interacting motif, and this interaction was required for effective phosphorylation of WRKY8 in plants. Phosphorylation of WRKY8 increased its DNA binding activity to the cognate W-box sequence. The phospho-mimicking mutant of WRKY8 showed higher transactivation activity, and its ectopic expression induced defense-related genes, such as 3-hydroxy-3-methylglutaryl CoA reductase 2 and NADP-malic enzyme. By contrast, silencing of WRKY8 decreased the expression of defense-related genes and increased disease susceptibility to the pathogens Phytophthora infestans and Colletotrichum orbiculare. Thus, MAPK-mediated phosphorylation of WRKY8 has an important role in the defense response through activation of downstream genes.

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death.

Silencing of WIPK and SIPK mitogen-activated protein kinases reduces tobacco mosaic virus accumulation but permits systemic viral movement in tobacco possessing the N resistance gene.

Infection of tobacco cultivars possessing the N resistance gene with Tobacco mosaic virus (TMV) results in confinement of the virus by necrotic lesions at the infection site. Although the mitogen-activated protein kinases WIPK and SIPK have been implicated in TMV resistance, evidence linking them directly to disease resistance is, as yet, insufficient. Viral multiplication was reduced slightly in WIPK- or SIPK-silenced plants but substantially in WIPK/SIPK-silenced plants, and was correlated with an increase in salicylic acid (SA) and a decrease in jasmonic acid (JA). Silencing of WIPK and SIPK in a tobacco cultivar lacking the N gene did not inhibit viral accumulation. The reduction in viral accumulation was attenuated by expressing a gene for an SA-degrading enzyme or by exogenously applying JA. Inoculation of lower leaves resulted in the systemic spread of TMV and formation of necrotic lesions in uninoculated upper leaves. These results suggested that WIPK and SIPK function to negatively regulate local resistance to TMV accumulation, partially through modulating accumulation of SA and JA in an N-dependent manner, but positively regulate systemic resistance.

Involvement of two rice ETHYLENE INSENSITIVE3-LIKE genes in wound signaling.

Ethylene and jasmonic acid (JA) have been proposed as key compounds for wound signaling in plants. In Arabidopsis, ETHYLENE INSENSITIVE3 (EIN3), which is an essential transcription factor for ethylene signaling, is regulated at the post-transcriptional level, while transcriptional regulation of EIN3 or EIN3-LIKE (EIL) genes has not been well documented. The expression of 6 rice EIL genes (OsEIL1-6) was analyzed and only OsEIL1 and 2 were found to be wound-inducible EIL. OsEIL2 was also induced by JA. Electrophoretic mobility shift assays showed that recombinant OsEIL1 and 2 proteins bound to specific DNA sequences that are recognized by a wound-inducible tobacco EIL. Accumulation of OsEIL1 and 2 transcripts reached a maximum at 1 and 0.5 h after wounding, respectively, and the corresponding DNA-binding activity in nuclear extracts of rice leaves was increased at 1 h after wounding. Candidates for OsEIL-target genes were selected by microarray analysis of wounded rice and by promoter sequence analyses of wound-inducible genes identified by microarray analysis. In OsEIL1- and/or 2-suppressed rice plants, the expression of at least four of 18 candidate genes analyzed was down-regulated. These results indicate the importance of inducible OsEILs in wound signaling in rice.

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.

A calmodulin-binding mitogen-activated protein kinase phosphatase is induced by wounding and regulates the activities of stress-related mitogen-activated protein kinases in rice.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are negative regulators of MAPKs. In dicotyledons such as Arabidopsis and tobacco, MKPs have been shown to play pivotal roles in abiotic stress responses, hormone responses and microtubule organization. However, little is known about the role of MKPs in monocotyledons such as rice. Database searches identified five putative MKPs in rice. We investigated their expression in response to wounding, and found that the expression of OsMKP1 is rapidly induced by wounding. In this study, we functionally characterized the involvement of OsMKP1 in wound responses. The deduced amino acid sequence of OsMKP1 shows strong similarity to Arabidopsis AtMKP1 and tobacco NtMKP1. Moreover, OsMKP1 bound calmodulin in a manner similar to NtMKP1. To determine the biological function of OsMKP1, we obtained osmkp1, a loss-of-function mutant, in which retrotransposon Tos17 was inserted in the second exon of OsMKP1. Unlike the Arabidopsis atmkp1 loss-of-function mutant, which shows no abnormal phenotype without stimuli, osmkp1 showed a semi-dwarf phenotype. Exogenous supply of neither gibberellin nor brassinosteroid complemented the semi-dwarf phenotype of osmkp1. Activities of two stress-responsive MAPKs, OsMPK3 and OsMPK6, in osmkp1 were higher than those in the wild type both before and after wounding. Microarray analysis identified 13 up-regulated and eight down-regulated genes in osmkp1. Among the up-regulated genes, the expression of five genes showed clear responses to wounding, indicating that wound responses are constitutively activated in osmkp1. These results suggest that OsMKP1 is involved in the negative regulation of rice wound responses.

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.

Nitrate reductase is responsible for elicitin-induced nitric oxide production in Nicotiana benthamiana.

Recent works have established a key role for nitric oxide (NO) in activating disease resistance in plants. Nitrate reductase (NR) is one of the enzymes that are capable of producing NO in plants. In a previous study, we reported that pathogen signals induce expression of NR genes in potato, suggesting the involvement of NR in NO production induced by pathogen signals. In this study, we cloned NR genes from Nicotiana benthamiana and investigated their involvement in NO production induced by INF1, a major elicitin secreted by Phytophthora infestans. Treatment of protoplasts prepared from N. benthamiana leaves with INF1 elevated NO production to a maximum level 1-3 h after treatment. INF1-induced NO generation was suppressed completely by an NO-specific scavenger, but partially by a nitric oxide synthase inhibitor. To investigate the involvement of NR in INF1-induced NO production, NR genes were silenced by virus-induced gene silencing. The NR-silenced plants showed yellowish leaves which resemble the characteristic of Arabidopsis NR double mutants. Silencing of NR genes significantly decreased both NO(2) (-)-producing activity and INF1-induced NO production, indicating that NR is involved in INF1-induced NO production. In contrast, overexpression of NbNR1 encoding N. benthamiana NR by Agrobacterium-mediated transient expression elevated NO(2) (-)-producing activity nine times over the control; however, INF1-induced NO production in protoplasts overexpressing NbNR1 was comparable with that in control protoplasts. These results suggest that NR is involved in INF1-induced NO production, and post-translational modification of NR or availability of substrate NO(2) (-) may be a rate-limiting step of NO production by NR.

Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance.

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.

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 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.

Catalytic activation of the plant MAPK phosphatase NtMKP1 by its physiological substrate salicylic acid-induced protein kinase but not by calmodulins.

MAPK phosphatases (MKPs) are negative regulators of MAPKs. Previously, we identified NtMKP1 as a novel calmodulin (CaM)-binding protein (Yamakawa, H., Katou, S., Seo, S., Mitsuhara, I., Kamada, H., and Ohashi, Y. (2004) J. Biol. Chem. 279, 928-936). In this study, we characterized the interaction of NtMKP1 with substrate MAPKs and CaM. NtMKP1 (produced by in vitro transcription/translation) inactivated salicylic acid-induced protein kinase (SIPK) through dephosphorylation of the TEY motif of SIPK. CaM bound but unexpectedly did not activate the phosphatase activity of NtMKP1. NtMKP1 has four characteristic domains, viz. a dual-specificity phosphatase catalytic domain, a gelsolin homology domain, a CaM-binding domain, and C-terminal domain. Deletion analysis revealed that the N-terminal non-catalytic region of NtMKP1 bound SIPK and was essential for inactivating SIPK, whereas the CaM-binding and C-terminal domains were dispensable. Moreover, the phosphatase activity of NtMKP1 was increased strongly by the binding of SIPK, but weakly by another MAPK, wound-induced protein kinase. Swapping and site-directed mutagenesis of SIPK and wound-induced protein kinase revealed that the strong activation of NtMKP1 phosphatase activity by SIPK partially depended on the putative common docking domain of SIPK. On the other hand, conversion of Lys(41) and Arg(43) of NtMKP1 to Ala (K41A/R43A) abolished the interaction with SIPK. Expression of constitutively active MAPK kinase in Nicotiana benthamiana induced activation of SIPK and cell death. Simultaneous expression of either NtMKP1 or NtMKP1 L443R, which was unable to bind CaM, compromised the constitutively active MAPK kinase-induced responses, whereas that of NtMKP1 K41A/R43A did not. These results indicate that the regulation of NtMKP1 activity by SIPK binding, but not by CaM binding, is important for the function of NtMKP1.

Plant MAPK phosphatase interacts with calmodulins.

A mitogen-activated protein kinase (MAPK) phosphatase gene, designated NtMKP1, was isolated as a candidate gene for a calmodulin (CaM)-binding protein from tobacco. NtMKP1 protein has four characteristic domains conserved among plant MAPK phosphatases reported so far, namely a dual specificity protein phosphatase catalytic domain, gelsolin-like domain, putative CaM-binding domain (CaMBD), and serine-rich region, indicating that NtMKP1 is the ortholog of Arabidopsis MKP1. The bacterially expressed NtMKP1 protein physically interacted with three plant-specific types of CaM in an overlay assay with labeled CaMs, showing high affinity to NtCaM1 and NtCaM3 but lower affinity to NtCaM13. The peptide for the putative CaMBD bound both NtCaM1 and NtCaM3 significantly but bound NtCaM13 only slightly. Moreover, CaM overlay assays with mutated CaMBDs revealed that Trp440 and Leu443 in the middle of the basic amphiphilic alpha-helix motif (amino acids 436-453) are critical for binding CaM. In comparison with the transient accumulation of a wound-induced MAPK, WIPK transcript, a prolonged activation of NtMKP1 expression was found in response to wounding and tobacco mosaic virus-induced hypersensitive reaction. In transgenic tobacco plants overexpressing NtMKP1, wound-induced activation of SIPK, salicylic acid-induced MAPK, and WIPK was inhibited. These results suggest that plant CaMs are involved in these stress-activated MAPK cascades via NtMKP1.