Salicylic Acid-induced Protein Kinase Research Articles

The MAPK-Alfin-like 7 module negatively regulates ROS scavenging genes to promote NLR-mediated immunity

Nucleotide-binding leucine-rich repeat (NLR) receptor-mediated immunity includes rapid production of reactive oxygen species (ROS) and transcriptional reprogramming, which is controlled by transcription factors (TFs). Although some TFs have been reported to participate in NLR-mediated immune response, most TFs are transcriptional activators, and whether and how transcriptional repressors regulate NLR-mediated plant defenses remains largely unknown. Here, we show that the Alfin-like 7 (AL7) interacts with N NLR and functions as a transcriptional repressor. Knockdown and knockout of AL7 compromise N NLR-mediated resistance against tobacco mosaic virus, whereas AL7 overexpression enhances defense, indicating a positive regulatory role for AL7 in immunity. AL7 binds to the promoters of ROS scavenging genes to inhibit their transcription during immune responses. Mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK) directly interact with and phosphorylate AL7, which impairs the AL7-N interaction and enhances its DNA binding activity, which promotes ROS accumulation and enables immune activation. In addition to N, AL7 is also required for the function of other Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeats (TNLs) including Roq1 and RRS1-R/RPS4. Our findings reveal a hitherto unknown MAPK-AL7 module that negatively regulates ROS scavenging genes to promote NLR-mediated immunity.

Pattern of changes in salicylic acid-induced protein kinase (SIPK) gene expression and salicylic acid accumulation in wheat under cadmium exposure.

Salicylic acid-induced protein kinase (SIPK) is known as a 'master switch' for stress responses in plants. It can be induced by salicylic acid (SA) and several stress factors. The main aim of the present study was to reveal the relationship between SA accumulation and the gene expression level of SIPK during 50 and 250µm Cd stress in wheat plants. Quantitative real-time PCR was used for determination of the gene expression level of SIPK. Salicylic acid content measurement was performed with an HPLC system equipped with a fluorescence detector. Cadmium treatment increased the endogenous SA level and expression level of SIPK in a concentration-dependent manner. Induction of SIPK expression preceded the accumulation of endogenous SA. Although SA treatment induced dramatic endogenous SA accumulation, its SIPK-inducing effect was moderate. In roots, higher induction of SIPK was observed than in leaves. The same tendency of SIPK expression was observed in both Cd- and SA-treated plants, as decisively the highest transcript level was detected after 30min of treatment, but thereafter the expression decreased rapidly to control level or even below. The induction of SIPK was transient in all cases, and even a very high SA level in either the leaves or roots was not able to maintain the elevated expression level of this gene. The results suggest that SIPK has a role in initiating Cd stress response and the exogenous SA-induced signalling process.

The role of SIPK signaling pathway in antioxidant activity and programmed cell death of tobacco cells after exposure to cadmium

Cadmium (Cd) toxicity induces oxidative burst and leads to programmed cell death (PCD) in plant cells. The role of salicylic acid-induced protein kinase (SIPK) signaling pathway in Cd-induced oxidative stress was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Barley 21). The cells were pretreated with 40 μM PD98059 (inhibitor of MAPKK) and then exposed to 50 μM Cd for 24 h. The percentages of cell viability, apoptosis, necrosis, and the content of reactive oxygen species (ROS) were monitored by flow cytometry. Expression of PCD related gene (Hsr203J) and the contents of certain signaling molecules were measured as well. The results showed that Cd increased the expression of SIPK, Hsr203J, and CAT genes, the activities of catalase and caspase-3-like enzymes. Addition of PD98059 inhibitor reduced the expression of Hsr203J and CAT genes, decreased CAT activity, but increased ROS and SA contents, and caspase-3-like activity and apoptosis rate. The highest apoptosis level was accompanied by the highest level of Hsr203J gene expression. From the results it can be suggested that upon treatment of tobacco cells with Cd, internal SA content increased and induced the SIPK signaling pathway, thereby inhibited the antioxidant system and led to PCD.

Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

Up-regulation of MPK4 increases the feeding efficiency of the green peach aphid under elevated CO2 in Nicotiana attenuata.

Previous research has shown that elevated CO2 reduces plant resistance against insects and enhances the water use efficiency of C3 plants, which improves the feeding efficiency of aphids. Although plant mitogen-activated protein kinases (MAPKs) are known to regulate water relations and phytohormone-mediated resistance, little is known about the effect of elevated CO2 on MAPKs and the cascading effects on aphids. By using stably transformed Nicotiana attenuata plants silenced in MPK4, wound-induced protein kinase (WIPK), or salicylic acid-induced protein kinase (SIPK), we determined the functions of MAPKs in plant-aphid interactions and their responses to elevated CO2. The results showed that among all plant genotypes, inverted repeat MPK4 plants had the largest stomatal apertures, the lowest water content, the strongest jasmonic acid (JA)-dependent resistance, and the lowest aphid numbers, suggesting that MPK4 affects plant responses to aphids by regulating stomatal aperture and JA-dependent resistance. Regardless of aphid infestation, elevated CO2 up-regulated MPK4, but not WIPK or SIPK, in wild-type plants. Elevated CO2 increased the number, mean relative growth rate, and feeding efficiency of aphids on all plant genotypes except inverted repeat MPK4. We conclude that MPK4 is a CO2-responsive plant determinant that regulates the molecular interaction between plants and aphids.

Identification of MAPKs as signal transduction components required for the cell death response during compatible infection by the synergistic pair Potato virus X-Potato virus Y

Systemic necrosis is one of the most severe symptoms caused in compatible plant-virus interactions and shares common features with the hypersensitive response (HR). Mitogen-activated protein kinase (MAPK) cascades are associated with responses to compatible and incompatible host-virus interactions. Here, we show that virus-induced gene silencing of the Nicotiana benthamiana MAPK genes salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), and the MAPK kinase (MAPKK) genes MEK1 and MKK1, partially compromised the HR-like response induced by the synergistic interaction of Potato virus X with Potato virus Y (PVX-PVY). Nevertheless, ameliorated cell death induced by PVX-PVY in the MAPK(K)-silenced plants did not facilitate virus accumulation in systemically infected leaves. Dual silencing of SIPK and of the oxylipin biosynthetic gene 9-Lipoxygenase showed that the latter was epistatic to SIPK in response to PVX-PVY infection. These findings demonstrate that SIPK, WIPK, MEK1 and MKK1 function as positive regulators of PVX-PVY-induced cell death.

Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco.

The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.

The type 3 effector NopL of Sinorhizobium sp. strain NGR234 is a mitogen-activated protein kinase substrate.

Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis.

Herbivore defense responses and associated herbivore defense mechanism as revealed by comparing a resistant wild soybean with a susceptible cultivar

Plants have evolved sophisticated defense mechanisms against herbivores to help them adapt to the environment. Understanding the defense mechanisms in plants can help us control insects in a more effective manner. In this study, we found that compared with Tianlong 2 (a cultivated soybean with insect susceptibility), ED059 (a wild soybean line with insect resistance) contains sharper pubescence tips, as well as lower transcript levels of wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), which are important mitogen-activated protein kinases involved in early defense response to herbivores. The observed lower transcript levels of WIPK and SIPK induced higher levels of jasmonic acid (JA), JA biosynthesis enzymes (AOC3) and some secondary metabolites in ED059. Functional analysis of the KTI1 gene via Agrobacterium-mediated transformation in Arabidopsis thaliana indicated that it plays an important role in herbivore defense in ED059. We further investigated the molecular response of third-instar Helicoverpa armigera (Hübner) larvae to Tianlong 2 and ED059. We found apoptotic cells only in the midguts of larvae that fed on ED059. Compared with larvae reared on the susceptible cultivar Tianlong 2, transcript levels of catalase (CAT) and glutathione S-transferase (GST) were up-regulated, whereas those of CAR, CHSB, and TRY were down-regulated in larvae that fed on the highly resistant variety ED059. We propose that these differences underlie the different herbivore defense responses of ED059 and Tianlong 2.

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.

Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

BackgroundHerbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown.ResultsHere, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses.ConclusionsOur results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0326-z) contains supplementary material, which is available to authorized users.

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.

MAPK signaling: a key element in plant defense response to insects.

Insects have long been the most abundant herbivores, and plants have evolved sophisticated mechanisms to defend against their attack. In particular, plants can perceive specific patterns of tissue damage associated with insect herbivory. Some plant species can perceive certain elicitors in insect oral secretions (OS) that enter wounds during feeding, and rapidly activate a series of intertwined signaling pathways to orchestrate the biosynthesis of various defensive metabolites. Mitogen-activated protein kinases (MAPKs), common to all eukaryotes, are involved in the orchestration of many cellular processes, including development and stress responses. In plants, at least two MAPKs, salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), are rapidly activated by wounding or insect OS; importantly, genetic studies using transgenic or mutant plants impaired in MAPK signaling indicated that MAPKs play critical roles in regulating the herbivory-induced dynamics of phytohormones, such as jasmonic acid, ethylene and salicylic acid, and MAPKs are also required for transcriptional activation of herbivore defense-related genes and accumulation of defensive metabolites. In this review, we summarize recent developments in understanding the functions of MAPKs in plant resistance to insect herbivores.

Reactive oxygen species in signalling the transcriptional activation of WIPK expression in tobacco

Plant mitogen-activated protein kinases represented by tobacco WIPK (wounding-induced protein kinase) and its orthologs in other species are unique in their regulation at transcriptional level in response to stress and pathogen infection. We previously demonstrated that transcriptional activation of WIPK is essential for induced WIPK activity, and activation of salicylic acid-induced protein kinase (SIPK) by the constitutively active NtMEK2(DD) is sufficient to induce WIPK gene expression. Here, we report that the effect of SIPK on WIPK gene expression is mediated by reactive oxygen species (ROS). Using a combination of pharmacological and gain-of-function transgenic approaches, we studied the relationship among SIPK activation, WIPK gene activation in response to fungal cryptogein, light-dependent ROS generation in chloroplasts, and ROS generated via NADPH oxidase. In the conditional gain-of-function GVG-NtMEK2(DD) transgenic tobacco, induction of WIPK expression is dependent on the ROS generation in chloroplasts. Consistently, methyl viologen, an inducer of ROS generation in chloroplasts, highly activated WIPK expression. In addition to chloroplast-originated ROS, H(2)O(2) generated from the cell-surface NADPH oxidase could also activate WIPK gene expression, and inhibition of cryptogein-induced ROS generation also abolished WIPK gene activation. Our data demonstrate that WIPK gene activation is mediated by ROS, which provides a mechanism by which ROS influence cellular signalling processes in plant stress/defence response.

Involvement of mitogen-activated protein kinase signalling in pearl millet–downy mildew interaction

Mitogen-activated protein kinase (MAPK) cascade-mediated signalling is essential in the establishment of resistance towards pathogens. The present study compared MAPK activities in a compatible and incompatible interaction between pearl millet [Pennisetum glaucum (L.) R. Br.] and downy mildew pathogen Sclerospora graminicola. Differential expression was observed with rapid and increased activation of MAPKs, PgMPK1 (48kDa) and PgMPK2 (44kDa), in the incompatible interaction; with a weak activity of only PgMPK1 in the compatible interaction. Immunoblot analysis showed PgMPK1 and PgMPK2 to be orthologs of salicylic acid-induced protein kinase and wound-induced protein kinase, respectively. Immunocytochemical analysis revealed pathogen-induced accumulation and nuclear localisation of PgMPKs only in the incompatible interaction with highest signals in the vascular tissues. Maximum PgMPKs activation correlated with the activation of several defence-related enzymes. In addition, inhibition of MAPK-activation by kinase cascade inhibitors correlated with the suppression of defence-related enzyme activities and pathogen-induced H2O2 accumulation. Treatment of pearl millet seedlings with abiotic and biotic elicitors led to a strong early induction of only PgMPK1. β-Amino butyric acid and H2O2 were found to be best activators of PgMPK1. These results suggest that in pearl millet MAPK signalling is involved in mediating several defence mechanisms in response to pathogen infection.

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.

The Nicotiana benthamiana Mitogen-Activated Protein Kinase Cascade and WRKY Transcription Factor Participate in Nep1Mo-Triggered Plant Responses

Many bacterial, fungal, and oomycete species secrete necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) that trigger programmed cell death (PCD) and innate immune responses in dicotyledonous plants. However, how NLP induce such immune responses is not understood. Here, we show that silencing of the MAPKKKα-MEK2-WIPK mitogen-activated protein kinase (MAPK) cascade through virus-induced gene silencing compromises hydrogen peroxide accumulation and PCD induced by Nep1(Mo) from Magnaporthe oryzae. WIPK interacts with NbWRKY2, a transcription factor in Nicotiana benthamiana, in vitro and in vivo, suggesting an effector pathway that mediates Nep1(Mo)-induced cell death. Unexpectedly, salicylic acid-induced protein kinase (SIPK)- and NbWRKY2-silenced plants showed impaired Nep1(Mo)-induced stomatal closure, decreased Nep1(Mo)-promoted nitric oxide (NO) production in guard cells, and a reduction in Nep1(Mo)-induced resistance against Phytophthora nicotianae. Expression studies by real-time polymerase chain reaction suggested that the MEK2-WIPK-NbWRKY2 pathway regulated Nep1(Mo)triggered NO accumulation could be partly dependent on nitrate reductase, which was implicated in NO synthesis. Taken together, these studies demonstrate that the MAPK cascade is involved in Nep1(Mo)-triggered plant responses and MAPK signaling associated with PCD exhibits shared and distinct components with that for stomatal closure.

Ethylene Signaling Pathway and MAPK Cascades Are Required for AAL Toxin–Induced Programmed Cell Death

Programmed cell death (PCD), known as hypersensitive response cell death, has an important role in plant defense response. The signaling pathway of PCD remains unknown. We employed AAL toxin and Nicotiana umbratica to analysis plant PCD. AAL toxin is a pathogenicity factor of the necrotrophic pathogen Alternaria alternata f. sp. lycopersici. N. umbratica is sensitive to AAL toxin, susceptible to pathogens, and effective in Tobacco rattle virus-based virus-induced gene silencing (VIGS). VIGS analyses indicated that AAL toxin-triggered cell death (ACD) is dependent upon the mitogen-activated protein (MAP) kinase kinase MEK2, which is upstream of both salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) responsible for ethylene (ET) synthesis. ET treatment of MEK2-silenced N. umbratica re-established ACD. In SIPK- and WIPK-silenced N. umbratica, ACD was compromised and ET accumulation was not observed. However, in contrast to the case of MEK2-silenced plants, ET treatment did not induce cell death in SIPK- and WIPK-silenced plants. This work showed that ET-dependent pathway and MAP kinase cascades are required in ACD. Our results suggested that MEK2-SIPK/WIPK cascades have roles in ET biosynthesis; however, SIPK and WIPK have other roles in ET signaling or another pathway leading to cell death by AAL toxin.

Hrip1, a novel protein elicitor from necrotrophic fungus, Alternaria tenuissima, elicits cell death, expression of defence‐related genes and systemic acquired resistance in tobacco

Here, we report the identification, purification, characterization and gene cloning of a novel hypersensitive response inducing protein secreted by necrotrophic fungus, Alternaria tenuissima, designated as hypersensitive response inducing protein 1 (Hrip1). The protein caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis-related events including DNA laddering. The protein-encoding gene was cloned by rapid amplification of cDNA ends (RACE) method. The sequence analysis revealed that the cDNA is 495 bp in length and the open reading frame (ORF) encodes for a polypeptide of 163 amino acids with theoretical pI of 5.50 and molecular weight of 17 562.5 Da. Hrip1 induced calcium influx, medium alkalinization, activation of salicylic acid-induced protein kinase and several defence-related genes after infiltration in tobacco leaves. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of defence-related genes was activated. After several days, systemic acquired resistance was also induced. The tobacco plant cells that perceived the Hrip1 generated a cascade of signals acting at local, short, and long distances, and caused the coordinated expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. Thus, Hrip1 represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance in necrotrophic fungi.

Involvement of protein kinases and calcium in the NO-signalling cascade for defence-gene induction in ozonated tobacco plants

This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O(3)) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca(2+) in defence-gene activation. NO was visualized with the NO probe DAF-FM. Using a pharmacological approach, the effects of different inhibitors on the expression profiles of NO-dependent defence genes were monitored using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O(3) stimulates a 48 kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O(3)-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. By contrast, protein dephosphorylation mediates pathogenesis-related protein 1a (PR1a) expression in O(3)-treated tobacco plants. Ca(2+) is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca(2+) transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca(2+) suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA, and the cellular messengers in this O(3)-induced defence gene activation is proposed.