Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in tobacco

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaic acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.     

Protein Phosphorylation Is Induced in Tobacco Cells by the Elicitor Cryptogein

Changes in plasmalemma ion fluxes were observed when tobacco (Nicotiana tabacum) cells were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. A strong alkalization of the culture medium, accompanied by a leakage of potassium, was induced within a few minutes of treatment. These effects reached a maximum after 30 to 40 min and lasted for several hours. This treatment also resulted in a rapid, but transient, production of activated oxygen species. All these physiological responses were fully sensitive to staurosporine, a known protein kinase inhibitor. Furthermore, a study of protein phosphorylation showed that cryptogein induced a staurosporine-sensitive phosphorylation of several polypeptides. These data suggest that phosphorylated proteins may be essential for the transduction of elicitor signals.     

MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response?

Hypoosmotic stress activates a phosphorylation-dependent oxidative burst. In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like. Apigenin, a MAP kinase and cyclin-dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst ( Cazaléet al. 1998 ; Plant Physiol. 116, 659–669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6-DMAP. However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst. Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.     

Questioning the role of salicylic acid and cytosolic acidification in mitogen-activated protein kinase activation induced by cryptogein in tobacco cells

Elicitors of plant defence reactions, oligogalacturonides and cryptogein, an elicitin produced by Phytophthora cryptogea, were previously shown to induce a rapid and transient activation of two mitogen-activated protein kinases (MAPKs) in cells of tobacco [ Nicotiana tabacum L. cv. Xanthi; A. Lebrun-Garcia et al. (1998) Plant J 15:773-781]. We verified that these two MAPKs correspond to the salicylic acid-induced protein kinase (SIPK) and the wound-induced protein kinase (WIPK). The involvement of salicylic acid (SA) in cryptogein-induced MAPK activation was investigated using transgenic NahG tobacco cells expressing the salicylate hydroxylase gene and thus unable to accumulate SA. The large and sustained activation of both MAPKs by cryptogein was maintained in transgenic cells compared with non-transgenic tobacco cells. Moreover, weak acids, namely SA, 4-hydroxybenzoic acid, an ineffective analogue of SA in plant resistance, and butyric acid acidified the cytosol, a physiological event also induced by cryptogein, but activated both MAPKs only slightly and transiently in tobacco cells. These results indicate that MAPK activation by cryptogein is not mediated by SA, that cytosolic acidification can be transduced by MAPKs, and that in cryptogein-treated cells, cytosolic acidification should contribute poorly to MAPK activation.     

Slow and prolonged activation of the p47 protein kinase during hypersensitive cell death in a culture of tobacco cells

To investigate the involvement of protein kinases in the signaling cascade that leads to hypersensitive cell death, we used a previously established system in which a fungal elicitor, xylanase from Trichoderma viride (TvX), induces a hypersensitive reaction in tobacco (Nicotiana tabacum) cells in culture (line XD6S). The elicitor induced the slow and prolonged activation of a p47 protein kinase, which has the characteristics of a family member of the mitogen-activated protein kinases. An inhibitor of protein kinases, staurosporine, and a blocker of Ca channels, Gd³⁺ ions, both of which blocked the TvX-induced hypersensitive cell death, inhibited the TvX-induced activation of p47 protein kinase. Moreover, an inhibitor of serine/threonine protein phosphatase alone induced both rapid cell death and the persistent activation of the p47 protein kinase. Thus, the p47 protein kinase might be a component of the signal transduction pathway that leads to hypersensitive cell death, and the regulation of the duration of activation of the p47 protein kinase might be important in determining the destiny of tobacco cells.     

Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells

Two cDNA clones, cATMPK1 and CATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined. Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level. ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%). The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2. Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds. Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome. Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coli, activating these enzymes. A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxinstarved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). Protein kinase activities which phosphorylated the recombinant ATMPK2 protein also increased rapidly after auxin treatment in the auxin-starved BY-2 cells. These results suggest that auxin may function as an activator of plant MAP kinase homologues, as do various mitogens in animal systems.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures

Elicitation of Arabidopsis thaliana (L.) Heynh. suspension cultures with the bacterial protein harpin (from Pseudomonas syringae pv. syringae) induced the activation of two kinases of 39 and 44 kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses in Arabidopsis thaliana. Received: 23 February 1999 / Accepted: 22 July 1999     

Characterization of fertilization-modulated myelin basic protein kinases from sea star: regulation of Mapk.

The myelin basic protein (MBP)-phosphorylating enzymes present during maturation and early embryogenesis of the sea star (Pisaster ochraceus) were investigated. The major maturation-activated MBP kinase (p45 Mapk) was molecularly cloned based on tryptic sequence information obtained with the purified enzyme and shown to be highly related to human Erk1 with 76% amino acid identity. Kinase assays and immunoblotting studies revealed that Mapk remained highly active until 12 h post-fertilization (PF), after which it declined. By 4 days PF, Mapk protein was no longer detectable. At 3 h PF, about half of the detectable MBP phosphotransferase activity could be attributed to a 75 kDa protein kinase that was distinct from Mapk. Like Mapk, this protein phosphorylated MBP mostly on threonine residues, but it failed to phosphorylate a peptide (APRTPGGRR) based upon the Thr-97 MAP kinase phosphorylation site in MBP. Rather, it phosphorylated a peptide (AAQKRPSQRTKYLA) patterned after the N-terminus of MBP. Our studies also showed a dramatic increase in MBP phosphotransferase activity occurred by 4 days PF that arose from a third kinase that phosphorylated MBP solely on serine residues. This kinase exhibited the following substrate substrate preference: AAQKRPSQRTKYLA, peptide substrate for S6 kinases (AKRRRLSSLRASTSKSESSQK) > MBP > histone H1 > prota-mine > casein > APRTPGGRR. This kinase was not appreciably affected by addition of phosphatidylserine/diacylglycerol, or the staurosporine analogue Roche Compound 3, but it was partly inhibited by a protein kinase C pseudosubstrate peptide. Gel filtration analysis revealed an apparent molecular mass of 41 kDa for the enzyme. Therefore, at least two novel MBP-phosphorylating enzymes distinct from Mapk are preferentially activated following fertilization and early embryogenesis of the sea star.     

Activation of 40-kDa Protein Kinases in Response to Hypo- and Hyperosmotic Shock in the Halotolerant Green Alga Dunaliella tertiolecta

Changes in protein kinase activities in Dunaliella tertiolectain response to hypo- and hyperosmotic shocks were assessed byassays of protein kinase activities on SDS-polyacrylamide gelsthat contained protein substrates, Hypoosmotic shock (transferfrom 0.5 to 0.2 M Nacl) transiently activated a 40-kDa proteinkinase (low osmotic pressure-activated protein kinase, LAP kinase)that phosphorylated myelin basic protein (MBP) and histone H1but not casein. By contrast, hyperosmotic shock (transfer from0.5 to 1.1 M NACl) rapidly activated another 40-kDa proteinkinase (high osmotic pressure-activated protein kinase, HAPkinase) that phosphorylated casein and histone H1 but not MBP.Inhibitors of protein kinases, namely, K-252a and staurosporine,suppressed the activation of both LAP kinase and HAP kinase.The protein kinase activities in extracts of osmotically shockedcells disappeared completely after treatment with calf intestinalalkaline phosphatase. Anti-phosphotyrosine monoclonal antibodiesdid not cross-react with either LAP kinase or HAP kinase. Theseresults indicate that at least two 40-kDa protein kinases, LAPkinase and HAP kinase, are activated by hyper- and hypoosmoticshock, respectively, and that their activities are regulatedvia phosphorylation by putative protein kinase(s) that act atan upstream position in the signaling cascade(s) that followsosmotic shock. (Received July 24, 1995; Accepted October 20, 1995)     

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.     

Effects of protein kinase and phosphatidylinositol-3 kinase inhibitors on growth and ultrastructure of Trypanosoma cruzi

An increasing number of protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. Some PK inhibitors display antiproliferative effects on protozoa. We tested three PK inhibitors on the growth and ultrastructure of epimastigotes of Trypanosoma cruzi and the effect of these drugs on intracellular amastigotes. They were staurosporine (serine/threonine kinase inhibitor), genistein (tyrosine kinase inhibitor), and wortmannin (phosphatidylinositol 3' (PI3) kinase inhibitor). All drugs inhibited epimastigote growth at the concentrations tested. Wortmannin inhibited parasite growth at the lowest concentrations. However, staurosporine was the most effective after 24 h treatment and genistein caused the stronger inhibition during the whole treatment (60-70% inhibition). The IC50 were: staurosporine: 6.43+/-1.28 microM; genistein: 6.54+/-1.86 microM; and wortmannin: 0.056+/-0.014 microM. These PK inhibitors had strong ultrastructural effects on the epimastigotes: abnormal chromatin condensation of the nucleus; loose flagellar membrane with the formation of blebs; incomplete cell division; autophagosomes and myelin-like figures. These drugs did not interfere with the division of intracellular amastigotes or with its differentiation to trypomastigotes. However, as trypanosomes have kinomes that contain a large set of protein kinases and phosphatases, PKs should not be disregarded as an important target for chemotherapy of Chagas disease.     

Real-time fluorogenic kinase assay using protein as substrate

A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C βII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K_m^ATP), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.     

The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in bright yellow-2 tobacco cells

The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. To define the nature of the stimulated endocytosis, we quantified clathrin-coated pits (CCPs) forming on the plasma membrane of BY-2 cells. A transitory stimulation of this morphological event by cryptogein occurs within the first 15 min. In the presence of cryptogein, increases in both FM4-64 internalization and clathrin-mediated endocytosis are specifically blocked upon treatment with 5 microm tyrphostin A23, a receptor-mediated endocytosis inhibitor. The kinetics of the transient increase in CCPs at the plasma membrane coincides with that of transitory reactive oxygen species (ROS) production occurring within the first 15 min after elicitation. Moreover, in BY-2 cells expressing NtrbohD antisense cDNA, which are unable to produce ROS when treated with cryptogein, the CCP stimulation is inhibited. These results indicate that the very early endocytic process induced by cryptogein in tobacco is due, at least partly, to clathrin-mediated endocytosis and is dependent on ROS production by the NADPH oxidase NtrbohD.     

Responses of Cultured Tobacco Cells to Cryptogein, a Proteinaceous Elicitor from Phytophthora cryptogea: Possible Plasmalemma Involvement

In culture, the phytopathogenic fungus Phytophthora cryptogea secretes a protein which elicits hypersensitive-like necroses and protects tobacco plants against invasion by the pathogen Phytophthora parasitica var. nicotianae. This protein, named cryptogein, has been purified and its amino acid sequence determined. In this work, we studied the effect of cryptogein on tobacco cell suspension cultures. Cryptogein was lethal at about 0.10 micromolar. When added at sublethal doses, it elicited the production of ethylene and phytoalexins. It also induced a rapid increase in pH and conductivity of the extracellular medium without affecting the integrity of the plasma membrane. Cryptogein reduced the fusicoccin-induced acidification of the extracellular medium. The concentration which inhibited the fusicoccin response by 50% was 0.8 nanomolar, while 1 micromolar erythrosine B, an ATPase inhibitor, was needed to produce the same inhibition. However, cryptogein did not inhibit the activity of a purified plasma membrane ATPase. Results of binding studies with whole cells suggested the presence of elicitor-binding sites with a high affinity for cryptogein. The involvement of the plasma membrane during the initial interaction between elicitor and cells is discussed.