Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities.

In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca(2+)-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca(2+)-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca(2+)-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.

Protein kinases in the plant defence response.

Protein kinases play a central role in signalling during pathogen recognition and the subsequent activation of plant defence mechanisms. Members of different kinase subfamilies, such as calcium-dependent protein kinases and MAP kinases, are involved. Nevertheless, often, only a single component of a signalling cascade in an experimental plant system has been characterised. The future challenge is to understand how these kinases work, which cellular responses they mediate, and how they fit into the bigger picture of defence signalling. This challenge has become increasingly feasible with the recent introduction of new techniques: these techniques include reverse genetics, which will allow the allocation of biological function to kinase isoforms, (phospho) proteomics combined with mass spectrometry, and transient expression of kinases in a (constitutively) active form, mimicking the induction of defence responses in a biological system.

Calcium-dependent protein kinases play an essential role in a plant defence response.

Calcium-dependent protein kinases (CDPKs) comprise a large family of serine/threonine kinases in plants and protozoans. We isolated two related CDPK cDNAs (NtCDPK2 and NtCDPK3) from Nicotiana tabacum. These CDPK transcripts are elevated after race-specific defence elicitation and hypo-osmotic stress. Transiently expressed myc-epitope-tagged NtCDPK2 in Nicotiana benthamiana and N.tabacum leaves showed a rapid transient interconversion to an activated form after elicitation and hypo-osmotic stress. The Avr9 race-specific elicitor caused a more pronounced and sustained response. This transition is due to phosphorylation of the CDPK. Immuno complex kinase assays with epitope-tagged NtCDPK2 showed that stress-induced phosphorylation and interconversion of NtCDPK2 correlates with an increase in enzymatic activity. The function of NtCDPK2 in plant defence was investigated by employing virus-induced gene silencing (VIGS) in N.benthamiana. CDPK-silenced plants showed a reduced and delayed hypersensitive response after race-specific elicitation in a gene-for-gene interaction, and lacked an accompanying wilting phenotype. Silencing correlated with loss of CDPK mRNA, whereas mRNA accumulation of mitogen-activated protein kinase WIPK remained unaltered.

No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation are discussed.

Identification of a target gene for the bE-bW homeodomain protein complex in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW that form a heterodimeric complex when both proteins originate from different alleles. The heterodimer is presumed to be the central regulator for pathogenicity genes. Here, we show that a translational fusion protein comprising specific domains of bE1 and bW2 remains biologically active and binds to a sequence motif in the promoter of lga2, a gene located in the a mating type locus. This b binding sequence 1 (bbs1) is also recognized by the native bE1-bW2 heterodimer in vivo and mediates the b-dependent regulation of the lga2 gene. Our data demonstrate that the bE-bW heterodimer can act as a positive transcriptional regulator.

Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response.

In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9-dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca(2)+ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9-dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.

Early signalling events in the Avr9/Cf-9-dependent plant defence response.

Abstract Resistance of tomato to the leaf mould fungus Cladosporium fulvum is controlled by the interaction between a plant-encoded resistance gene (Cf-9) and pathogen-encoded avirulence (Avr9) gene. Our objective is to understand the underlying molecular mechanisms that transmit the Cf-9/Avr9-dependent pathogen perception event and activate the plant defence response. Our approach toward the understanding of Cf-function is based on the analysis of early Cf-9/Avr9-mediated responses and signalling events. Because Cf-9 transgenically expressed in tobacco retains its specificity and activity to the Avr9 elicitor, signalling experiments were conducted in the heterologous system using these transgenic lines or derived Cf9 tobacco cell cultures. Among the earliest responses to the Avr9/Cf-9 elicitation event were rapid changes in ion-fluxes, the synthesis of active oxygen species (AOS), probably catalysed by a plant NADPH-oxidase, and the transient activation of two MAP kinases. These kinases were identified as WIPK (wounding-induced protein kinase) and SIPK (salicylic-acid induced kinase) from tobacco. Studies with pharmacological inhibitors suggested that the MAP kinases are located in an independent signalling pathway from the Avr9/Cf-9-dependent synthesis of AOS. SIPK and WIPK were involved in pathogen-related elicitation processes as well as in abiotic stress responses. This indicates that the plant defence is triggered via a signalling network that shares components with pathways originating from abiotic environmental stress stimuli.

Regulation of pathogenic development in the corn smut fungus Ustilago maydis.

Abstract In Ustilago maydis, the b mating type locus constitutes the central regulatory domain for pathogenic development. The b locus encodes two homeodomain proteins that are thought to function as transcriptional regulators. Here we describe our current view of the different regulatory pathways in which the b locus is involved. We emphasize on the development of tools for the isolation of genes that are directly regulated by b homeodomain proteins.

Rapid Avr9- and Cf-9 -dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses.

The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.

Single-chain fusions of two unrelated homeodomain proteins trigger pathogenicity in Ustilago maydis.

Pathogenic and sexual development of the fungus Ustilago maydis, the causal agent of corn smut disease, is regulated by heterodimerization of two unrelated homeodomain proteins bE and bW, both encoded by the multi-allelic b mating-type locus. This complex can only be formed if the two proteins are derived from different alleles. The heterodimer is believed to function as a transcriptional regulator that binds to target sites upstream of developmentally regulated genes. We have synthesized a translational fusion in which bE is tethered to bW by a designed flexible kink region. U. maydis strains expressing this synthetic b-fusion become pathogenic for corn illustrating that the single-chain fusion substitutes for the active bE/bW heterodimer. Synthetic b-fusions in which bE and bW originate from the same allele as well as fusions deleted for the dimerization domains were shown to be active while both homeodomains were required for function. Such active fusion proteins are expected to be instrumental in the identification of pathogenicity genes.

Control of mating and development in Ustilago maydis.

In the fungus Ustilago maydis, the ability to distinguish between partners that are of the same or of different mating type is controlled by two mating-type loci. One locus allows extracellular recognition though a pheromone-based system. After cell fusion, the other mating-type locus, which exists in multiple alleles, determines intracellular recognition. Each allele encodes a pair of homeodomain proteins that are active only in pairwise combinations in which the two partners originate from different alleles of the locus. Recent discoveries suggest that the underlying molecular recognition mechanism is the ability to form heterodimers. Whereas the proteins in all different allelic combinations interact, it is a specific feature of proteins from the same allele not to interact. This suggests the existence of a code for protein-protein recognition.

Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis.

In the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides. Only combinations of bE and bW of different allelic origin are active. We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon. By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles. Dimerization involves the N-terminal variable domains. Different point mutants of bE2 were isolated that function in combination with bW2. The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system. Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins. Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization.

Penicillin-binding protein 7/8 of Escherichia coli is a DD-endopeptidase.

Penicillin-binding protein 7 (PBP7) and its proteolytic degradation product PBP8 are shown to be soluble proteins, which can be set free from whole cells of Escherichia coli by an osmotic shock. The proteins are loosely associated with the membranes and are totally released into the supernatant in the presence of 1 M NaCl. Partial purification of PBP8 was accomplished by hydroxyapatite, heparin-Sepharose and MonoS chromatography. Murein meso-diaminopimelate-D-alanine DD-endopeptidase activity was demonstrated for both PBP7 and PBP8, which specifically hydrolyse the DD-diaminopimelate-alanine bonds in high-molecular-mass murein sacculi but fail to cleave these bonds in isolated dimeric muropeptides. The enzyme is inhibited by the 'penem' beta-lactam antibiotic CGP31608 at a concentration of 0.25 micrograms/ml by 50%. Thus besides PBP4 and the mepA gene product, a third endopeptidase exists in E. coli.

Specific interaction of penicillin-binding proteins 3 and 7/8 with soluble lytic transglycosylase in Escherichia coli.

Soluble lytic transglycosylase 70 (Slt70), one of the better characterized murein hydrolases of Escherichia coli, was covalently bound to CNBr-activated Sepharose and used as a specific tool to screen for proteins showing an affinity for Slt70. Several proteins were specifically enriched by Slt-Sepharose affinity chromatography. Two of them were identified as the penicillin-binding proteins (PBP)3 and PBP7/8. Thus, the bifunctional synthase PBP3, specifically involved in septum formation, and PBP7/8, recently shown to be a DD-endopeptidase, bind to Slt70 in vitro. In addition, PBP7/8 was found not only to stabilize but also to stimulate the enzymatic activity of Slt70 by a protein-protein interaction. It is concluded that Slt70, PBP7/8, and PBP3 may form a multienzyme complex in vivo.

Characterization of three different lytic transglycosylases in Escherichia coli.

Two lytic transglycosylases, releasing 1,6-anhydromuropeptides from murein sacculi are present in a mutant deleted for the soluble lytic transglycosylase 70 (Slt70). Thus, there are three different lytic transglycosylases in Escherichia coli. One of the remaining enzymes is soluble and one is a membrane protein that can be solubilized by 2% Triton X-100 in 0.5 M NaCl. Both enzymes are exo-muramidases. Only the membrane enzyme, but not the soluble ones, hydrolyses isolated murein glycan strands (poly-GlcNAc-MurNAc). While the soluble enzymes are inhibited by the muropeptide TetraTriLysArg(dianhydro), the membrane enzyme is not. The antibiotic bulgecin that inhibits Slt70 does not inhibit the lytic transglycosylases present in the slt70 deletion mutant.

Murein chemistry of cell division in Escherichia coli.

The length distribution of the glycan strands of murein has been analysed with a novel method in filamentous and spherical cells of Escherichia coli, as well as during septum formation and cell separation. A shift to the longer glycan strands was observed in the murein of furazlocillin-induced filaments. In contrast, shorter glycan strands were increased in the murein of mecillinam-induced spherical cells. During septum formation in a chain-forming envA mutant that is defective in the splitting process of the septum, a shift to the shorter glycan strands was detected that was not seen in wild type E. coli cells. It is concluded that septum-specific murein structures of rather short glycan strands are released during splitting of the septum. This intermediate material remains present in the septum of the envA mutant. The splitting process of the septum was investigated by analysing the murein during penicillin-induced bacteriolysis, which is known to take place by strictly localized murein degradation in the equatorial zone of the cell. No changes in the length distribution of the glycan strands could be detected during penicillin-induced lysis, with the exception of an increase in disaccharides, the shortest glycan strands possible. This is explained by the action of exo-muramidases progressively digesting glycan strands, leaving disaccharide units covalently linked to the remaining murein at the sites of murein cross-linkage. It is proposed that this "zipper-like" mechanism represents the normal cutting process of the septum during cell separation.