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.

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.

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.

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.