RESUMO
A novel protein elicitor (PaNie(234)) from Pythium aphanidermatum (Edson) Fitzp. was purified, microsequenced, and the corresponding cDNA was cloned. The deduced amino acid sequence contains a putative eukaryotic secretion signal with a proteinase cleavage site. The heterologously expressed elicitor protein without the secretion signal of 21 amino acids (PaNie(213)) triggered programmed cell death and de novo formation of 4-hydroxybenzoic acid in cultured cells of carrot (Daucus carota). Programmed cell death was determined using the tetrazolium assay and DNA laddering. Infiltration of PaNie(213) into the intercellular space of leaves of Arabidopsis (Columbia-0, wild type) resulted in necroses and deposition of callose on the cell walls of spongy parenchyma cells surrounding the necrotic mesophyll cells. Necroses were also formed in tobacco (Nicotiana tabacum cv Wisconsin W38, wild type) and tomato (Lycopersicon esculentum Mill.) but not in maize (Zea mays), oat (Avena sativa), and Tradescantia zebrina (Bosse), indicating that monocotyledonous plants are unable to perceive the signal. The reactions observed after treatment with the purified PaNie(213) were identical to responses measured after treatment with a crude elicitor preparation from the culture medium of P. aphanidermatum, described previously. The availability of the pure protein offers the possibility to isolate the corresponding receptor and its connection to downstream signaling-inducing defense reactions.
Assuntos
Proteínas de Algas/fisiologia , Arabidopsis/fisiologia , Daucus carota/fisiologia , Proteínas Fúngicas/fisiologia , Nicotiana/fisiologia , Pythium/fisiologia , Zea mays/fisiologia , Adaptação Fisiológica , Proteínas de Algas/genética , Proteínas de Algas/isolamento & purificação , Sequência de Aminoácidos , Apoptose/genética , Apoptose/fisiologia , Arabidopsis/microbiologia , Células Cultivadas , Cromatina/metabolismo , Clonagem Molecular , DNA Complementar , Daucus carota/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Regulação da Expressão Gênica de Plantas , Biblioteca Genômica , Glucanos/metabolismo , Dados de Sequência Molecular , Parabenos/metabolismo , Transdução de Sinais , Nicotiana/microbiologia , Zea mays/microbiologiaRESUMO
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.
Assuntos
Cladosporium/genética , Cladosporium/patogenicidade , Proteínas Fúngicas/genética , Genes Fúngicos , Genes de Plantas , Glicoproteínas de Membrana/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Animais , Células COS , Linhagem Celular , Proteínas Fúngicas/metabolismo , Solanum lycopersicum/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Genéticos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Ressonância de Plasmônio de Superfície , Nicotiana/genética , Nicotiana/metabolismo , Virulência/genéticaRESUMO
Harpin from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola (harpin(Psph)) elicits the hypersensitive response and the accumulation of pathogenesis-related gene transcripts in the nonhost plant tobacco. Here, we report the characterization of a nonproteinaceous binding site for harpin(Psph) in tobacco plasma membranes, which is assumed to mediate the activation of plant defense responses in a receptor-like manner. Binding of 125I-harpin(Psph) to tobacco microsomal membranes (dissociation constant = 425 nM) and protoplasts (dissociation constant = 380 nM) was specific, reversible, and saturable. A close correlation was found between the abilities of harpin(Psph) fragments to elicit the transcript accumulation of the pathogenesis-related tobacco gene HIN1 and to compete for binding of 125I-harpin(Psph) to its binding site. Another elicitor of the hypersensitive response and HIN1 induction in tobacco, the Phytophthora megasperma-derived beta-elicitin beta-megaspermin, failed to bind to the putative harpin(Psph) receptor. In contrast to activation by beta-megaspermin, harpin(Psph)-induced activation of the 48-kD salicylic acid-responsive mitogen-activated protein kinase (MAPK) and HIN1 transcript accumulation were independent of extracellular calcium. Moreover, use of the MAPK kinase inhibitor U0126 revealed that MAPK activity was essential for pathogenesis-related gene expression in harpin(Psph)-treated tobacco cells. Thus, a receptor-mediated MAPK-dependent signaling pathway may mediate the activation of plant defense responses induced by harpin(Psph).