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 Ca 2 ؉ 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. INTRODUCTIONThe capacity of plants to stop the growth of pathogens and parasites depends on early warning of invasion, followed by the activation of defense mechanisms. Disease resistance ( R ) genes are part of the plant's surveillance system and, in so-called gene-for-gene interactions, confer resistance to pathogens that carry the corresponding avirulence ( Avr ) genes (Flor, 1971). The R gene product is generally considered as a receptor for the matching Avr protein (Staskawicz et al., 1995). Despite the isolation of an increasing number of plant R genes (see below), little is known about the signal transduction chains that follow the R/Avr recognition event. A different picture emerges from the analysis of plant responses after nonspecific elicitation with bacterial or fungal oligosaccharides, proteins, or peptides that are not part of a matching Avr / R gene pair. To date, only one receptor, a 70-kD transmembrane  -glucan elicitor binding protein from soybean, has been characterized, and the corresponding gene has been cloned (Umemoto et al., 1997). In parsley, a 91-kD transmembrane protein, which binds the fungal protein elicitor fro...
The tomato Cf-9 gene confers resistance to races of the fungal pathogen Cladosporium fulvum expressing the Avr9 gene. cDNA amplified fragment length polymorphism analysis was used to display transcripts whose expression is rapidly altered during the Avr9-and Cf-9 -mediated defense response in tobacco cell cultures. Diphenyleneiodonium was used to abolish the production of active oxygen species during gene induction. Of 30,000 fragments inspected, 290 showed altered abundance, of which 263 were induced independently of active oxygen species. cDNA clones were obtained for 13 ACRE (for Avr9/Cf-9 rapidly elicited) genes. ACRE gene induction occurred in the presence of cycloheximide. Avr9 induced ACRE gene expression in leaves. Surprisingly, ACRE genes were also rapidly but transiently induced in leaves in response to other stresses. The amino acid sequences of some ACRE proteins are homologous to sequences of known proteins such as ethylene response element binding protein transcription factors, the N resistance protein, a calcium binding protein, 13-lipoxygenase, and a RING-H2 zinc finger protein. Rapid induction of ACRE genes suggests that they play a pivotal role during plant defense responses. INTRODUCTIONDisease resistance in plants often involves recognition of invading pathogens followed by activation of a defense response. Such incompatible interactions are dependent on the presence of a resistance ( R ) gene in the host and an avirulence ( Avr ) gene in the pathogen (Flor, 1971;Keen, 1990). Many plant R genes have been identified. Their products have motifs consistent with potential roles in pathogen detection and subsequent signal transduction (Bent, 1996). However, the signal transduction activated by R gene products is still poorly understood.Cladosporium fulvum is a biotrophic fungus that causes leaf mold disease of tomato. The tomato Cf-9 gene confers resistance to C. fulvum races expressing the corresponding Avr9 gene . The Avr9 protein is secreted by the fungus and is processed to a cystine knot peptide of 28 amino acids, which can be retrieved in intercellular washing fluid (IF) from infected leaves (De Wit and Spikman, 1982;Van den Ackerveken et al., 1993). Infiltration of Cf9 tomato or transgenic Cf9 tobacco with Avr9 leads to necrosis within 24 hr.This response is faster in tobacco than it is in tomato (Hammond-Kosack et al., 1998). Cell suspension cultures derived from Cf9 tobacco plants, when challenged with Avr9, rapidly produce active oxygen species (AOS) (Piedras et al., 1998) and activate two mitogen-activated protein (MAP) kinases (Romeis et al., 1999) and a calcium-dependent protein kinase (Romeis et al., 2000).The mode of action of the Cf-9 protein is not known. Changes in gene expression are likely to be important for activation of defense mechanisms, and transcriptional changes have been reported in several plant-pathogen interaction systems (Rushton and Somssich, 1998). The nonhost resistance responses of parsley and tobacco cells to elicitors from cultures of Phytophthora spp have be...
The tomato Cf-9 gene confers resistance to races of Cla-dosporium fulvum expressing the corresponding avirulence gene Avr9. The availability of transgenic tobacco lines carrying Cf-9, and the well-characterized 28 amino acid Avr9 elicitor, make this an excellent system to study resistance gene function. In this paper we establish tobacco suspension cultures derived from transgenic tobacco plants containing Cf-9, as well as from plants without Cf-9. Cultures derived from Cf9 tobacco produce active oxygen species (AOS) within 5 min of treatment with pure or synthetic Avr9. This enabled us to perform biochemical and pharmacological analysis in cell culture of the very earliest events in resistance gene function. In addition to AOS production, an increase in oxygen uptake was detected in the Avr9-treated Cf9 cells. Both phenomena were inhibited by low concentrations of diphenyleneiodonium (DPI). Additional pharmacological inhibitor studies suggest that uptake of calcium, activation of protein kinases, and probably phospholipase A2 (PLA2) activity are intermediates in the Cf-9- and Avr9-dependent signaling pathway that leads to AOS production. Interestingly, those defense responses did not result in plant cell death.
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 Ca 2 ؉ 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. INTRODUCTIONThe capacity of plants to stop the growth of pathogens and parasites depends on early warning of invasion, followed by the activation of defense mechanisms. Disease resistance ( R ) genes are part of the plant's surveillance system and, in so-called gene-for-gene interactions, confer resistance to pathogens that carry the corresponding avirulence ( Avr ) genes (Flor, 1971). The R gene product is generally considered as a receptor for the matching Avr protein (Staskawicz et al., 1995). Despite the isolation of an increasing number of plant R genes (see below), little is known about the signal transduction chains that follow the R/Avr recognition event. A different picture emerges from the analysis of plant responses after nonspecific elicitation with bacterial or fungal oligosaccharides, proteins, or peptides that are not part of a matching Avr / R gene pair. To date, only one receptor, a 70-kD transmembrane  -glucan elicitor binding protein from soybean, has been characterized, and the corresponding gene has been cloned (Umemoto et al., 1997). In parsley, a 91-kD transmembrane protein, which binds the fungal protein elicitor fro...
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