RESUMO
Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.
Assuntos
Nicotiana/metabolismo , Oxirredutases/fisiologia , Proteínas de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , DNA Antissenso , Proteínas Fúngicas/farmacologia , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Microscopia Eletrônica de Transmissão , Oxirredutases/análise , Oxirredutases/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Espécies Reativas de Oxigênio/análise , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Nicotiana/ultraestruturaRESUMO
Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.
Assuntos
Lipídeos/química , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Cromatografia Líquida , Dicroísmo Circular , Ciclopentanos/química , Relação Dose-Resposta a Droga , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Ligantes , Metabolismo dos Lipídeos , Espectrometria de Massas , Oxilipinas , Phytophthora/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P2(1)2(1)2(1) with one molecule per asymmetric unit. The crystals diffract to a resolution of 1.6 angstroms.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Transporte/química , Proteínas de Plantas/química , Arabidopsis/imunologia , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a Ácido Graxo , Transdução de SinaisRESUMO
Elicitins and lipid-transfer proteins are small cysteine-rich lipid-binding proteins secreted by oomycetes and plant cells, respectively, that share some structural and functional properties. In spite of intensive work on their structure and diversity at the protein and genetic levels, the precise biological roles of lipid-transfer proteins remains unclear, although the most recent data suggest a role in somatic embryogenesis, in the formation of protective surface layers and in defence against pathogens. By contrast, elicitins are known elicitors of plant defence, and recent work demonstrating that elicitins and lipid-transfer proteins share the same biological receptors gives a new perspective to understand the role played by lipid binding proteins, mainly the early recognition of intruders in plants.
Assuntos
Proteínas de Algas/fisiologia , Proteínas de Transporte/fisiologia , Imunidade Inata/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Algas/química , Antígenos de Plantas , Proteínas de Transporte/química , Ergosterol/química , Proteínas Fúngicas/fisiologia , Lipídeos/fisiologia , Lisofosfatidilcolinas/química , Oomicetos/crescimento & desenvolvimento , Oomicetos/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/fisiologia , Conformação Proteica , Proteínas , Estresse MecânicoRESUMO
Elicitation of defense reactions in tobacco by cryptogein, triggered a production of active oxygen species (AOS) via the NADPH oxidase, NtrbohD, and an accumulation of beta1din, a defense induced beta-type subunit of 20S proteasome. The proteasome inhibitor, MG132, stimulated this AOS production. Tobacco cells transformed with sense constructs of beta1din showed an inhibition of the AOS production following elicitin treatment, whereas the antisense transformed cells showed a strongly enhanced AOS production. In cells transformed with sense construct of beta1din, the NtrbohD transcripts failed to be induced by cryptogein as observed in control and antisense transformed cells. Conversely, in tobacco cells transformed with antisense constructs for NtrbohD, beta1din transcripts remained at a low level after elicitation. These results constitute the first demonstration of proteasome comprising beta1din acting as a negative regulator of NtrbohD and contributes to the regulation of AOS generation during plant defense reactions.
Assuntos
NADPH Oxidases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Leupeptinas/metabolismo , Oligonucleotídeos Antissenso , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inibidores de Proteassoma , Espécies Reativas de Oxigênio/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plants have evolved efficient mechanisms to resist pathogens. The earliest defense response is the hypersensitive response (HR) considered as the main step leading to plant systemic acquired resistance (SAR) that protects the whole plant against a large spectrum of pathogens. We showed previously that elicitation of defense reactions in tobacco cells by cryptogein, a proteinaceous elicitor of plant defense reactions, leads to a rapid and differential accumulation of transcripts corresponding to genes encoding defense-induced (din) subunits of 20S proteasome: beta1din, alpha3din and alpha6din.Here, expression of these three subunits was investigated by Northern blotting and by Western blotting using specific antibodies synthesized against two peptides deduced from the beta1din, alpha3din or alpha6din encoding sequence. Kinetics of mRNA and protein accumulation in various defense models showed a simultaneous accumulation of beta1din, alpha3din and alpha6din corresponding mRNAs and proteins only in plants developing a systemic acquired resistance. Inhibition by diphenyleneiodonium of the oxidative burst induced in defense reactions blocked the expression of beta1din, alpha3din and alpha6din. Using 2D gel electrophoresis and Western blotting, we showed multiple spots for each induced subunit suggesting the possible existence of multigenic families confirmed by genomic DNA analysis. These results suggest a complex regulation of induced subunits tightly correlated with the activation of plant defense reactions. beta1din, alpha3din and alpha6din subunits could probably replace the corresponding constitutive subunits in 20S proteasome leading to "plant defense proteasomes" which could play an important role in plant defense reactions.
Assuntos
Cisteína Endopeptidases/biossíntese , Complexos Multienzimáticos/biossíntese , Nicotiana/enzimologia , Proteínas de Algas/farmacologia , Northern Blotting , Western Blotting , Cisteína Endopeptidases/fisiologia , Eletroforese em Gel Bidimensional , Indução Enzimática , Proteínas Fúngicas , Expressão Gênica , Oxigenases de Função Mista/biossíntese , Complexos Multienzimáticos/fisiologia , Peptídeo Hidrolases/biossíntese , Folhas de Planta/enzimologia , Complexo de Endopeptidases do Proteassoma , Proteínas , Nicotiana/virologia , Vírus do Mosaico do Tabaco/patogenicidadeRESUMO
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.
Assuntos
Ergosterol/farmacologia , Nicotiana/metabolismo , Estresse Oxidativo , Fosfolipases A/metabolismo , Proteína Quinase C/metabolismo , Sulfonamidas , Proteínas de Algas/farmacologia , Sobrevivência Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Proteínas Fúngicas , Isoquinolinas/farmacologia , Neomicina/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Pirrolidinonas/farmacologia , Transdução de Sinais , Fatores de Tempo , Nicotiana/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1-1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1-lipid complex were both determined by fluorescence and X-ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side-by-side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways.
Assuntos
Antígenos de Plantas/química , Proteínas de Arabidopsis/química , Arabidopsis/genética , Proteínas de Transporte/química , Proteínas de Plantas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Sequência Consenso , Cisteína/química , Dissulfetos/química , Proteínas de Ligação a Ácido Graxo , Interações Hidrofóbicas e Hidrofílicas , Ponto Isoelétrico , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutação , Peptídeos/química , Fosfolipídeos/metabolismo , Filogenia , Pichia/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/genética , Zinco/químicaRESUMO
The regulation of the system responsible for the production of reactive oxygen species (ROS) during plant-micro-organism interaction is still largely unknown. The protein NtrbohD has been recently demonstrated as the plasma membrane oxidase responsible for ROS production in elicited tobacco cells. Here, its C-terminus part was used as a bait in a two-hybrid screen in order to identify putative regulators of this system. This led to the isolation of a cDNA coding for a member of the 14-3-3 protein family. The corresponding transcript was induced after infiltration of tobacco leaves with the fungal elicitor cryptogein. Tobacco cells transformed with an antisense construct of this 14-3-3 no longer accumulated ROS, which constitutes a functional validation of the two-hybrid screen. This work provides new insights to the understanding of the regulation of ROS production in a signalling context and gives a new light to the possible role of 14-3-3 proteins in plant-micro-organisms interactions.
Assuntos
Proteínas 14-3-3/metabolismo , Nicotiana/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Sequência de Aminoácidos , DNA Complementar , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Nicotiana/citologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
The structure of sylvaticin, a 10 kDa major pythin protein excreted by the parasitic oomycete Pythium sylvaticum, has been determined. Although closely related to alpha-elicitins in its biological response, toxicity and overall structure, sylvaticin presents a number of structural features that make it an unusual member of the elicitin class. Elicitins possess a large hydrophobic cavity and the mechanism of the systemic acquired resistance induced in planta is known to proceed through lipid transport and complexation within this cavity. Unlike other elicitins, sylvaticin contains tryptophan residues, one of which points inwards towards the central cavity, thus limiting access to sterols. In the case of sylvaticin, the sterol-transport mechanism is likely to be of less importance compared with other members of the elicitin family and still remains to be fully characterized.
Assuntos
Proteínas de Algas/química , Furanos/química , Pythium/metabolismo , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X/métodos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas , Solventes/química , Esteróis/químicaRESUMO
Cadmium is suspected to exert its toxic action on cells through oxidative damage. However, the transition metal is unable to directly generate reactive oxygen species (ROS) via redox reactions with molecular oxygen in a biological environment. Here, we show that bright yellow-2 (BY-2) tobacco cells exposed to millimolar concentrations of CdCl(2) developed cell death within 2-3 h. The death process was preceded by two successive waves of ROS differing in their nature and subcellular localization. Firstly, these consisted in the transient NADPH oxidase-dependent accumulation of H(2)O(2) followed by the accumulation of O(2) (-*) in mitochondria. A third wave of ROS consisting in fatty acid hydroperoxide accumulation was concomitant with cell death. Accumulation of H(2)O(2) was preceded by an increase in cytosolic free calcium concentration originating from internal pools that was essential to activate the NADPH oxidase. The cell line gp3, impaired in NADPH oxidase activity, and that was unable to accumulate H(2)O(2) in response to Cd(2+), was nevertheless poisoned by the metal. Therefore, this first wave of ROS was not sufficient to trigger all the cadmium-dependent deleterious effects. However, we show that the accumulation of O(2) (-*) of mitochondrial origin and membrane peroxidation are key players in Cd(2+)-induced cell death.
Assuntos
Cádmio/farmacologia , Nicotiana/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sequência de Bases , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citoplasma/metabolismo , Primers do DNA , Homeostase , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , NADPH Oxidases/metabolismo , Processamento Pós-Transcricional do RNA , Nicotiana/citologia , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismoRESUMO
A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in these physiological functions.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/análise , Nicotiana/metabolismo , Proteínas de Plantas/análise , Proteoma/análise , Transporte Biológico , Parede Celular/metabolismo , Células Cultivadas , Espectrometria de Massas , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Octoxinol/química , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the alpha-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development.
Assuntos
Proteínas de Transporte/química , Oxirredutases Intramoleculares/química , Lipoxigenase/química , Animais , Cromatografia Gasosa-Espectrometria de Massas , Hordeum , Hibridomas/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Químicos , Modelos Moleculares , Especificidade por Substrato , Fatores de TempoRESUMO
We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.
Assuntos
Proteínas de Algas/síntese química , Proteínas de Algas/genética , Ergosterol/análogos & derivados , Mutagênese Sítio-Dirigida , Nicotiana/microbiologia , Phytophthora/genética , Phytophthora/patogenicidade , Proteínas de Algas/metabolismo , Proteínas de Algas/toxicidade , Dicroísmo Circular , Simulação por Computador , Ergosterol/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas , Metabolismo dos Lipídeos , Micotoxinas/síntese química , Micotoxinas/genética , Micotoxinas/toxicidade , NADPH Oxidases/biossíntese , Fenilalanina Amônia-Liase/biossíntese , Doenças das Plantas/microbiologia , Proteínas de Plantas/biossíntese , Ligação Proteica/genética , Proteínas , Relação Quantitativa Estrutura-Atividade , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Nicotiana/citologia , Nicotiana/enzimologia , Nicotiana/metabolismoRESUMO
A cDNA encoding a protein, NtrbohD, located on the plasma membrane and homologue to the flavocytochrome of the neutrophil NADPH oxidase, was cloned in tobacco. The corresponding mRNA was accumulated when tobacco leaves and cells were treated with the fungal elicitor cryptogein. After elicitation with cryptogein, tobacco cells transformed with antisense constructs of NtrbohD showed the same extracellular alkalinization as the control, but no longer produced active oxygen species (AOS). This work represents the first demonstration of the function of a homologue of gp91-phox in AOS production in elicited tobacco cells.
Assuntos
Proteínas de Algas/farmacologia , Membrana Celular/química , Nicotiana/citologia , Nicotiana/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Proteínas Fúngicas , Perfilação da Expressão Gênica , Genes de Plantas/genética , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oxirredutases/química , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Nicotiana/efeitos dos fármacos , Nicotiana/genéticaRESUMO
Five cDNAs encoding Rac protein homologues to the Rho-related proteins from plants (Rop) were isolated in tobacco, and the function of one of them, Ntrac5, was studied. The Ntrac5 mRNA is repressed when tobacco leaves and cells are treated with the fungal elicitor cryptogein. Tobacco cells were transformed with sense constructs of Ntrac5 or Ntrac5V15, encoding the native GTP/GDP-bound form of this Rac protein homologue or the constitutively active mutant in its GTP-bound form, respectively. Immunological studies indicate that the corresponding protein is continuously located on the plasma membrane (PM). Both types of transformed cells show the same extra-cellular alkalinization as the control, but a high decrease in the active oxygen species (AOS) production after elicitation with cryptogein. Moreover, the regulation of NtrbohD, the oxidase involved in AOS production upon elicitation, is affected at both transcriptional and translational levels in cells overexpressing Ntrac5. Thus, Ntrac5 could be considered as a negative regulator of NtrbohD.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Nicotiana/fisiologia , Oxirredutases/genética , Proteínas de Plantas/genética , Explosão Respiratória/genética , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA Complementar/química , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Oxirredutases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nicotiana/enzimologia , Nicotiana/genética , Transformação Genética , Proteínas rac de Ligação ao GTP/classificaçãoRESUMO
Sylvaticin belongs to the elicitin family. These 10 kDa oomycetous proteins induce a hypersensitive response in plants, including necrosis and cell death, but subsequently leading to a non-specific systemic acquired resistance (SAR) against other pathogens. Sylvaticin has been crystallized using PEG 2000 MME as a precipitant agent in the presence of nickel chloride. The crystals belong to space group C2, with unit-cell parameters a = 99.29, b = 25.67, c = 67.45 A, beta = 99.66 degrees. Diffraction data were recorded to 2.1 A resolution at a synchrotron-radiation source.
Assuntos
Proteínas de Algas/química , Furanos/química , Pythium/metabolismo , Cristalografia por Raios X , Furanos/isolamento & purificação , Níquel/química , Polietilenoglicóis/química , Conformação Proteica , Proteínas , Solventes/química , SíncrotronsRESUMO
Cryptogein is a small 10 kDa elicitor produced by the phytoparasitic oomycete Phytophthora cryptogea. The protein also displays a sterol carrier activity. The native protein crystallizes in space group P4(1)22, with unit-cell parameters a = b = 46.51, c = 134.9 A (diffraction limit: 2.1 A). Its complex with cholesterol crystallizes in space group C222(1), with unit-cell parameters a = 30.96, b = 94.8, c = 65.3 A and a resolution enhanced to 1.45 A. The large inner non-specific hydrophobic cavity is able to accommodate a large variety of 3-beta-hydroxy sterols. Cryptogein probably acts as a sterol shuttle helping the pathogen to grow and complete its life cycle.