RESUMEN
Infection of plants, particularly by a necrotizing pathogen, usually induces a long-lasting, broad-based, systemic resistance to secondary pathogen attack. Many studies implicate salicylic acid as an essential signal in the development of such systemic acquired resistance in several plant species. Salicylic acid appears to mediate plant defence by binding to and inhibiting catalase, thus increasing the concentration of H(2)O(2) and other active oxygen species. Active oxygen species may then act as second messengers that induce plant defence gene expression, analogous to their activation of gene expression in mammalian cells.
RESUMEN
A complementary DNA encoding a salicylic acid (SA)-binding protein has been cloned. Its properties suggest involvement in SA-mediated induction of systemic acquired resistance (SAR) in plants. The sequence of the protein is similar to that of catalases and the protein exhibits catalase activity. Salicylic acid specifically inhibited the catalase activity in vitro and induced an increase in H2O2 concentrations in vivo. H2O2 or compounds, such as SA, that inhibit catalases or enhance the generation of H2O2, induced expression of defense-related genes associated with SAR. Thus, the action of SA in SAR is likely mediated by elevated amounts of H2O2.
Asunto(s)
Proteínas Portadoras/metabolismo , Catalasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Nicotiana/metabolismo , Enfermedades de las Plantas , Proteínas de Plantas , Plantas Tóxicas , Salicilatos/farmacología , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Catalasa/antagonistas & inhibidores , Genes de Plantas , Hidroxibenzoatos/farmacología , Datos de Secuencia Molecular , Oxidación-Reducción , Salicilatos/metabolismo , Ácido Salicílico , Nicotiana/genéticaRESUMEN
Some cultivars of tobacco are resistant to tobacco mosaic virus (TMV) and synthesize pathogenesis-related (PR) proteins upon infection. In a search for the signal or signals that induce resistance or PR genes, it was found that the endogenous salicylic acid levels in resistant, but not susceptible, cultivars increased at least 20-fold in infected leaves and 5-fold in uninfected leaves after TMV inoculation. Induction of PRl genes paralleled the rise in salicylic acid levels. Since earlier work has demonstrated that treatment with exogenous salicylic acid induces PR genes and resistance, these findings suggest that salicylic acid functions as the natural transduction signal.
RESUMEN
Increases in endogenous salicylic acid (SA) levels and induction of several families of pathogenesis-related genes (PR-1 through PR-5) occur during the resistance response of tobacco to tobacco mosaic virus infection. We found that at temperatures that prevent the induction of PR genes and resistance, the increases in SA levels were eliminated. The addition of exogenous SA to infected plants at these temperatures was sufficient to induce the PR genes but not the hypersensitive response. However, when the resistance response was restored by shifting infected plants to permissive temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. The majority of the conjugates appeared to be SA glucosides. The same glucoside was formed when plants were supplied with exogenous SA. These results provide further evidence that endogenous SA signals the induction of certain defense responses and suggests additional complexity in the modulation of this signal.
RESUMEN
Immunofluorescence microscopy and in situ hybridization were used to examine the expression of genes encoding C4 photosynthetic enzymes during early leaf development in the C4 dicotyledonous grain plant amaranth. During early developmental stages, the chloroplast-encoded large subunit and nuclear-encoded small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) were expressed in both bundle sheath and mesophyll cells in a C3-type pattern. The RuBPCase proteins and mRNAs became specifically localized to bundle sheath cells in the characteristic C4-type pattern as the leaves continued to expand and develop. Changes in the localization of the RuBPCase proteins corresponded closely with changes in the localization of their mRNAs, indicating that the cell-specific expression of genes encoding RuBPCase is controlled, at least in part, at the level of transcript accumulation. Genes encoding pyruvate orthophosphate dikinase were expressed specifically in mesophyll cells at all developmental stages examined. Immunolocalization with antibodies raised against phosphoenolpyruvate carboxylase (PEPCase) showed that this enzyme is present only in leaf mesophyll cells, even though RNA sequences with homology to PEPCase gene sequences were present in both bundle sheath and mesophyll cells. These results suggest that the regulation of genes encoding PEPCase in amaranth is complex and could involve the differential expression of divergent PEPCase genes or possibly regulation at the post-transcriptional level.
RESUMEN
The adenovirus DNA-binding protein (DBP) is an abundant multifunctional protein located primarily in the nuclei of infected cells. To define sequences involved in nuclear transport of DBP, a series of point and small deletion mutants were constructed via oligonucleotide-directed mutagenesis. Two short stretches of basic amino acids located in the amino-terminal domain (amino acids 42 to 46 and 84 to 89) were identified. Their importance, however, depended on the context in which DBP was expressed. Disruption of either site prevented nuclear localization after transient expression in transfected 293 cells, implying that two nuclear localization signals are necessary for transport of this nuclear protein. In contrast, the mutant DBPs synthesized during viral infection were located either primarily in the nucleus or in the nucleus and cytoplasm, depending on the mutation and the stage of the viral infection. Thus, the nuclear localization defect could be complemented by viral infection, perhaps through the interaction of the mutant polypeptide with a virus-encoded or -induced factor(s).
Asunto(s)
Adenovirus Humanos/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Proteínas E2 de Adenovirus , Secuencia de Aminoácidos , Transporte Biológico/genética , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Transfección , Replicación Viral/fisiologíaRESUMEN
Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.
Asunto(s)
Adenovirus Humanos/genética , Empalme del ARN/fisiología , ARN Viral/genética , Secuencia de Bases , Exones/fisiología , Vectores Genéticos , Humanos , Mutación , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Virus 40 de los Simios/genética , Transactivadores/fisiología , Transfección , Proteínas ViralesRESUMEN
In monkey cells abortively infected with human adenovirus serotype 2, the synthesis of the fiber polypeptide of the virion capsid is reduced by at least a factor of 100 when compared with that in monkey cells productively infected with a host range mutant of adenovirus serotype 2 (Ad2hr400). However, the steady-state level of fiber-encoding mRNA present in abortively infected monkey cells is only reduced by a factor of 5 to 10. When mRNA isolated from abortively and productively infected monkey cells was microinjected into the cytoplasms of uninfected or abortively infected monkey cells, no differences in the efficiency of translation of the fiber messages from these two sources were observed. These results suggest that the block to synthesis of the fiber polypeptide in abortively infected monkey cells does not reside in the translational machinery of the abortively infected cells themselves but may involve compartmentalization of the fiber message within the cells or an altered processing of the fiber message which prevents correct presentation to the ribosomes.
Asunto(s)
Adenovirus Humanos/genética , Proteínas de la Cápside , Cápside/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Línea Celular , Chlorocebus aethiops , MicroinyeccionesRESUMEN
The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.
Asunto(s)
Adenovirus Humanos/genética , Transformación Celular Viral , Quimera , Proteínas de Unión al ADN/genética , Genes Virales , Línea Celular , ADN Viral/genética , Células HeLa/fisiología , Humanos , Mutación , Temperatura , Proteínas Virales/genéticaRESUMEN
The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.
Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Proteínas de Plantas/genética , Ribulosa-Bifosfato Carboxilasa/genética , Genes , Luz , Magnoliopsida/genética , Magnoliopsida/efectos de la radiación , Proteínas de Plantas/biosíntesis , Procesamiento Postranscripcional del ARN/efectos de la radiación , ARN Mensajero/análisis , Ribulosa-Bifosfato Carboxilasa/biosíntesis , Transcripción Genética/efectos de la radiaciónRESUMEN
The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation.
Asunto(s)
Regulación de la Expresión Génica , Luz , Plantas/enzimología , Biosíntesis de Proteínas , Ribulosa-Bifosfato Carboxilasa/genética , Electroforesis en Gel de Poliacrilamida , Plantas/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
The PR1 family of pathogenesis-related proteins from tobacco (Nicotiana tabacum L.) leaves is induced by a variety of pathogenic and chemical agents and is associated with resistance to tobacco mosaic virus. The majority of the PR1 proteins did not copurify with mesophyll protoplasts (the major cell type of the leaf) isolated from tobacco mosaic virus-infected N. tabacum cv. Xanthi-nc leaves. However, these isolated protoplasts were capable of synthesizing and selectively secreting the PR1 proteins. Using monoclonal antibodies for immunofluorescence microscopy, we localized these proteins to the extracellular spaces predominantly in regions adjacent to viral lesions as well as in xylem elements of infected leaves.
Asunto(s)
Plantas/microbiología , Virus del Mosaico del Tabaco/genética , Proteínas Virales/biosíntesis , Anticuerpos Monoclonales , Técnica del Anticuerpo Fluorescente , Plantas Tóxicas , Nicotiana/microbiología , Proteínas Virales/aislamiento & purificaciónRESUMEN
Molecular, genetic and biochemical studies have identified key players in the signaling pathways regulating growth and development, as well as defense responses in plants. Recently, nitric oxide (NO) - the versatile and powerful effector of animal redox-regulated signaling and immune responses - was shown to mediate plant defense responses against pathogens. Interestingly, several key components involved in NO-mediated signaling in animals also appear to be operative in plants.
Asunto(s)
Óxido Nítrico/fisiología , Fenómenos Fisiológicos de las Plantas , Transducción de Señal/fisiología , Animales , Modelos Químicos , Óxido Nítrico Sintasa/metabolismo , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
The Arabidopsis genome encodes approximately 20 different mitogen-activated protein kinases (MAPKs) that are likely to be involved in growth, development and responses to endogenous and environmental cues. Several plant MAPKs are activated by a variety of stress stimuli, including pathogen infection, wounding, temperature, drought, salinity, osmolarity, UV irradiation, ozone and reactive oxygen species. Recent gain-of-function studies show that two tobacco MAPKs induce the expression of defense genes and cause cell death. By contrast, loss-of-function studies of other MAPK pathways revealed negative regulation of disease resistance. This 'push-and-pull' regulation by different MAPK pathways might provide a more precise control of plant defense responses.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Plantas/metabolismo , Apoptosis , Proteínas Quinasas Activadas por Mitógenos/clasificación , Proteínas Quinasas Activadas por Mitógenos/genética , Estrés Oxidativo , Enfermedades de las PlantasRESUMEN
Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.
Asunto(s)
Óxido Nítrico/fisiología , Plantas/metabolismo , Transducción de Señal , Aconitato Hidratasa/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , GMP Cíclico/metabolismo , Mamíferos , Óxido Nítrico/biosíntesis , Óxido Nítrico/química , Óxido Nítrico Sintasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Salicílico/metabolismoRESUMEN
Improvements in transformation techniques and the isolation of many genes whose transcripts or protein products either have antimicrobial or insecticidal activity or are involved in the synthesis of products with such activities have provided valuable tools for engineering resistance in plants. Future exploitation of this technology should provide an environmentally friendly alternative to traditional disease and pest control measures.
Asunto(s)
Toxinas Bacterianas , Ingeniería Genética , Enfermedades de las Plantas , Plantas Modificadas Genéticamente , Antiinfecciosos , Antivirales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas , Control de Insectos , Insecticidas , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/virología , Virus de Plantas/patogenicidadRESUMEN
Salicylic acid (SA) is a key component in the signal transduction pathway(s), leading to the activation of certain defense responses in plants after pathogen attack. Previous studies have identified several proteins, including catalase and ascorbate peroxidase, through which the SA signal might act. Here we describe a new SA-binding protein. This soluble protein is present in low abundance in tobacco (Nicotiana tabacum) leaves and has an apparent molecular weight of approximately 25,000. It reversibly binds SA with an apparent dissociation constant of 90 nM, an affinity that is 150-fold higher than that between SA and catalase. The ability of most analogs of SA to compete with labeled SA for binding to this protein correlated with their ability to induce defense gene expression and enhanced resistance. Strikingly, benzothiadiazole, a recently described chemical activator that induces plant defenses and disease resistance at very low rates of application, was the strongest competitor, being much more effective than unlabeled SA. The possible role of this SA-binding protein in defense signal transduction is discussed.
RESUMEN
Recently, it has been demonstrated that the salicylic acid (SA)-binding protein (SABP) from tobacco (Nicotiana tabacum) is a SA-inhibitable catalase (Z. Chen, H. Silva, D.F. Klessig [1993] Science 262: 1883-1886). Here we report the presence of SABP and SA-inhibitable catalase activity in Arabidopsis, tomato, and cucumber. The cucumber SABP has properties similar to the tobacco SABP, including binding affinity and specificity for SA.
RESUMEN
We previously proposed that salicylic acid (SA)-sensitive catalases serve as biological targets of SA in plant defense responses. To further examine the role of SA-sensitive catalases, we have analyzed the relationship between SA levels and SA sensitivity of catalases in different rice (Oryza sativa) tissues. We show here that, whereas rice shoots contain extremely high levels of free SA, as previously reported (I. Raskin, H. Skubatz, W. Tang, B.J.D. Meeuse [1990] Ann Bot 66: 369-373; P. Silverman, M. Seskar, D. Kanter, P. Schweizer, J.-P. Metraux, I. Raskin [1995] Plant Physiol 108: 633-639), rice roots and cell-suspension cultures have very low SA levels. Catalases from different rice tissues also exhibit differences in sensitivity to SA. Catalase from rice shoots is insensitive to SA, but roots and cell-suspension cultures contain SA-sensitive catalase. The difference in SA sensitivity of catalases from these different tissues correlates with the tissue-specific expression of two catalase genes, CatA and CatB, which encode highly distinctive catalase proteins. CatA, which encodes a catalase with relatively low sequence homology to the tobacco SA-sensitive catalases, is expressed at high levels exclusively in the shoots. On the other hand, in roots and cell-suspension cultures, with northern analysis we detected expression of only the CatB gene, which encodes a catalase with higher sequence homology to tobacco catalases. The role of catalases in mediating some of the SA-induced responses is discussed in light of these results and the recently defined mechanisms of catalase inhibition by SA.
RESUMEN
Potato (Solanum tuberosum) plants contain a high basal level of salicylic acid (SA), the role of which in disease resistance is currently unclear. Here we report that, in spite of a drastic reduction in total SA levels in transgenic potato plants expressing the bacterial salicylate hydroxylase gene (nahG), there was no significant increase in disease severity when infected by Phytophthora infestans. Therefore, the high basal level of SA does not lead to constitutive resistance in healthy potato plants. However, in contrast to control plants, arachidonic acid failed to induce systematic acquired resistance (SAR) in nahG plants against P. infestans, indicating an essential role of SA in potato SAR. These results suggest that in potato the development of SAR against P. infestans may involve increased sensitivity of the plant to SA.