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PubMed1989

Pea early browning virus RNA1 encodes four polypeptides including a putative zinc-finger protein.

We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.

Amino Acid Sequence↗
PubMed1978

Studies on spin labeled ribonucleic acids encapsulated by viral proteins.

Spin labeled poly rA (sl-poly rA) was encapsulated by the coat proteins of two plant viruses having different morphologies: TMV, a rigid rod and CCMV, an icosahedral sphere. Electron microscopy showed that the resultant particles were morphologically similar to the parent virus from which the coat protein was obtained. Encapsulation produced progressive immobilization of the spin label. The motion of the spin label attached to TMV-sl-poly rA appears anisotropic with a correlation time about the long axis of approximately 5 x 10(-6) sec. Exogenous nuclease had no effect on the epr spectrum of this nucleo-protein complex. The epr spectrum of CCMV-sl-poly rA was isotropic with a correlation time less than 5 x 10(-7). CCMV-sl-poly rA was partially degraded by T2 ribonuclease. Theoretical calculations of correlation times for the motion of the nucleo-protein particles were similar to the experimentally derived values suggesting that the nucleo-protein particles are tightly packed with little potential for internal motion.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Interaction of tobacco mosaic virus protein with synthetic polynucleotides containing a fluorescent label: optical properties of poly(A,epsilonA) and poly(C,epsilonC) copolymers and energy migration from the tryptophan to 1,N6-ethenoadenine or 3,N4-ethenocytosine residues in RNP.

A spectrophotometric method for determination of the modification degrees and molar extinction coefficients for poly(A, epsilonA) and poly(C, epsilonC) copolymers has been developed. Dependence of some absorption and fluorescence parameters of the copolymers on the modification degree has been studied. Distribution of modified residues in copolymers differs from random and depends on modification conditions. Interaction between the TMV protein and copolymers has been investigated. The protein interacts with poly(A, epsilonA) of low or medium modification degree and displays no activity with respect to poly(epsilonA). On the contrary, introduction of epsilonC to the polynucleotide promotes complex formation between poly(C, epsilonC) and TMV protein. Analysis of the fluorescence emission and excitation spectra has revealed energy transfer from tryptophan to epsilonA or epsilon C in the RNP to occur and permits one to estimate the average distance between Trp (presumably Trp 52)o and the RNA base binding region in the virus to be 17 to 20 A.

Adenine↗
PubMed1980

Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.

Animals↗
PubMed2002

The 5'-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F.

The 5'-leader sequence (called Omega) of tobacco mosaic virus (TMV) functions as a translational enhancer in plants. A poly(CAA) region within Omega is responsible for the translation enhancement and serves as a binding site for the heat shock protein, HSP101, which is required for the translational enhancement. Genetic analysis of the HSP101-mediated enhancement of translation from Omega-containing mRNA suggested that two eukaryotic initiation factors (eIFs), i.e. eIF4G and eIF3, were necessary. In this study, the functional interaction between Omega and other RNA elements known to participate in the recruitment of eIF4G, i.e. the 5'-cap and the poly(A) tail, was examined. Omega exhibited functional overlap with the 5'-cap and the poly(A) tail but not with the native TMV 3'-UTR which contains an independent translational enhancer. Consistent with the role of HSP101 in mediating the translational function of Omega, the enhancement afforded by Omega increased following a heat shock, which elevates expression of HSP101. The use of a fractionated translation lysate revealed that of the two eIF4F proteins present in plants, eIF4F was specifically required for the activity of Omega. The data suggest that Omega is functionally similar to a 5'-cap and a poly(A) tail in that it serves to recruit eIF4F in order to enhance translation from an mRNA.

3' Untranslated Regions↗
PubMed1989

Synchrotron X-ray scattering study of chromatin condensation induced by monovalent salt: analysis of the small-angle scattering data.

Small-angle X-ray scattering experiments were carried out on rat thymus chromatin in "native" and "H1-depleted" states at various NaCl concentrations using synchrotron radiation. From the analysis of cross-sectional Guinier plots, the radius of gyration of the cross section (Rc) and the mass per unit length (Mc) of native chromatin were evaluated. In the absence of NaCl, the cross section of chromatin filament has a radius of gyration of 3.44 nm, suggesting the structure corresponding to the "10 nm" filament. With increasing NaCl concentration, the Rc value increases steeply to 6.74 nm at 5 mM NaCl and then gradually to 8.82 nm at 50 mM NaCl, whereas the Mc value, which is determined relative to that of tobacco mosaic virus (TMV), increases steadily from 1.58 nucleosomes per 10 nm in the absence of NaCl to 7.66 nucleosomes per 10 nm at 50 mM NaCl. However, since calibration with TMV tends to overestimate the Mc value, the actual Mc values may be less than those values. Above about 40 mM NaCl, aggregation of chromatin is suggested. Similar analysis of H1-depleted chromatin confirmed that H1-depleted chromatin takes a more disordered structure than native chromatin at low ionic strength and does not undergo a definite structure change upon further addition of NaCl.

Animals↗
PubMed1998

Biotic and abiotic stress-related expression of 1-aminocyclopropane-1-carboxylate oxidase gene family in Nicotiana glutinosa L.

Three full length 1-aminocyclopropane-1-carboxylate (ACC) oxidase cDNA clones (pNG-ACO1, 1,254 bp; pNG-ACO2, 1,198 bp; and pNG-ACO3, 1,053 bp) were isolated from the TMV-treated leaf cDNA library of Nicotiana glutinosa plant. They share a high degree of sequence identity (78-81%) throughout the coding regions but are divergent within the 3'-untranslated regions. The gene-specific probes were prepared using these regions to investigate the differential expression of the ACC oxidase gene family in various organs and in response to a multitude of biotic and abiotic stresses in N. glutinosa plants. All three genes were transcriptionally active displaying unique patterns of expression. Both the pNG-ACO1 and pNG-ACO3 transcripts highly accumulated during the senescence of leaves, while the pNG-ACO2 mRNA was constitutively present. In addition, the NG-ACO1 and NG-ACO3 transcripts were predominantly found in roots whereas the NG-ACO2 mRNA was mainly in stems. Upon TMV infection, both NG-ACO1 and NG-ACO3 were markedly induced, but in mock treatment which has an effect of mild wounding, only the NG-ACO3 gene was induced. Furthermore, salicylic acid and CuSO4 treatments of leaves increased the level of NG-ACO1 and NG-ACO3 transcripts, while they did not affect the NG-ACO2 gene expression. Results showed that both the NG-ACO1 and NG-ACO3 genes were highly inducible by ethylene and methyl jasmonate treatments, with NG-ACO3 being more responsive. By contrast, NG-ACO2 did not respond to these growth regulators. Thus, it appears that there are two groups of ACC oxidase transcripts expressed in leaf tissue of N. glutinosa, either stress-induced or constitutive. The possible molecular mechanism of differential regulation of ACC oxidase gene expression and its physiological significance are discussed.

Amino Acid Oxidoreductases↗
PubMed2004

Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage.

Different biotic stresses yield specific symptoms, owing to their distinct influence on a plant's physiological status. To monitor early changes in a plant's physiological status upon pathogen attack, chlorophyll fluorescence imaging (Chl-FI) and thermography, which respectively visualize photosynthetic efficiency and transpiration, were carried out in parallel for two fundamentally different plant-pathogen interactions. These non-destructive imaging techniques were able to visualize infections at an early stage, before damage appeared. Under growth-room conditions, a robotized set-up captured time series of visual, thermal and chlorophyll fluorescence images from infected regions on attached leaves. As a first symptom of the plant-virus interaction between resistant tobacco and tobacco mosaic virus (TMV), thermal imaging detected a local rise in temperature while Chl-FI monitored a co-localized increase in fluorescence intensity. Chl-FI also revealed pre-symptomatic high-intensity spots for the plant-fungus system sugar beet-Cercospora beticola. Concomitantly, spots of lower temperature were monitored with thermography, in marked contrast with our observations on TMV-infection in tobacco. Knowledge of disease signatures for different plant-pathogen interactions could allow early identification of emerging biotic stresses in crops, facilitating the containment of disease outbreaks. Presymptomatic monitoring clearly opens perspectives for quantitative screening for disease resistance, either on excised leaf pieces or attached leaves.

Ascomycota↗
PubMed2005

Defense and resistance-inducing activities in tobacco of the sulfated beta-1,3 glucan PS3 and its synergistic activities with the unsulfated molecule.

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.

Cells, Cultured↗
PubMed2005

Accumulation of the two transcripts of the N gene, conferring resistance to tobacco mosaic virus, is probably important for N gene-dependent hypersensitive cell death.

The N gene is a Toll/interleukin-1 receptor (TIR)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR)-type resistance (R) gene that generates two alternative transcripts, N(S) and N(L). N(S) encodes the full-length N protein while N(L) is predicted to encode a truncated form of the protein lacking most of the LRR region. We found that the two transcripts were accumulated at 20 degrees C, a permissive temperature, but not at 30 degrees C, a non-permissive temperature for the N gene, in tobacco mosaic virus (TMV)-inoculated leaves. When N gene-dependent cell death was triggered by transient 20 degrees C treatment for 2-6 h, considerable levels of the transcripts were accumulated just before cell death, although the levels of N(S) were always higher. The accumulation was induced by transient expression of the 50 kDa helicase domain (p50) of TMV replicase which is the Avr component of N, but not by transient expression of NtMEK2 (DD)-mediated cell death or N gene-independent hypersensitive cell death. These results suggest that the accumulation of N(S) and N(L) is associated with the function of N and, above a certain threshold, triggers N-mediated hypersensitive cell death.

Cell Death↗
PubMedDate not supplied

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals.

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.

Amino Acid Sequence↗
PubMedDate not supplied

Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.

Amino Acid Sequence↗
PubMedDate not supplied

Virus-induced gene expression for enzymes of ethylene biosynthesis in hypersensitively reacting tobacco.

A full-length cDNA clone (cEFE-26) encoding ethylene-forming enzyme (EFE) was isolated from a cDNA library, prepared from leaves of tobacco mosaic virus (TMV)-infected tobacco cultivar Samsun NN. The cDNA clone encodes a protein with 90% amino acid sequence similarity to established EFEs of tomato and other plants. By using cEFE-26 cDNA and the insert from cDNA clone pACC13 (B. A. Bailey, A. Avni, N. Li, A. K. Mattoo, and J. D. Anderson, Plant Physiol. 100:1615-1616, 1992) encoding tobacco 1-aminocyclopropane-1-carboxylic acid synthase as probes, it was established that tobacco contains small gene families for these proteins. Furthermore, RNA blot analyses indicated that transcript levels in leaves for the two ethylene pathway genes were elevated after infection with TMV. The results are discussed in relation to a possible signalling role of ethylene in induced resistance and gene expression for pathogenesis-related proteins.

Amino Acid Sequence↗
PubMed2002

Potato spindle tuber viroid strains of different pathogenicity induces and suppresses expression of common and unique genes in infected tomato.

Viroids are the smallest plant pathogens. These RNAs do not encode proteins and are not encapsidated, and yet they can replicate autonomously, move systemically, and cause diseases in infected plants. Notably, strains of a viroid with subtle differences in nucleotide sequences can cause dramatically different symptoms in infected plants. These features make viroids unique probes to investigate the role of a pathogenic RNA genome in triggering host responses. We conducted a comprehensive analysis of the differential gene expression patterns of tomato plants at various stages of infection by a mild and severe strain of Potato spindle tuber viroid (PSTVd). We also compared tomato gene expression altered by the PSTVd strains with that altered by Tobacco mosaic virus (TMV). Our analyses revealed that the two PSTVd strains altered expression of both common and unique tomato genes. These genes encode products involved in defense/stress response, cell wall structure, chloroplast function, protein metabolism, and other diverse functions. Five genes have unknown functions. Four genes are novel. The expression of some but not all of these genes was also altered by TMV infection. Our results indicate that viroids, although structurally simple, can trigger complex host responses. Further characterization of viroid-altered gene expression in a host plant should help understand viroid pathogenicity and, potentially, the mechanisms of RNA-mediated regulation of plant gene expression.

Gene Expression Regulation, Developmental↗
PubMed2003

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus.

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.

Cells, Cultured↗
PubMed2004

The premammillary hypothalamic area of the ewe: anatomical characterization of a melatonin target area mediating seasonal reproduction.

Recent evidence suggests that the ovine premammillary hypothalamic area (PMH) is an important target for the pineal hormone, melatonin, and its role in seasonal reproduction. In rodents, the PMH is a complex region consisting of several cell groups with differing neurochemical content and anatomical connections. Therefore, to obtain a better understanding of the potential neural targets for melatonin in this area of the sheep brain, we have undertaken a detailed anatomical characterization of the PMH, including its nuclear divisions and the location of neuropeptide/neurotransmitter cells within them. By combining immunocytochemistry for NeuN, a neuronal marker, with Nissl staining in anestrous, ovariectomized, estradiol-treated ewes, we identified three nuclei within the PMH: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus (PMv), and the ventral tuberomammillary nucleus (TMv). The cARC contained neurons that were immunoreactive for tyrosine hydroxylase, dynorphin, estrogen receptor alpha, cocaine- and amphetamine-regulated transcript peptide (CART), and nitric oxide synthase (NOS). The PMv was also characterized by the presence of cells that contained NOS and CART, although the size of these cells was larger than that of their corresponding phenotype in the cARC. By contrast, in the TMv, of the markers examined in the present study, only fibers immunoreactive for orexin were seen. Thus, the ovine PMH is a heterogeneous region comprised of three subdivisions, each with distinct morphological and neurochemical characteristics. This anatomical map of the PMH provides a basis for future studies to determine the functional contribution of each component to the influence of melatonin on seasonal reproduction.

Animals↗
PubMed1980

Structure and function in neuropeptides.

This report reviews some aspects of the organization of molecular information, structure-activity relations and receptor interactions of three members of a family of neuroactive and hormonal peptides, adrenocorticotrophin (ACTH), alpha-melanotrophin (alpha-MSH) and the enkephalins, as they are presently being investigated in the author's laboratory (in collaboration with H. W. Kosterlitz, D. Schulster and P. W. Schiller). It has been established that ACTH acts on two different steroidogenically responsive receptors in rat adrenocortical cells: one that stimulates steroidogenesis without cyclic AMP synthesis and one that induces cyclic AMP production. This finding is a further example of the pleiotropic action of the opiocortin gene through different mechanisms. Another recent discovery, also related to the organization of ACTH information, reveals an unexpected interaction of ACTH(1-24) with lipid bilayer membranes: the molecule binds to the membranes in a reversible fashion and penetrates them. Parts of the hormonal message are exposed on the side of the membrane opposite to that to which the hormone was added. New 'fat' amino acids like carboranylalanine and adamantylalanine, as well as t-butylglycine, have been used to establish structure-activity relations and the influence of strongly enhanced lipophilicity on the biological activity of the enkephalins. An address/potentiator function of the two C-terminal amino acids is suggested. Tobacco mosaic virus (TMV) has been used as a carrier for covalently attached peptide hormones. Attachment of large numbers of hormone molecules endows these conjugates with the properties of superpotency, superaffinity and prolonged action on the target cell. Some of these properties are illustrated by TMV-enkephalin conjugates.

Adrenocorticotropic Hormone↗
PubMed1999

Tobacco mosaic virus particle structure and the initiation of disassembly.

The structure of an intact tobacco mosaic virus (TMV) particle was determined at 2.9 A resolution using fibre diffraction methods. All residues of the coat protein and the three nucleotides of RNA that are bound to each protein subunit were visible in the electron density map. Examination of the structures of TMV, cucumber green mottle mosaic virus and ribgrass mosaic virus, and site-directed mutagenesis experiments in which carboxylate groups were changed to the corresponding amides, showed that initial stages of disassembly are driven by complex electrostatic interactions involving at least seven carboxylate side-chains and a phosphate group. The locations of these interactions can drift during evolution, allowing the viruses to evade plant defensive responses that depend on recognition of the viral coat protein surface.

Computer Simulation↗