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PubMed2003

Role of reactive oxygen species and antioxidants in plant disease resistance.

Membrane damage caused by the non-specific fungal toxin fusaric acid was less on pretreated than on control leaves when tobacco leaves were pretreated with anti-senescence plant hormones, such as kinetin, benzyladenine or the anti-ozonant N-[2-(2-oxo-1-imidazolidinyl)]ethyl-N'-phenylurea. Similarly, the necrosis caused by mercuric chloride was reduced by the above anti-senescence agents. In addition, in in vitro tests, leaves from selected paraquat-tolerant tobacco plants were less sensitive to Alternaria alternata (Fr) Keissler infection than those of the control paraquat-sensitive tobacco leaves. Paraquat-tolerant Conyza canadensis (L) Cronq weeds naturally selected in vineyards in Hungary showed similar inhibition of senescence to paraquat-tolerant tobacco, expressed as more green leaves and slower development. In accordance with this, the paraquat-tolerant Conyza leaves remained almost symptomless, while paraquat-sensitive plants showed severe symptoms after infection with Botrytis cinerea Pers. Oxidative burst (accumulation of hydrogen peroxide) was attenuated in TMV-infected leaves of Xanthi-nc tobacco as a result of treatment with salicylic acid or in leaves where systemic acquired resistance (SAR) had been induced by a previous TMV infection. Accordingly, superoxide dismutase (SOD) activity was higher in Xanthi tobacco leaves with SAR than without SAR. However, in NahG tobacco, in which SAR cannot develop, there was no augmentation of SOD activity. All the above data support the significance of delayed senescence and antioxidants in the resistance of plants to biotic and abiotic necrosis-inducing agents.

Adaptation, Physiological↗
PubMed2002

Electrophoretic mobility of rigid rodlike particles in dilute aqueous and glycerol suspensions: comparison between theory and experiment.

The electrophoretic mobilities of rigid rodlike tobacco mosaic virus (TMV) particles solvated in water and in 80% glycerol/20% water solutions with a wide range of electrical double-layer thicknesses have been measured by Deggelmann et al. (Deggelmann, M., Graf, C., Hagenbuchle, M., Hoss, U., Johner, C., Kramer, H., Martin, C., and Weber, R., J. Phys. Chem.98, 364-368 (1994)) and the present authors, respectively. The measured mobilities have a dependence on the electrical double-layer thickness much weaker than that predicted by theories for the mobility of cylinders of infinite length. To interpret the experiments, we develop an approximate theory for the mobility of finite aspect ratio particles with arbitrary ratios of the double-layer thickness to the rod length and diameter. This uniformly valid approximation interpolates between previous theories for infinite rod length and for infinitesimal rod thickness. A comparison of the uniformly valid theory with the experimentally measured mobilities indicates that TMV has a negative charge of 1000e in water and 400e in the glycerol solution.

Journal Article↗
PubMed1994

Structure-function relationship between tobacco mosaic virus coat protein and hypersensitivity in Nicotiana sylvestris.

Alterations in the structure of the tobacco mosaic virus (TMV) coat protein affect the elicitation of the N' gene hypersensitive response (HR) in Nicotiana sylvestris. To investigate this structure-function relationship, amino acid substitutions with predicted structural effects were created throughout the known structure of the TMV coat protein. Substitutions that resulted in the elicitation of the HR resided within and would predictably interfere with interface regions located between adjacent subunits in ordered aggregates of coat protein. Substitutions that did not result in the elicitation of the HR were either conservative or located outside these interface regions. In vitro analysis of coat protein aggregates demonstrated HR-eliciting coat proteins to have reduced aggregate stability in comparison with non-HR-eliciting coat proteins and a correlation existed between the strength of the elicited HR and the ability of a substitution to interfere with ordered aggregate formation. This finding corresponded with the predicted structural effects of HR-eliciting substitutions. Radical substitutions that predictably disrupted coat protein tertiary structure were found to prevent HR elicitation. These findings demonstrate that structural alterations that affect the stability of coat protein quaternary structure but not tertiary structure lead to host cell recognition and HR elicitation. A model for HR elicitation is proposed, in which disassembly of coat protein aggregates exposes a host "receptor" binding site.

Amino Acids↗
PubMed2001

Automated identification of filaments in cryoelectron microscopy images.

Since the foundation for the three-dimensional image reconstruction of helical objects from electron micrographs was laid more than 30 years ago, there have been sustained developments in specimen preparation, data acquisition, image analysis, and interpretation of results. However, the boxing of filaments in large numbers of images--one of the critical steps toward the reconstruction at high resolution--is still constrained by manual processing even though interactive interfaces have been built to aid the tedious and sometimes inaccurate boxing process. This article describes an accurate approach for automated detection of filamentous structures in low-contrast images acquired in defocus pairs using cryoelectron microscopy. The performance of the approach has been evaluated across various magnifications and at a series of defocus values using tobacco mosaic virus (TMV) preserved in vitreous ice as a test specimen. By integrating the proposed approach into our automated data acquisition and reconstruction system, we are now able to generate a three-dimensional map of TMV to approximately 10-A resolution within 24 h of inserting the specimen grid into the microscope.

Algorithms↗
PubMed2000

A hypercycle theory of proliferation of viruses and resistance to the viruses of transgenic plant.

A set of dynamical equations for the proliferation of two typical viruses TMV and PVY has been derived from the reaction equations describing their replication, assembly and translation. These equations can be seen as the generalization of hypercycle theory to the system. The quantitative explanation on the phenomena of proliferation of plant virus and the mechanism of resistance to the disease of transgenic plant is offered. The phenomenon of specific cessation of minus-strand RNA accumulation in the proliferation of TMV, the cross-protection of plant viruses and the mechanism of resistance to viruses of transgenic plant are discussed based on the computer simulation of the proliferation of viruses and the prediction of the secondary structure of the genomic RNA.

Computer Simulation↗
PubMed1995

A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine: airway epithelial function and mucus changes in dog.

A newly synthesized mucolytic agent, N-acetylcysteine L-lysinate (Nacystelyn) was studied. Tracheal mucus velocity (TMV), transepithelial potential difference (PD), rheological properties, and ion content of collected airway secretions were evaluated in six healthy mongrel dogs after placebo, Nacystelyn (NAL) and acetylcysteine (NAC) metered dose inhaler (MDI) aerosols. Although TMV was increased and viscoelasticity decreased after both treatments, the treatment effect with NAL was significantly greater. Furthermore, NAL increased the negative PD and CI- content of secretions in the trachea, an effect not observed after NAC. Both compounds increased ciliary beat frequency (CBF) on the frog palate at a concentration range similar to that approximated in dog airways. The increased mucociliary clearance could be partially explained by favourable rheological changes combined with stimulation of CBF. Since both compounds break disulfide bonds in mucus polymers, the greater change in mucus rheology and clearance rate after NAL, without change in water content, could be explained by the increase in CI- content. Nacystelyn appears to combine different modes of action which synergistically cause an increase in the clearance rate of airway secretions.

Acetylcysteine↗
PubMed1999

Effects of dextran on tracheal mucociliary velocity in dogs in vivo.

We have previously shown that dextran (molecular weight 4kDa) is a potential mucolytic agent, reducing the viscoelasticity and spinnability of cystic fibrosis (CF) sputum and improving its mucociliary clearability during in vitro testing. We wished to see whether low molecular weight (LMW) dextran had similar effects on mucus rheology when administered by aerosol to living dogs, and whether the administration of dextran increased the rate of mucociliary clearance. Healthy mongrel dogs were anesthetized with pentobarbital and intubated. After a 30-min Ringer aerosol delivery during spontaneous breathing, tracheal mucociliary velocity (TMV by charcoal marker particle transport) was measured under bronchoscopic control, and mucus for viscoelasticity analysis (magnetic rheometer) was collected by the endotracheal tube method. Then LMW dextran in Ringer vehicle was delivered by aerosol via the endotracheal tube, followed by the same procedures. We performed eight experiments in eight dogs, involving 30 min administrations of dextran aerosol; all dogs received inhalations of 20 mg/ml, 65 mg/ml, and 200 mg/ml dextran. Compared with Ringer control, TMV increased to 145% of control (P=0.0417) at 65 mg/ml dextran. Mucus viscoelasticity (G*) significantly decreased to 19% of control (P=0.0426) at 65 mg/ml. This in vivo study supports our previous in vitro testing, that LMW dextran decreases the mucus viscoelasticity and increases the rate of mucociliary clearance. We estimate the dosage received by aerosol at 65 mg/ml to be within the effective concentration range studied in vitro, i.e. 10-15 mg/ml final concentration. The results are consistent with the proposed mechanism that the saccharide moieties in LMW dextran compete for hydrogen bonding sites with other mucous glycoproteins. These new hydrogen bonds are structurally and rheologically ineffective, thus reducing the overall cross-link density, and making the mucus more easily cleared by ciliary and cough mechanisms.

Administration, Inhalation↗
PubMed1993

Low level of genetic drift in foreign sequences replicating in an RNA virus in plants.

The accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) RNA virus, tobacco mosaic tobamovirus (TMV). Neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase II) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. Four hybrid virus populations, two of each foreign sequence, were taken through 9-10 passages in whole plants of Nicotiana benthamiana. Sequences were sampled from these populations by conversion to cDNA, amplification by the polymerase chain reaction, and sequencing resulting bacterial clones. The background mutation rate contributed by the enzymes of this assay system allowed viral mutation rates greater than 10(-4) mutations per base per passage to be measured. Surprisingly, all native and foreign genes accumulated mutations at a very low rate, lower than could be detected by the assay procedure. This low mutation accumulation rate of < or = 10(-4) mutations per base per passage may be due to replicase fidelity or populational "bottlenecking." Sequence drift should not be a practical limitation to most uses of TMV as a vector, although deletion phenomena observed in this study may present difficulties.

Base Sequence↗
PubMed1995

Kinetic and functional mapping of viral epitopes using biosensor technology.

Some monoclonal antibodies (Mabs) that react with the extremity of the tobacco mosaic virus (TMV) particle containing the 5' end of the RNA are able to block the disassembly of TMV by ribosomes while others are totally devoid of such activity. No correlation could be established between the binding kinetics and affinity of the Mabs and their inhibitory capacity. An epitope map of the Mab binding sites was constructed on the basis of kinetic two-site binding assays with the viral monomeric protein (TMVP) performed using biosensor technology (BlAcore). Mabs possessing inhibitory activity were found to bind to the part of the TMVP surface closest to the central axis in the polymerized particle. As this part of the subunit is known to interact with the viral RNA, it seems that inhibitory Mabs act by sterically preventing the interaction between virus and ribosomes. This study illustrates the advantages of the biosensor technology for locating conformational epitopes in viral proteins.

Antibodies, Monoclonal↗
PubMed1995

Identification of the cauliflower mosaic virus movement protein RNA-binding domain.

The in vitro RNA-binding activity of the movement protein (P1) of cauliflower mosaic virus was studied after its expression in Escherichia coli, purification, and uv-crosslinking to a radioactive probe. It was found that insoluble P1 aggregates were involved in RNA-binding activity. A series of deletion mutants were used to identify a domain within P1 required for binding activity. The RNA-binding domain is located between amino acids 120 and 197 and includes the region of homology between P1 and the movement protein (P30) of tobacco mosaic virus (TMV). The homologous region corresponds to part of RNA-binding domain "A" in TMV P30, but unlike domain A, the P1 domain is able to bind RNA out of the context of the complete protein. The P1 RNA-binding domain shows some structural similarity with RNA-binding domains of other proteins. The conservation of this domain in the caulimo- and badnaviruses provides support for the view that this activity has biological relevance.

Amino Acid Sequence↗
PubMed1995

A single nucleotide change in the coat protein gene of tobacco mosaic virus is involved in the induction of severe chlorosis.

YSI/1 is a mutant of the common strain (U1) of tobacco mosaic virus (TMV) which induces a severe yellow mosaic in Nicotiana tabacum instead of the light green/dark green mosaic induced by its parental U1 virus. Although there was less coat protein (CP) in whole leaf extracts of YSI/1-infected leaves than in U1-infected leaves, severalfold more CP was found in the chloroplasts, most of which was associated with the thylakoids. Sequencing the CP genes of both viruses showed the presence of nucleotide differences at viral RNA positions 5770 and 6127, both of which result in amino acid replacements; YSI/1 has an Asp-->Val change at amino acid 19 and a Ser-->Phe change at amino acid 138. A common strain TMV engineered to contain the YSI/1 3' end sequences, including the CP, induced the severe yellow mosaic of the YSI/1 mutant. A chimeric virus with the change only at nucleotide 5770 (amino acid 19) in the CP induced a severe yellow mosaic, showing that this replacement is involved in the induction of chlorosis by YSI/1. A second isolate of the same chimera also induced severe yellow mosaic symptoms; sequencing showed that it had gained the change at nucleotide 6127. However, a chimeric virus with the change only at nucleotide 6127 (amino acid 138) in the CP was unable to induce the severe yellow mosaic.

Base Sequence↗
PubMed1996

Comparison of the nucleic acid- and NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus.

The cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins into Escherichia coli, isolating the E. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4 M NaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either in E. coli or in planta from RNA 3 of CMV was tagged at its C-terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni(2+)-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified from E. coli and zucchini squash, both were able bind CMV RNA 3 in vitro.

Base Sequence↗
PubMed1997

A tobamovirus genome that contains an internal ribosome entry site functional in vitro.

Most eukaryotic mRNAs are translated by a "scanning ribosome" mechanism. We have found that unlike the type member of the genus Tobamovirus, translation of the 3'-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhov et al., 1993; 1994) occurred in vitro by an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translated in vitro: (i) "MP-CP-3'NTR" transcripts contained movement protein (MP) gene, CP gene and the 3'-nontranslated region of crTMV RNA. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamoviruses in vivo. (ii) "deltaNPT-CP" transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) "CP-GUS" transcripts contained the first CP gene and the gene of Escherichia coli beta-glucuronidase (GUS) at the 3'-proximal position. The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRES(CP)) promoting internal initiation of translation in vitro. Dicistronic IRES(CP), containing chimeric mRNAs with the 5'-terminal stem-loop structure preventing translation of the first gene (MP, deltaNPT, or CP), expressed the CP or GUS genes despite their 3'-proximal localization. The capacity of crTMV IRES(CP) for mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation. Two mutants were used to study structural elements of IRES(CP). It was concluded that integrity of IRES(CP) was essential for internal initiation. The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs.

Base Sequence↗
PubMed1998

Deletion of internal sequences results in tobacco mosaic virus defective RNAs that accumulate to high levels without interfering with replication of the helper virus.

Deletion of certain internal sequences of the tobacco mosaic tobamovirus (TMV) genome was required to create replication-defective RNAs (dRNA) that were replicated in trans by TMV. All dRNAs that accumulated to detectable levels were missing nucleotides 3420-4902, which appeared to constitute a core region that inhibited replication in trans. Deletion of additional sequences resulted in dRNAs that varied tremendously in ability to be replicated from none to levels exceeding that of the helper viral RNA. Accumulation of dRNA negative- and positive-stranded RNAs of each dRNA paralleled those of the helper virus. Negative-stranded RNA accumulation of both helper and dRNA ceased at the same early time in the infection while synthesis of both positive-stranded RNAs continued, suggesting that both dRNAs and helper virus RNAs were synthesized from the same pool of replicase complexes. Positive- to negative-stranded RNA ratios for the dRNAs were similar to, or slightly greater than the wild-type helper virus. Full-length dRNAs were not supported in trans by a replication-competent helper virus. Even though some of the artificially constructed dRNAs accumulated to levels exceeding the level of the helper virus, none appreciably affected the replication of the helper virus, suggesting that the dRNAs are produced from "excess" replicase capacity.

DNA-Directed DNA Polymerase↗
PubMed2002

Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins.

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

Blotting, Western↗
PubMed2002

A nuclear localization signal and a membrane association domain contribute to the cellular localization of the Tobacco mosaic virus 126-kDa replicase protein.

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

Amino Acid Sequence↗
PubMed1993

Tobacco proteinase inhibitor I genes are locally, but not systemically induced by stress.

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.

Amino Acid Sequence↗
PubMed1991

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2.

The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.

Amino Acid Sequence↗