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At least 109 records · Page 6Linked to original sources
PubMed2002

Molecular cloning of cDNAs encoding ribonuclease-related proteins in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection.

We earlier isolated a cDNA clone (NGR1) encoding a wound-inducible ribonuclease (RNase NW) from leaves of Nicotiana glutinosa [Kariu et al. Biosci. Biotechnol. Biochem., 62, 1144-1151 (1998)]. In this study, two distinct cDNA clones, NGR2 and NGR3, encoding proteins with a ribonuclease-related sequence in the N. glutinosa leaves, were amplified and sequenced. The nucleotide sequences of NGR2 and NGR3 consist of 1244 bp and 1069 bp, and have open reading frames encoding 277 (RNase NGR2) and 236 (RNase NGR3) amino acid residues, respectively. The deduced amino acid sequences of the putative RNases NGR2 and NGR3 showed 33% and 58% amino acid sequence identity, respectively, with that of RNase NW and 32% identity with each other. Sequence comparison showed that NGR2 is similar to RNase RNS2 (61%) from Arabidopsis thaliana, while NGR3 is related to RNase LX (84%) from tomato (Lycopersicon esculentum). RNA gel blot analysis showed that the RNase NGR2 gene is constitutively expressed to measurable levels; it is not increased by either wounding or TMV infection. In contrast, the expression of the NGR3 gene is induced after 48 h upon TMV infection.

Amino Acid Sequence↗
PubMed2001

Purification and Characterization of AAVP, a Protein Inhibitor of TMV Infection, from the Edible Fungus, Agrocybe aegerita.

A protein with activity of inhibiting TMV infection on Nicotiana glutinosa, named AAVP, was isolated and purified from the fruiting bodies of edible fungus Agrocybe aegerita by precipitation of 40%--80% saturation of (NH(4))(2)SO(4) followed by DEAE-Fast Flow and S-200 column chromatography. The protein was proved to be homogeneous by SDS-PAGE and IEF. Analysis of the composition of amino acids showed that this protein contained no cysteine, poor in methionine and phenylalanine and rich in acidic and hydroxyl amino acids. The inhibition of TMV was 84.32% when the concentration of AAVP was 200 mg/L. N-terminus of this protein was blocked by pyroglutamyl, and the N-terminal sequence was QGVNIYNIVAGA. On potato-sucrose-agar medium, the purified protein did not inhibit the growth of three plant pathogen fungi, Trichoderma viride, Colletotrichum musae and Fusarium oxysporum.

Journal Article↗
PubMed1998

[Ultrastructural differences of RMV and TMV infected Nicotiana tabacum mesophyll cells for distinguishing virus strains].

Nine strains of ribgrass mosaic virus (RMV) and tobacco mosaic virus(TMV) from cruciferous and solanaceous plants in different geographic regions of China were used to inoculate 4-leave-old plants of Nicotiana tobacum for observation of ultrastructually pathological changes due to infection of different strains. The samples of the mesophyll cells of the plants 15 d after inoculation were prepared in a routine and examined using transmission electronic microscopy. Characteristic virion arrangement, inclusion forms, and cell structural aberrations were found different among the various strains. The virions were accumulated in cluster, arranged in ring scattered, irregularly congregated with membrane, and aggregated in angle layer or parallel layer in cytoplasm and vacuoles whereas they were not found in organelles including nuclei, chloroplasts, and mitochondria. The inclusions with bacilliform, club-shaped or granulous X-tubule varied in number and form among the viral strains investigated. The abnormality of cell wall including thickening, collapsing and distortions were also observed among the cells infected by the strains. Other distinguishable changes attributed to the strains included the number, size, shape, and clumping pattern of chloroplasts, and of starch grains and osmiphilic globules chroloplasts of mesophyll cells infected only by two of the 9 strains examined. Additionally, the increased numbers of mitochondria and endoplasmic reticulum were observed in the cells infected by some of the strains. The results indicate that the ultrastructural changes due to viral infection may be useful for distinguishing various RMV and TMV strains.

Microscopy, Electron↗
PubMed1976

Separation studies of TMV replicase.

By means of affinity chromatography RNA-agarose a partial purification of a soluble TMV replicase is obtained in addition to a poly(U) polymerase when extracts of fresh, systemically TMV-infected tobacco leaves are used. An active replicase fraction always shows a certain amount of poly(U) polymerase. The replicase is inactivated by electrophoresis and the enzyme shows a harmful adsorption to certain supporting materials. When a purified replicase fraction, or an extract from frozen infected leaves, was gel filtrated on Sephadex G-75 in 50 mM Tris-HC1 pH 7.2, three peaks of replicase activity could be detected. Peak I was found in the void volume, peak II was elute with about 1.8 times the void volume and peak III with about 2.6 times the void volume. The activities of peaks II and III had very short half-times. Peak II was not obtained with extracts of frozen healthy leaves.

Chromatography, Affinity↗
PubMedDate not supplied

[TMV-infection localization and development of induced virus resistance in Nicotiana sanderae Hort., Datura stramonium L. and Datura metel].

The localization of virus infection and development of local and systemic induced resistance in plants Nicotiana sanderae Hort., Datura stramonium L. and D. metel L. infected by TMV have been studied. It was shown that acquired resistance to the second infection is induced in hypersensitive plants infected by TMV independent of localization mechanism. No definite correlation exists between these defense reactions.

Datura↗
PubMed1984

UAG readthrough during TMV RNA translation: isolation and sequence of two tRNAs with suppressor activity from tobacco plants.

The hypothetical replicase or replicase subunit cistron in the 5'-proximal part of tobacco mosaic virus (TMV) RNA yields a major 126-K protein and a minor 183-K ;readthrough' protein in vivo and in vitro. Two natural suppressor tRNAs were purified from uninfected tobacco plants on the basis of their ability to promote readthrough over the corresponding UAG termination codon in vitro. In a reticulocyte lysate the yield of 183-K readthrough protein increases from 10% in the absence of added tobacco plant tRNA up to 35% in the case of pure tRNA added. Their amino acid acceptance and anticodon sequence (GpsiA) identifies the two natural suppressor tRNAs as the two normal major cytoplasmic tyrosine-specific tRNAs. tRNA(1) has an A:U pair at the base of the TpsiC stem and an unmodified G(10), whereas tRNA(2) contains a G:C pair in the corresponding location and mG in position 10. This is the first case that, in a higher eukaryote, the complete structure is known of both the natural suppressor tRNAs and the corresponding viral RNA on which they exert their function. The corresponding codon-anticodon interaction, which is not in accordance with the wobble hypothesis, and the possible biological significance of the readthrough phenomenon is discussed.

Journal Article↗
PubMed1984

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts.

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAs, both with GPsiA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, 85% of the tRNA from wheat germ contains a QPsiA anticodon and 15% has a GPsiA anticodon. Otherwise the sequences of tRNAs from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNA(GPsiA) and not at all by tRNA(QPsiA). The tRNAs from wheat leaves were also sequenced. This revealed that adult wheat contains tRNA(GPsiA) only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.

Journal Article↗
PubMed1998

Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors.

The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.

Arachis↗
PubMed1981

Nucleotide sequence of a cloned cDNA copy of TMV (cowpea strain) RNA, including the assembly origin, the coat protein cistron, and the 3' non-coding region.

The cloned cDNA derived from the 3' end of cowpea strain (Cc) RNA of tobacco mosaic virus (TMV) has been sequenced. Substantial sequence information of 1,060 nucleotides from the 3' end of the RNA reveals some interesting features: (1) the coat protein cistron corresponds to residues 210-701 from the 3' end. Some errors in the amino acid sequence previously reported have been corrected and the revised total length of the coat protein is 162 amino acid residues. The capping site of the coat protein mRNA is at residue 711 from the 3' end of genome RNA. (2) The assembly origin of reconstitution is positioned within the coat protein cistron at residue 369-461 which can be formed into a highly base-paired hairpin loop structure. The sequence, GAXGUUG, in the loop region and a triplet-repeated purine base tract surrounding the loop are found. These structural features are common to assembly origins of both Cc and vulgare strains. (3) We find the sequence highly homologous to, but distinct from, the genuine assembly origin. It will be called the pseudo-assembly origin, which is located in the corresponding region to the assembly origin of the vulgare strain, outside the coat protein cistron. There is also the sequence, GAXGUUG, in the middle of the region. (4) In the 5' flanking region of the coat protein cistron, a long reading frame, probably of 30 K protein, is found. The coding region is terminated in the coat protein cistron and thus the 30 K protein and the coat protein cistrons overlap. (5) The 3' non-coding region is 209 residues long and can be folded into a possible tRNA-like structure. Surprisingly, we find that the 3' terminal sequence of Cc RNA is not very similar to that of vulgare RNA but extensively homologous to that of turnip yellow mosaic virus (TYMV) RNA.

Amino Acid Sequence↗
PubMed1986

Subcellular localization of the 30K protein in TMV-inoculated tobacco protoplasts.

We investigated the intracellular localization of the 30K protein in TMV-inoculated tobacco protoplasts by means of pulse-labeling and pulse-chase experiments with [35S]methionine. Protoplasts were lysed with a nonionic detergent and the extracts were centrifuged to yield soluble and crude nuclear fractions. Most of the 30K protein was found in the crude nuclear fraction. The nuclear fraction was further purified by centrifugation in a step-wise Percoll gradient. Nuclei and the 30K protein were found in the same fractions. The results of pulse-chase experiments indicated that the 30K protein is synthesized in the soluble fraction and then translocated to the crude nuclear fraction. The 30K protein of Ls1, a temperature-sensitive (ts) mutant affecting the cell-to-cell viral transport function, was also found in the nuclei, even at a nonpermissive temperature. These results suggested that the 30K protein has to be localized in the nuclei to function, and that impaired translocation of the 30K protein to the nuclei is not responsible for the ts lesion of mutant Ls1.

Centrifugation, Density Gradient↗
PubMed1990

Time course of TMV 30K protein accumulation in intact leaves.

The kinetics of accumulation of the 30K and coat proteins of tobacco mosaic virus (TMV) which are expressed via subgenomic mRNAs were determined in near-synchronously infected cells of intact tobacco leaves. The 30K protein accumulated to maximal levels during the early stages of the infection as previously reported in tobacco protoplasts. However, in intact leaves, 30K protein accumulated for longer periods and attained higher concentrations than in protoplasts, remaining detectable in all later time samples. Detection of the coat protein, and also the 126K and 183K proteins, began at approximately the same time as the 30K protein (9-12 hr after initiation of replication), but their accumulation continued throughout the 72-hr sampling period. Most of the 30K protein accumulated before 24 hr, whereas most of the coat protein accumulated after 24 hr. This confirms that these two genes are regulated differently, both temporally and quantitatively.

Blotting, Western↗
PubMed1990

Study of TMV assembly with heterologous RNA containing the origin-of-assembly sequence.

The assembly of tobacco mosaic virus (TMV) is initiated by a specific reaction between a capsid protein oligomer and an origin-of-assembly region (OAS) located 900 nucleotides from the 3' terminus of virion RNA. Packaging is then completed by rod elongation both in the 5' and 3' directions. The temporal order of the direction of elongation and the characteristics of the reaction were studied by analysis of the in vitro assembly reaction between strain U1 protein oligomers and transcripts containing a strain U1 OAS embedded at different positions in heterologous RNA. The results confirm that elongation in the 5' direction starts very soon after the initiation reaction and is completed rapidly, within minutes. Packaging in the 3' direction is slower and does not appear to commence until 5' rod formation is complete. The reaction of strain U2 protein with the strain U1 OAS initiates rapidly, but elongation occurs only in the 5' direction; 3' packaging does not occur except when the OAS is at or near the 5' terminus, in which case elongation in the 3' direction initiates without delay with either the U1 or U2 protein. Pauses occur during elongation in the 3' direction at an average of 320 nucleotides, indicating a packaging periodicity of about six to eight helical turns.

Capsid↗
PubMed1986

The chemical induction of PR (b) proteins and resistance to TMV infection in tobacco.

Aspirin injected into Xanthi-nc tobacco leaves induces the production of PR protein and resistance to TMV. The concentration of PR protein and resistance increases with increasing aspirin concentration, up to a plateau. 2-Thiouracil and dioxohexahydrotriazine also induce PR protein when injected into tobacco leaves. Barium and manganese salts induced PR protein, but those of eight other cations did not. Certain salts were phytotoxic but did not induce PR protein, confirming that the production of PR protein is not a non-specific stress response.

Aspirin↗
PubMed1992

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.

Bacteriorhodopsins↗
PubMed1999

Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture.

A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.

Animals↗
PubMed1997

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV).

Plasmodesmatal gating in epidermal cells of Nicotiana tabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic 'halo' shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.

Green Fluorescent Proteins↗