Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean

Incubation of protoplasts with polyethylene glycol (PEG) and plasmid DNA containing the coding region for aminoglycoside phosphotransferase gene (NPT II) proved to be a simple transformation method for Nicotiana tabacum and Vigna aconitifolia, a drought-tolerant grain legume. In both plant species examined, the plant cultivar was an important factor, which clearly influenced transformation rates. The use of different expression signals derived from gene VI of the cauliflower mosaic virus (plasmid pABD1) or from the nopaline synthase gene of Agrobacterium tumefaciens (plasmid pLGV neo2103) also resulted in different frequencies of stably transformed colonies. Plants could be regenerated from kanamycin-resistant line of tobacco and moth bean.     

Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (Liposomes)

Summary Protoplasts were isolated from tobacco suspension cultures using a new cellulase preparation. Tobacco mosaic virus (TMV) RNA was encapsulated in reverse-phase evaporation vesicle (REV) liposomes of phosphatidylserine and cholesterol, and was successfully introduced into tobacco protoplasts by treatment of the REV/protoplast mixture with polyvinyl alcohol or polyethylene glycol followed by washing with high pH-high Ca buffer. Delivery of TMV-RNA was monitored by determining the number of infected protoplasts using the immunofluorescence technique. Production of TMV particles in the infected protoplasts was also confirmed by electron microscopy. Because of the high encapsulation efficiency of REV liposomes the amount of TMV-RNA necessary to cause infection in the majority of protoplasts could be reduced to 1/10 to 1/5 that required in the previous study (Fukunaga et al. 1981). The usefulness of the REV-mediated delivery of nucleic acids for genetic manipulation of plant protoplasts is discussed.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc.Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein.Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.     

Mitotic protoplasts and their infection with tobacco mosaic virus RNA encapsulated in liposomes

M-phase and S-phase protoplasts were prepared from tobacco cells in suspension culture after a high degree of synchronization using aphidicolin, a specific inhibitor for eukaryotic DNA polymerase. When TMV-RNA was introduced into these protoplasts mediated by REV liposomes, 37% of M-phase and 26% of S-phase protoplasts were infected as determined by the fluorescent antibody technique. After the 24 hr interval between the introduction of TMV-RNA into protoplasts and the determination of infection, half of the infected mitotic protoplasts formed dumbell-shaped daughter cells. The significance of synchronized protoplasts in genetic engineering of plant cells is discussed in reference to the delivery of DNA into the nucleus.Abbreviation LS medium, Linsmaier and Skoog medium - PEG polyethylene glycol - REV reversephase evaporation vesicles - TMV tobacco mosaic virus     

Leadzyme formed in vivo interferes with tobacco mosaic virus infection in Nicotiana tabacum

We developed a new method for inhibiting tobacco mosaic virus infection in tobacco plants based on specific RNA hydrolysis induced by a leadzyme. We identified a leadzyme substrate target sequence in genomic tobacco mosaic virus RNA and designed a 16-mer oligoribonucleotide capable of forming a specific leadzyme motif with a five-nucleotide catalytic loop. The synthetic 16-mer RNA was applied with nontoxic, catalytic amount of lead to infected tobacco leaves. We observed inhibition of tobacco mosaic virus infection in tobacco leaves in vivo due to specific tobacco mosaic virus RNA cleavage effected by leadzyme. A significant reduction in tobacco mosaic virus accumulation was observed even when the leadzyme was applied up to 2 h after inoculation of leaves with tobacco mosaic virus. This process, called leadzyme interference, is determined by specific recognition and cleavage of the target site by the RNA catalytic strand in the presence of Pb(2+).     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples.

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

SAP-TRANSMISSIBLE MOSAIC DISEASES OF SOLANACEOUS CROPS IN TRINIDAD

Four sap-transmissible viruses were isolated from cultivated Solanaceae in Trinidad: (1) tobacco mosaic virus, from tobacco, tomato and sweet pepper; (2) cucumber mosaic virus, from tobacco and petunia; (3) 'pepper vein-banding virus', probably related to pepper mosaic viruses in Puerto Rico and Brazil, from peppers and tobacco; (4) 'egg-plant mosaic virus', possibly related to the tobacco ring-spot virus, from egg-plant and tomato. Pepper vein-banding virus causes leaf-crinkling and vein-banding in Physalis floridana , petunia, various Nicotiana spp. and most peppers; the Large Bell Hot pepper is killed; tomato and egg-plant are immune. Egg-plant mosaic virus produces mosaic, ring-spotting, or both, on different solanaceous species. It also gives local and systemic ring-spotting on Chenopodium hybridum and necrotic local lesions on the primary leaves of cowpea (var. Black-eye); cucumber is a symptomless carrier. Only cucumber mosaic virus was found naturally infecting non-solanaceous hosts, cucumber and certain common wild plants.
The thermal inactivation point of pepper vein-banding virus is 62° C, its dilution end-point 2×10^-5 and its longevity in vitro 6 day s at 23–30° C.; corresponding values for egg-plant mosaic virus are 78° C., 10^-6 and over 3 weeks. Aphisgossypii transmits cucumber mosaic and pepper vein-banding, but not egg-plant mosaic, of which Epitrix sp. is an occasional vector. Tobacco mosaic, as elsewhere, probably has no regular insect vectors in Trinidad.     

Isolation of Viruses by Electro-Extraction of Infected Plants

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.     

3. Isolation of Viruses by Electro-Extraction of Infected Plants

ABSTRACT

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.     

Presence of Plant Viruses in some Rivers of Southern Italy

Water samples were collected from some rivers of Southern Italy whose water is normally used for irrigation and checked for the presence of plant viruses. The assayes were carried out by centrifuging at 5000x g one liter of water, resuspending the sediments in phosphate buffer and testing their infectivities on Chenopodium quinoa Willd. It was possible to isolate tobacco mosaic virus, cucumber mosaic virus and two more viruses not identified yet.     

The interaction of sodium dodecyl sulfate with cucumber green mottle mosaic virus protein and tobacco mosaic virus protein

The binding of sodium dodecyl sulfate to coat protein subunits of cucumber green mottle mosaic virus and tobacco mosaic virus was studied by equilibrium dialysis. The amount of dodecyl sulfate bound to the cucumber virus protein in 0.1 m phosphate buffer (pH 7.2) was found to be 1.55 g/g, which was the same value as that obtained with the tobacco virus protein. The presence of 8 m urea markedly decreased the degree of binding of dodecyl sulfate to the proteins. The amount of binding to the cucumber virus protein was reduced to 0.56 g/g, and that to the tobacco virus protein decreased to 0.8 g/g. The net charges of both proteins were negative at neutral pH and the amount of negative charge of the cucumber virus protein, obtained from the potentiometric titration curves, was larger than that of the tobacco virus protein, either in the native state or in the denatured state. In dodecyl sulfate/polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. On the other hand, in the presence of 8 m urea, the electrophoretic migration rate of the cucumber virus protein was equal to that of the tobacco virus protein. Sedimentation equilibrium experiments in 6 m guanidinium chloride gave molecular weights of 17,700 and 17,200 for the tobacco mosaic virus and the cucumber virus proteins, respectively. These results suggest that the effective negative charge density of the cucumber virus protein-dodecyl sulfate complex is higher than that of the tobacco virus proteindodecyl sulfate complex in 0.1% dodecyl sulfate solution. The conformation of both proteins was investigated by circular dichroism measurements. Both proteins have a slightly higher degree of α-helix content in dodecyl sulfate solution than in the native state. The addition of 8 m urea to both proteins while in this solution induced a change in conformation to one having a much smaller degree of ordered structure, although the change in the cucumber virus protein was more intense than that in the tobacco virus protein.     

A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts

Summary Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the -glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low -glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.Abbreviations CaMV cauliflower mosaic virus - PCR polymerase chain reaction - PEG polyethylene glycol - Mu 4-methyl umbelliferone     

Identification of soybean mosaic, southern bean mosaic and tobacco ringspot viruses from soybean in the People's Republic of China

Samples of soybean plants with virus-like symptoms were collected from several locations in the People's Republic of China in 1981. These samples were used to prepare inocula for mechanical inoculation to soybean. Twenty-one virus cultures were obtained, the identities of which were determined by serology, symptomatology and host range. Sixteen cultures contained only soybean mosaic virus, four of which were more pathogenic than any previously studied; one culture contained only tobacco ringspot virus, another only southern bean mosaic virus, and three other cultures mixed infections of soybean mosaic and southern bean mosaic viruses. This is the first report of the occurrence of tobacco ringspot virus and southern bean mosaic virus in soybean in the People's Republic of China.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

麻疯树逆境蛋白(curcin 2)基因在烟草中的表达

麻疯树（Jatropha curcas）幼苗在干旱、高低温胁迫和真菌浸染下,其叶片中诱导产生了一种新的毒蛋白curcin 2。这意味着curcin 2在其它植物中的异源表达可能会增强植物对外界胁迫的抵抗。curcin 2 cDNA的两个片断：cur2p片断（编码前成熟蛋白）和cur2m片断（编码成熟蛋白）,通过农杆菌的介导分别转化烟草并获得转基因植株。但是,只有在插入了cur2p片断的烟草中检测到了curcin 2蛋白的表达。同时,curcin 2在烟草中的表达增强了植株对烟草花叶病毒（TMV）的抗性。     

In vitro methylation of tobacco mosaic virus RNA with ribothymidine-forming tRNA methyltransferase. Characterization and specificity of the reaction.

A novel method has been developed for the detection and study of tRNA-like moieties in viral RNAs. Tobacco mosaic virus RNA is an acceptable substrate for crude Escherichia coli ribothymidine-forming tRNA methyltransferase. Under optimum reaction conditions at least 85% of the methylation product is ribothymidine (rT). The reaction is essentially quantitative, 1 mol of rT being formed per mol of tobacco mosaic virus RNA. The optimum reaction conditions include the presence of 6.6 micrometers S-adenosyl-L-[Me-3H]methionine, 25 micrometers spermine, 25 mM ammonium acetate, and 50 mM HEPES, pH 8.0. Sequence analysis of (Me-3H)-labeled tobacco mosaic virus RNA shows that all of the methylation occurs at a single site and strongly suggests that this site is the 32nd residue from the 3'-end of tobacco mosaic virus RNA. This site closely resembles the normal position of rT in transfer RNA.