Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Phenylalanine ammonia-lyase levels in protoplasts isolated from hypersensitive tobacco pre-infected with tobacco mosaic virus

Leaves of tobacco varieties carrying the N gene for hypersensitiviy react to tobacco mosaic virus (TMV) infection by forming necrotic lesions and by localizing the virus in the vicinity of these lesions. These changes are accompanied in the host by an increased metabolic activity, in particular by an increased production of phenolic compounds derived from phenylalanine. Necrogenesis apparently destroys cells which have become heavily infected despite this strong defense reaction. However, it has been demonstrated previously (Otsuki et al., 1972) that protoplasts derived from leaves which normally respond in vivo to virus inoculation by forming necrotic local lesions, show no such response when inoculated in vitro. In the present study we have investigated the effect of pre-infecting hypersensitive leaves with TMV on the production or the non-production of the factor(s) of necrosis at the level of either protoplasts or mesophyll cells isolated from these preinfected leaves. Phenylalanine ammonia-lyase (PAL), whose rate of synthesis has been shown (Duchesne et al., 1977) to increase in stimulated cells of infected leaves, was used as a biochemical marker in the search for the stimulus preceding necrogenesis. We found that this stimulus concerning PAL activity was never elicited in either protoplasts or mesophyll cells which were prepared just before the appearance of necrotic local lesions. This result did not depend on the conditions of pre-infection or on the methods used to isolate the protoplasts or mesophyll cells. We also assayed samples derived from pre-infected leaves that were already carrying local lesions, i.e., in which the stimulus and necrogenesis were already operating: not only did the isolated protoplasts and mesophyll cells not sustain the stimulus concerning PAL activity, but the stimulated enzyme activity decreased abruptly and, in most of the experiments, had disappeared within the time necessary for maceration. Evidence is presented showing that the non-elicitation or the abrupt decrease of stimulated PAL activity could not result from a selection of unstimulated cells or from a preferential destruction of stimulated cells during maceration of the leaves.Our results support the view that hypertonic osmotic pressure is responsible for the non-occurence of the hypersensitive response by acting according to one or both of the following processes: it suppresses the contacts through plasmodesmata between neighboring cells and, hence, it also suppresses the cell-to-cell diffusion of the factor(s) eliciting the stimulus; and/or since hypertonic osmotic pressure causes striking differences between leaf cells and protoplasts in total RNA and protein synthesis, these differences might include the suppression of synthesis of the elicitor of hypersensitivity.Abbreviations OMT O-methyltransferase - PAL phenylalanine ammonia-lyase - TMV Tobacco mosaic virus     

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

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     

Reproduction of sugar beet mosaic and tobacco mosaic viruses in anthocyanized beet plants

During our studies on the interaction of anthocyanins and plant virus diseases, reproduction of sugar beet mosaic (SBMV) and tobacco mosaic viruses (TMV) was investigated. Experiments were carried out in leaves of sugar beet,Beta vulgaris cv. Dobrovicka N and its spontaneous anthocyanized mutant. SBMV induces a systemic infection while TMV is responsible for primary local symptoms in sugar beet leaves only. Our quantitative analyses onAmaranthus caudatus L. andChenopodium quinoa Wilid. showed a significant decrease in concentration of SBMV in juice extracted from anthocyanized beet plants as compared with extracts from normal green infected plants. Significant differences were also obtained when SBMV — containing juice was tested in mixtures with healthy extracts from anthocyanized and normal green plants. Also the intensity of TMV symptoms in beet leaves was considerably decreased in leaves of antho-eyanized plants.     

A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus

Summary A high molecular weight protein unrelated to the viral coat was detected in tobacco mesophyll protoplasts infected by tobacco mosaic virus.     

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.     

Genetic analysis of helper virus-specific selective amplification of cucumber mosaic virus satellite RNAs

Satellite RNAs (sat-RNAs) are small molecular parasites associated with a number of plant RNA viruses. The cucumber mosaic virus (CMV) sat-RNAs are ca. 335 nucleotides and have evolved to produce a large number of closely related sat-RNAs. Different cucumoviruses can act as helper viruses in the amplification of CMV sat-RNAs. We have found that different helper viruses show a preference for a particular sat-RNA in a mixed infection. In this study the specificity of WL47 sat-RNA amplification by LS-CMV and of D4 sat-RNA amplification by tomato aspermy virus in mixed infections was examined. Recombinant cDNA clones of D4 sat-RNA and WL47 sat-RNA were used to map the sat-RNA sequences responsible for the helper virus selection of a specific sat-RNA for amplification.Correspondence to: M.J. Roossinck