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.     

Ribonucleic acids of healthy and tobacco mosaic virus infected tobacco leaves

A comparison between RNA content and RNA base composition of comparable tobacco mosaic virus infected and non-infected but water-rubbed leaves of Nicotiana tabacum var. Samsun was made from 0 to 32 h after inoculation. The amount of RNA in the infected leaves increased in the period of 2 to 9 h after inoculation, whereas at the same time the RNA content of the water-rubbed controls decreased. The increase amounted to about 25 %, the decrease to about 30 %. Between 13 and 23 h following inoculation, the RNA content of both infected and non-infected leaves changed back nearly to the level measured at zero-time and then rose again in the infected leaves. In spite of the considerable increase in RNA content in the infected leaves between 2 and 9 h after inoculation there was no remarkable shift in the base composition of the RNA from the C-G type of normal leaf RNA to the A-U type of tobacco mosaic virus RNA, as could be demonstrated in later stages of infection.     

Analysis of polypeptides of virus-specific informosomes induced by tobacco mosaic virus and potato virus X

Informosome-like virus-specific ribonucleoprotein (vRNP) of tobacco mosaic virus (TMV) comprise a set of four major polypeptides having molecular weights of 17 500, 31 000, 37 000 and 39 000. Of the minor polypeptides, those of apparent molecular weights 25 000, 55 000, 68 000 and 70 000 had electrophoretic mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. Polypeptide with mol.wt. 175 000 is TMV coat protein so far as: a) vRNP was precipitated with immunoglobulins against TMV and TMV coat protein; b) it had electrophoretic mobility similar to mobility of TMV coat protein; c) the peptide map of polypeptides with mol.wts 31 000, 37 000 and 39 000 are probably virus-specific-products. This is supposed because they are not present in cell informosomes protein, and they are not revealed in vRNP induced in cells after infection with potato virus X (PVX). Electrophoresis of vRNP-PVX protein reveals polypeptides of 23 000 (PVX coat protein), 55 000, 70 000, 78 000, 95 000, 120 000 and 145 000.     

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.     

A hydrogen bond study in tobacco mosaic virus using Moessbauer spectroscopy

The Moessbauer method was applied to obtain information on a suggested hydrogen bond in tobacco mosaic virus (TMV), between the hydroxyl group of Tyr 139 and a carboxyl oxygen of Glu 22 in a neighbouring subunit. Spectra of ¹²⁹I were taken of 3,5-di-iodo-l-tyrosine as a free amino acid and in situ in TMV. The increase of the pK value of 3,5-di-iodo-l-tyrosine by 0.8 units at position 139 in TMV compared to the free value is a strong argument in favour of the existence of a hydrogen bond via the relevant hydroxyl group.The reported study demonstrates the surprising sensitivity of the observable Moessbauer parameters to details of the electronic configuration in the neighbourhood of the probe nucleus.     

High-sensitivity express immunochromatographic method for detection of plant infection by tobacco mosaic virus

A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object — tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2–4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1–0.2 ml tested solution (extract from 10–20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.     

Difference in the mechanism of self-assembly in vitro in tobacco mosaic virus and potato virus X

The effects of 254 nm UV-irradiation of tobacco mosaic virus (TMV) and potato virus X (PVX) RNA preparations on the RNA ability to self-assembly in vitro with the viral coat proteins were studied. It was found that while TMV RNA ability to assemble with the homologous protein is rapidly inactivated by the UV-irradiation, PVX RNA ability to be encapsidated by the PVX coat protein is quite resistant to the irradiation. More than that, the irradiation of TMV RNA with the dose strongly inhibiting its assembly with the homologous protein, did not result in any significant inhibition of this RNA ability to be coated with the PVX protein. The results testify to the profound differences in the mechanisms of RNA-protein interactions in the processes of self-assembly in vitro of tobamoviruses and potexviruses.     

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.     

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 movement of tobacco mosaic viruses and potato virus X through tomato plants

Tomato aucuba mosaic virus, tobacco mosaic virus and potato virus X took 3'5-4, 5 and 3 days respectively to move from inoculated tomato leaflets into the petioles and stems
On reaching the stem each virus usually first moved downward, but in some plants both upward and downward movement occurred simultaneously and in a few
upward movement occurred first.
All three viruses travelled through the stem at approximately the same rate. Each was capable of travelling more than 80 cm. during the first 12 hr. after entering the stem, giving a minimal average rate of about 8 cm. per hr.
Uninfected pieces of stem invariably occurred between infected pieces. Maximum lengths of stem through which virus particles had apparently passed without causing infection, were 44.5, 49 and 39 cm. for the three viruses.