Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

Changes in glucose-6-phosphate dehydrogenase (G6P DH; EC 1.1.1.49) activity caused by infection of tobacco ( Nicotiana tabacum L.) leaves with potato virus Y (PVY), cucumber mosaic virus, potato virus X, tobacco rattle virus and turnip mosaic virus, the subcellular localisation of G6P DH isozymes in mesophyll protoplasts derived from healthy and PVY-infected tobacco leaves, as well as G6P DH control and the relationship of its isozymes with the degree of tobacco resistance to PVY multiplication, were studied. The activities of G6P DH were markedly increased in locally and systemically infected leaves and the time courses of the activity linearly correlated with those of virus multiplication. In leaves infected with PVY, the activity time courses of the crude and the partially purified G6P DH were coincident. This probably indicates the involvement of coarse regulation of the enzyme. PVY content linearly correlated with enhanced G6P DH activity in leaf discs derived from susceptible, tolerant and resistant cultivars of tobacco. The increased activity of the enzyme in infected protoplasts and plant tissues was predominantly caused by the increased activity of chloroplastic isozymes. This was confirmed by the specific staining of isozymes after electrophoretic separation of chloroplastic proteins of tobacco leaves. These findings enable the degree of resistance to virus multiplication to be quantified for the use of gene manipulation and breeding.     

Labeling of antibodies with radioactive isotopes in plant virus serology

Antibodies isolated from antiserum against plant viruses were labeled with the isotope³⁵S as follows: the mixture of antibodies with radioactive cysteine hydrochloride was allowed to stand for half an hour, run on a Sephadex G-25 column and individual fractions were collected. Sephadex G-50 bed was equilibrated and washed with saline (0,85 % NaCl) phosphate buffer (0,01 m) pH 7,2. Fractions showing the highest radioactivity and at the same time the most evident serological reaction were combined and used as³⁵S labeled antibodies. The labeled antibodies were used for rubbing leaves; the leaves were afterwards incubated, washed, killed, dried and then subjected to autoradiography. The method of indirect serological reaction also proved to be very good. Using this method, pig gamma globulin against rabbit gamma globulin was labeled with³⁵ S; this labeled gamma globulin was then used to detect serological reaction on leaves between the virus and homologous rabbit antiserum and/or antibodies. The results of those reactions were also determined by autoradiography. Exact procedure for labeling antibodies, carrying out serological reactions and autoradiography is desribed.     

Activity, isoenzyme composition, thermostability and molecular weight of peroxidase from intact and virus infected tobacco plant leaves]

The enzyme peroxidase was isolated from the leaves of the tobacco plant Xanthi (intact and infected with weakly (XY) and highly (XT) pathogenic strains of potato X-virus) and partially purified. The original extract (the 30,000 g supernatant) was purified by ammonium sulfate at 30--80% of saturation and by gel filtration through Sephadex G-25 and G-100 in 0.05 M tris-HCl buffer, pH 7.4 containing 17% sucrose. Disc electrophoresis revealed that both intact and infected plants contain 10 isoperoxidases. The electrophoregrams of isoenzymes from infected plants with the Rf values of 0.1, 0.48, 0.53 and 0.59 stained with benzidine produced a more intensive colouring as compared to the corresponding isoenzymes from intact plants. The total enzymatic activity for the plants infected with the XY and XT strains made up to 180% and 240% of that for the intact plants, respectively. The molecular weights of the peroxidase isoenzymes were found to be the same and equal to 40,000. Study of the thermostability at 60 degrees C and pH 7.0 showed that after 90 min the enzyme activity was 12.4% and 5.1% of the original one in intact and infected plants, respectively. The data obtained suggest that the activity, thermostability and synthesis of some peroxidase isoenzymes in tobacco plant leaves are affected by viral infection.     

THE DISTRIBUTION OF VIRUSES IN LEAVES AND THEIR INACTIVATION BY ULTRA-VIOLET IRRADIATION

The infectivity of sap expressed from the lower epidermis stripped from leaves systemically infected with potato virus Y , henbane mosaic virus or tobacco mosaic virus was compared with that of sap from the underlying mesophyll. Results suggested that the concentration of virus in each of the two tissues was about the same.
Ultra-violet irradiation of leaves infected with potato virus Y or henbane mosaic virus greatly reduced the infectivity of sap expressed from subepidermal tissues.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

HEAT-THERAPY OF VIRUS-INFECTED PLANTS

Virus-free plants were produced from parents systemically infected with the following five viruses: tomato bushy stunt, carnation ring spot, cucumber mosaic, tomato aspermy and Abutilon variegation. The leaves formed while the infected plants were kept at 36°C. were free from symptoms, and test plants inoculated from these remained uninfected. When cuttings were taken from the infected plants at the end of the treatment most grew into healthy plants. The treated plants themselves usually developed symptoms after varying lengths of time at 20°C, but some that before treatment were infected with tomato aspermy, cucumber mosaic or Abutilon variegation viruses, remained permanently healthy.
The same method failed to cure plants infected with tomato spotted wilt, potato virus X and tobacco mosaic virus, although it decreased their virus content. Heat-therapy seems not to be correlated with the thermal inactivation end point of the virus in vitro.     

The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-¹⁴C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.     

Detection of potato leafroll and potato mop-top viruses by immunosorbent electron microscopy

Attachment of virus particles to antiserum-coated electron microscope grids (immunosorbent electron microscopy) provided a test that was at least a thousand times more sensitive than conventional electron microscopy for detecting potato leafroll (PLRV) and potato mop-top (PMTV) viruses. The identity of the attached virus particles was confirmed by exposing them to additional virus antibody, which coated the particles.
PLRV particles (up to 50/μm² of grid area) were detected in extracts of infected potato leaves and tubers, infected Physalis floridana leaves, and single virus-carrying aphids. On average, Myzus persicae yielded 10–30 times more PLRV particles than did Macrosiphum euphorbiae .
PMTV particles (up to 10/μm² of grid area) were detected in extracts of inoculated tobacco leaves, and of infected Arran Pilot potato tubers with symptoms of primary infection. Particles from tobacco leaves were of two predominant lengths, about 125 nm or about 290 nm, and fewer particles of other lengths were found than in previous work, in which partially purified or purified preparations of virus particles were examined, using grids not coated with antiserum.     

PHOTOSYNTHESIS AND RESPIRATION RATES OF LEAVES OF NICOTIANA GLUTINOSA INFECTED WITH TOBACCO MOSAIC VIRUS AND OF N. TABACUM INFECTED WITH POTATO VIRUS X

The rates of respiration and of photosynthesis of tobacco leaves infected with potato virus X were not affected until the leaves showed symptoms; the respiration rate was then increased by more than 30% and the photosynthesis rate decreased by 20%. When local lesions appeared on the leaves of Nicotiana glutinosa infected with tobacco mosaic virus, but not before, the respiration rate was increased by an amount, up to 30%, that varied with the number of lesions. The photosynthesis rate was decreased by 20%, but there was no effect on photosynthesis or respiration until symptoms appeared. These results differ from those previously reported for tobacco leaves infected with tobacco mosaic virus, in which both respiration and photosynthesis were affected within 1 hr. of inoculation. The validity of extrapolating arguments based on the results obtained with other combinations to this commonly used combination and vice-versa is questioned.     

SEROLOGICALLY RELATED STRAINS OF POTATO VIRUS Y THAT ARE NOT MUTUALLY ANTAGONISTIC IN PLANTS

Tobacco veinal necrosis virus is serologically related to potato viruses Y and C. It does not protect tobacco, Nicotiana glutinosa , or potato plants from infection by them, and tobacco and N. glutinosa plants infected with either virus Y or C are still susceptible to it. There is some evidence that it does not multiply normally in potato plants infected with virus Y and that it is sometimes suppressed in such plants.
The possession of antigenic groups in common with viruses Y and C is considered to justify identifying tobacco veinal necrosis virus as a strain of virus Y , and to be of greater taxonomic significance than failure to protect plants against other strains. A further difference from other strains is that it is more virulent towards tobacco than towards potato.     

Electrolyte leakage in relation to viral and abiotic stresses inducing necrosis in cowpea leaves

Different types of stress, such as hypersensitive reaction to viruses or necrotic response induced by chemical and mechanical injuries, caused similar patterns in early electrolyte leakage from discs of cowpea leaves. The electrolyte leakage, suggestive of permeability alterations, always occurred in advance of cell death. Altered permeability can therefore be considered as an aspecific response to stress and a marker of necrogenesis. No correlation was found between permeability alterations and the mechanism for virus localization, the main characteristic of the hypersensitive reaction, because tobacco rattle virus induced similar alterations in either incompatible cowpea or compatible tobacco plants. Early Ca²⁺ losses occurred following viral and abiotic necrogenic stresses. The external supply of Ca²⁺ to cowpea during the course of the hypersensitivity to viruses failed to effect pathogenesis, assessed as the number and size of the resulting necrotic local lesions, and the release of electrolyte leakage from the infected leaves. However, the external supply of Ca²⁺ counteracted the negative effect of EDTA, a chelating agent, on the resistance of cowpea leaves to cell-to-cell spread of viruses, suggesting that a Ca²⁺-deficiency may weaken the mechanism of defense of the cells against the localized viral infection.     

Effects of Salicylate on Virus-Infected Tobacco Plants

Salicylate watered onto the soil of tobacco plants in pots reduced the antigen accumulation and local lesion growth of tobacco necrosis virus mechanically inoculated on the leaves. It also retarded the growth of the necrotic centres of lesions and, in parallel, inhibited ethylene production from infected leaves. However, the therapeutic index of salicylate was very small and the chemical had to be applied in advance of, or at the same time as virus inoculation to give good levels of resistance. The number of lesions and their rate of appearance were not affected by salicylate. In addition, it did not induce resistance against multiplication, systemic spread or symptom severity in tobacco plants inoculated with a necrotic strain of potato virus Y. These findings suggest that salicylate is not likely to prove useful as polyvalent chemotherapeutic agent.     

THE EFFECT OF INFECTION WITH TOBACCO ETCH VIRUS ON THE RATES OF RESPIRATION AND PHOTOSYNTHESIS OF TOBACCO LEAVES

Unlike tobacco mosaic virus, which increases the respiration of tobacco leaves within an hour of their being inoculated, a virulent strain of tobacco etch virus did not change respiration rates until leaves showed external symptoms. The respiration rates of inoculated or systemically infected leaves with symptoms rose to 40% above that of healthy leaves, three times the increase produced by tobacco mosaic virus. The increased respiration rate occurred at all times of the year and was maintained through the life of the leaves.
Leaves infected with tobacco etch virus and showing symptoms had a photo-synthetic rate 20% lower than that of healthy leaves.     

SOME EFFECTS OF HOST-PLANT NUTRITION ON THE MULTIPLICATION OF VIRUSES

The amounts of tobacco mosaic virus present in systemically infected tobacco plants varied greatly with the mineral nutrition of the plants and were related to the effects on plant growth. With plants in soil, supplements of phosphorus produced the greatest increases in plant size, in virus concentration of expressed sap, and in total virus per plant; nitrogen increased plant size only when phosphorus was also added, and only then increased virus concentration and total virus per plant. Combined supplements of phosphorus and nitrogen doubled the virus concentration of sap and increased the total virus per plant by factors up to forty. Potassium slightly reduced the virus concentration of sap, though it usually increased plant size and total virus per plant. From all plants, only about one-third of the virus contained in leaves was present in sap. Virus production seemed to occur at the expense of normal plant proteins, and the ratio of virus to other nitrogenous materials was highest in plants receiving a supplement of phosphorus but not of nitrogen.
The effects of host nutrition on the production of virus in inoculated leaves resembled those in systemically infected leaves, but were more variable.
No evidence was obtained, with plants grown in soil or sand, that host nutrition had any consistent effect on the intrinsic infectivity of tobacco mosaic virus.
The concentration of virus in sap from potato plants systemically infected with two strains of potato virus X was not consistently affected by fertilizers; the chief effect of host nutrition on virus production was indirect by altering plant size.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

Establishing a Corky Ringspot Disease Plot for Research Purposes

A method to establish two experimental corky ringspot disease (CRS) plots that had no prior CRS history is described. CRS is a serious disease of potato in the Pacific Northwest caused by tobacco rattle virus (TRV) and transmitted primarily by Paratrichodorus allius. ‘Samsun NN’ tobacco seedlings were inoculated with viruliferous P. allius in the greenhouse before they were transplanted into the field soil at the rate of 3,000 plus seedlings/ha. Care was taken to keep soil around plants in the greenhouse and transplants in the field moist to avoid vector mortality. The vector population in the soil of one of the fields was monitored by extraction, examination under microscope and bioassay on tobacco seedlings to ascertain that they were virus carriers. Presence of virus in tobacco bioassay plants was determined by visual symptoms on tobacco leaves and by testing leaves and roots using ELISA. Although TRV transmission was rapid, there was loss of infectivity in the first winter which necessitated a re-inoculation. After two years of planting infected tobacco seedlings, 100% of soil samples collected from this field contained viruliferous P. allius. In the second field, all five commercial potato cultivars, known to be susceptible, expressed symptoms of CRS disease indicating that the procedure was successful.     

Glucose-6-Phosphate Dehydrogenase/6-Phosphogluconate Dehydrogenase Ratio and the Glucose-6-Phosphate, 6-Phosphogluconate and Fructose-6-Phosphate Contents in Tobacco Plants Infected with Potato Virus Y

The ratio of activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G6P DH/6PG DH), and the contents of glucose-6-phosphate (G6P), 6-phosphogluconate (6PG) and fructose-6-phosphate (F6P) were studied at various stages of potato virus Y (PVY) multiplication in Nicotiana tabacum cv. Samsun. G6P DH/6PG DH increased through the experiment from 0.42 to 0.53 in leaves of healthy tobacco, and up to 0.59 in PVY systemically infected leaves. However, these ratios in the ruptured protoplast preparations, and the chloroplast and cytosol fractions of healthy protoplasts were similar to that from infected ones. The ratio lower than 1, found in the healthy and/or PVY- infected leaf tissues and in the infected protoplasts as well, confirms the assumption that G6P DH is the control enzyme of oxidative pentosephosphate pathway not only in the healthy but also in the infected plants. The contents of G6P, 6PG and F6P in the period of the highest PVY multiplication were strongly decreased (to 30 – 50 % when compared with control healthy leaves) and were negatively correlated with the G6P DH and 6PG DH activities.     

Changes in non-protein nitrogen metabolism during tobacco mosaic virus biosynthesis

1. Discs cut from tobacco leaf tissue infected with tobacco mosaic virus and cultured in water contain less non-protein nitrogen than comparable uninfected discs during the time at which TMV is formed. This deficiency disappears when virus formation ceases. Discs cultured in nutrient solution form about twice as much TMV as discs cultured in water. The maximum non-protein nitrogen deficiency is comparable in magnitude to the amount of virus synthesized. 2. The largest difference between injected and uninfected tissue occurs in the ammonia content. Smaller, but significant differences in amide content are found. Infected discs cultured in water show no significant differences from control discs in free amino acid content; infected discs cultured in nutrient solution develop a small deficiency in amino acid nitrogen. 3. The general patterns of change in composition of the pool of soluble nitrogen are similar in both infected and uninfected discs. 4. The data indicate that the bulk of the nitrogen incorporated into virus protein is withdrawn from the leaf's pool of soluble nitrogen; virus is formed de novo from ammonia nitrogen and non-nitrogenous carbon sources. The effect of virus infection on host nitrogen metabolism appears to be due to the formation of virus rather than to its presence.     

Stimulation by abscisic acid of RNA synthesis in discs from healthy and tobacco mosaic virus-infected tobacco leaves

Uptake of abscisic acid from the culture medium by discs of healthy and tobacco mosaic virus-infected tobacco leaves was measured. Small (two to five-fold) increases in abscisic acid concentration in discs caused increases in rates of [³H]uridine and [³H]adenine incorporation into total nucleic acid, virus RNA and host ribosomal RNA. Net accumulation of virus RNA was also enhanced by abscisic acid. This evidence for stimulation of RNA synthesis is compared with previous reports showing inhibition of RNA synthesis in other tissues. It is suggested that the increase in endogenous abscisic acid caused by tobacco mosaic virus infection may be at least partly responsible for observed increases in rates of RNA synthesis after infection.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus