The non-virus proteins associated with tobacco mosaic virus

1. Exhaustive fractionation of leaves from tobacco plants systemically infected with TMV has led to the isolation of two non-virus proteins, B3 and B6, and the detection of a third, A4, which do not occur in comparable uninfected plants. 2. Components B3 and B6 have been found consistently in a series of ten extracts from plants grown over an 18 month period in all seasons of the year. It is concluded that these components are as characteristic of the infected plant as TMV itself. 3. As they occur in the initial extracts, the non-virus proteins are of low molecular weight (S₂₀ ca. 3). On treatment, each component tends to form a high molecular weight polymer with an electrophoretic mobility considerably greater than that of the starting material. The high molecular weight derivatives of A4, B3, and B6 have been designated A8, B8, and B7 respectively. There is no evidence that these high molecular weight components occur as such in the infected leaf. 4. The non-virus proteins are free of nucleic acid and are not infectious. They cross-react immunochemically with TMV. 5. Compared with TMV content, the amounts of the non-virus proteins found in infected leaf are relatively small, falling in the range of 10 to 150 micrograms per gm. of tissue.     

The effects of tobacco mosaic virus synthesis on the free amino acid and amide composition of the host

1. Changes in concentrations of free amino acids and amides have been determined in TMV-infected tobacco leaf discs and in comparable uninfected discs during the time of virus formation. 2. During the period of rapid virus formation the infected discs show a transitory deficiency (as compared to uninfected discs) in glutamine, asparagine, aspartic acid, glutamic acid, serine, and to a lesser extent in valine, threonine, and proline. About 100 hours before this time smaller deficiencies in the concentrations of these components also occur. The latter effect is probably associated with the early synthesis of a non-virus protein in infected tissue. 3. Comparison of the above effects with the known amino acid composition of TMV indicates that it is unlikely that the virus protein is synthesized by condensation of appropriate free amino acids. Rather, the deficiencies observed appear to result from removal of ammonia from the nitrogen pool during synthesis of new proteins in infected tissue. Equilibrium shifts resulting from ammonia withdrawal probably account for the observed deficiencies in amides and free amino acids. TMV protein, therefore, appears to be synthesized de novo, from non-protein nitrogen, probably ammonia. 4. It is suggested that the changes in free amino acid concentrations induced by virus formation may account for some of the symptoms observed in infected plants.     

Localization of Pathogenesis-Related Proteins in the Epidermis and Intercellular Spaces of Tobacco Leaves after Their Induction by Potassium Salicylate or Tobacco Mosaic Virus Infection

The localization of pathogenesis-related (PR) proteins inducedin tobacco leaves by treatment with potassium salicylate ora hypersensitive response to tobacco mosaic virus (TMV) infectionwas studied using immunochemical methods. Total PR protein levelsincreased with time after these treatments. The proportion ofPR proteins in the intercellular spaces to the total contentin the leaf discs rapidly rose in the later stage of the treatmentto about 75% on the 9th day after salicylate treatment and tomore than 80% on the 6th to 9th day after TMV inoculation. After5 days of salicylate treatment, the amounts of PR proteins inthe peeled leaf epidermis were two fold those in the mesophylltissue. Only five percent or less of the total PR proteins inthe epidermal and mesophyll tissues of salicylate-treated leaveswere detected in the isolated epidermal and mesophyll protoplasts.The sugar content in highly purified PR la, lb and lc was lessthan one mole of monosaccharide per mole of each protein. Theseresults show that the PR proteins are non-glycoproteins secretedinto the intercellular spaces. (Received January 16, 1987; Accepted July 14, 1987)     

Relationships between tobacco mosaic virus biosynthesis and the nitrogen metabolism of the host

1. Comparisons of the nitrogen content of TMV-infected and uninfected tobacco leaf discs at various times after inoculation show that virus synthesis is associated with a net increase in protein content. This excess protein is due to: (a) TMV, (b) an excess in insoluble protein which develops soon after inoculation and ends about 100 hours before cessation of TMV synthesis, and (c) an excess in soluble non-virus protein, which is variable in size and which only occurs during the time of virus synthesis. A deficiency in non-protein nitrogen occurs during the time when virus appears. 2. Isotope experiments with N¹⁵-labelled nutrient show that: (a) The bulk of TMV nitrogen is derived from the free ammonia of the host tissue. (b) Amino acid residues of TMV protein are not derived from the corresponding free amino acids in the host. (c) The appearance of TMV is preceded by the synthesis of an insoluble precursor of the virus which is then converted into TMV or some soluble intermediate protein. This effect is associated with a cell particulate which represents a small fraction of the total insoluble protein. (d) Infected tissue synthesizes de novo small amounts of soluble non-virus protein, which may represent intermediates in TMV synthesis. (e) Infected tissue fails to synthesize a rapidly turned-over soluble protein which is synthesized in comparable uninfected tissue. (f) TMV synthesis is preceded by a temporary enhancement of the metabolic stability of an insoluble protein component. 3. The results lead to the conclusion that TMV formation is due to diversion of some part of the host's protein-synthesizing apparatus from its normal course.     

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.     

Observations and experiments on the use of an avirulent mutant strain of tobacco mosaic virus as a means of controlling tomato mosaic

In 1973 tobacco mosaic virus (TMV) strain M II-16 was successfully used by growers in the United Kingdom to protect commercial tomato crops against the severe effects of naturally occurring strains of TMV. However, plants in many crops had mosaic leaf symptoms which were occasionally severe, so possible reasons for symptom appearance were examined. The concentration of the mutant strain in commercially produced inocula (assessed by infectivity and spectrophotometry) ranged from 28 to 1220 μg virus/ml; nevertheless all samples contained sufficient virus to infect a high percentage of inoculated tomato seedlings. Increasing the distance between the plants and the spray gun used for inoculation from 5 to 15 cm resulted in a significant decrease in the number of tomato seedlings infected. When M II-16 infected tomato plants were subsequently inoculated with each of fifty-three different isolates of TMV, none showed severe symptoms of the challenging isolates within 4 wk, although some isolates of strain o induced atypically mild leaf symptoms. In a further experiment, M II-16 infected plants showed conspicuous leaf symptoms only 7 wk after inoculation with a virulent TMV isolate. M II-16 multiplied more slowly in tomato plants and had a lower specific infectivity than a naturally occurring strain of TMV. More than 50% of plants in crops inoculated with strain M II-16 which subsequently showed conspicuous leaf mosaic contained TMV strain 1 or a form intermediate between strains o and 1. It is suggested that the production of TMV symptoms in commercial crops previously inoculated with strain M II-16 may result from an initially low level of infection, due to inefficient inoculation, which allows subsequent infection of unprotected plants by virulent strains. Incomplete protection by strain M II-16 against all naturally occurring strains may also be an important factor.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.     

Influence of Auxin-Like Herbicides on Tobacco Mosaic Virus Multiplication

Tobacco (Nicotiana tabacum L., cv. Samsun) leaf discs inoculated with tobacco mosaic virus (TMV) were treated with auxin-like herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), 3-amino-1,2,4-triazol (Amitrol) and 6-chloro-2-ethylamino-4-isopropylamino-1,3,5-triazine (Atrazin). All herbicides in the concentration of 10^–7 M enhanced the virus content (MCPA to 227.4 %, Amitrol to 218.1 % and Atrazin to 257.3 % of values found in TMV-infected, herbicide untreated discs). The 2,4-D alone did not affect the activity of the glucose-6-phosphate dehydrogenase and ribonucleases, but the 2,4-D treatment together with TMV infection raised their activities twice as high as in the untreated control discs. Polyacrylamide gel electrophoresis of acidic extracellular proteins washed from leaf discs treated with 2,4-D did not prove the induction of PR-proteins.     

两个缺陷型TMV粒子在烟草中的互补表达

根据对ＴＭＶ高效复制和基因表达的顺式作用元件的分析,在体外重组包装了２个缺失型ＴＭＶ粒子：ＴＭＶＲＰ和ＴＭＶＣＰ。前者缺失了ＴＭＶ外壳蛋白ＣＰ基因的３′端及后序区域,后者缺失了大部分复制酶基因。把两者分别或共同电击感染烟草原生质体：１．用ＣＰ抗体进行免疫印渍检测,单独感染的原生质体内的ＣＰ在１６小时内无增加,而在共同感染的原生质体内,ＣＰ在感染２小时后就开始明显增加。２．用ＲＴ一两次ＰＣＲ法专一地检测新生负链ＲＮＡ的合成情况,在单独感染的原生质体内没有检测到,但在混合感染的原生质体内在感染１小时后就检测到ＣＰ基因特异的负链ＲＮＡ的形成,并用Ｓｏｕｔｈｅｒｎ杂交得到进一步验证。这些结果表明,复制酶缺失型ＴＭＶＣＰ内的ＣＰ基因不能表达,但可以在ＴＭＶＲＰ存在时,通过其所表达的复制酶互补作用得到复制从而有效表达．     

HydroxyproUne-rich Glycoprotein Accumulation in TMV-infected Tobacco Showing Systemic Acquired Resistance to Powdery Mildew

Tobacco cv. Havana 425, with systemic-acquired resistance (SAR) to an otherwise compatible Erysiphe cicho-racearum DC. race after TMV infection, was infected with TMV basaily (4^th and 5^th leaves) and the hydroxyproline-rich glycoproteins (HRGPs) were determined in two experiments (experiment 1 and experiment 2) by analysing cell wall hydroxyproline (Hyp) in the 6^th leaf. In basal TMV infected plants Hyp content (μmol^-1 FW) was greater than in controls in both experiments: the increase was significant at all times except 16 days after TMV inoculation in experiment 1; the pool of data of experiment 2 was also significantly increased. In TMV protected + E. cichoracearum challenged leaves compared to untreated controls significant increases in Hyp were also noted between the pools of data in both experiments. No differences were found between Hyp content in protected compared to protected + challenged leaves in both experiments. These results show accumulation of HRGPs in Havana tobacco associated with TMV infection and SAR activation against E, cichoracearum. The accumulation appears to be due to the inducer, since no further increase was detected in protected leaves after challenging.     

Influence of some herbicides on the reproduction of tobacco mosaic virus inNicotiana tabacum cv. ‘Samsun’

The application of 10^?3 g 1^?1 of 2,4 dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 3-amino-1,2,4-triazole and 6-chloro-2-ethylamino-4-isopropyl-amino-1,3,5-triazine toNicotiana tabacum cv. ‘Samsun’ plants inoculated with tobacco mosaic virus results in an increase in the content of this virus in the tissues. When whole plants are used, TMV content increases by 20% after herbicide application; when leaf dises are used, the amount of the virus can be doubled by the herbicide in comparison with control untreated inoculated discs. The results clearly show that the used non-selective herbicides act as stimulators of virus biosynthesis, probably via enhanced pentose phosphate cycle activity which enables an enhanced formation of viral RNA.     

Xanthine Oxidase Activity in the Susceptible and Hypersensitive Responses of Tobacco Leaves to Tobacco Mosaic Virus Infection

A superoxide-producing xanthine oxidoreductase was isolated and quantified after polyacrylamide disc gel electrophoresis of tobacco leaf extracts. The results obtained indicate that, like uricase activity, a slight increase in tobacco xanthine oxidase activity takes place in the susceptible interaction with tobacco mosaic virus (TMV). In contrast, out of three hypersensitive tobacco cultivars tested, only two showed the same slight increase m activity during the late stage of hypersensitive response.
Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] a specific and potent in vitro and in vivo inhibitor of xanthine oxidoreductase, applied to tobacco plants by root absorption, starting about 8 days before the inoculation, did not affect the hypersensitive response but weakened the hypersensitivity-linked virus localization and promoted the movement of a certain amount of TMV particles and/or virus related material from necrotic lesions which induced systemic necrotic symptoms in uninoculated leaves. However, due to the inefficacy of allopurinol in preventing necrotic lesion development, all results are consistent with the hypothesis that xanthine oxidoreductase, the first enzyme in purine oxidative degradation, plays only a secondary role during induction of primary hypersensitive cell death in TMV infected tobacco leaves.     

An Analysis of the Effects of Tobacco Mosaic Virus on Growth and the Changes in the Free Amino Compounds in Young Tomato Plants

Tomato plants at the four-leaf stage were inoculated on thefirst leaf with TMV in a growth room and the effects studiedin systemically-mfected leaves with reference to growth, virusmultiplication and changes in water, nitrogen, nitrate and chlorophyllcontents. Parallel changes in the free amino compounds werealso studied in the growth room (incident radiation 152 mwhcm^-2 day^-1) and in two experiments in a glasshouse (352 and226 mwh cm^-2 day^-1) Dry matter accumulation and leaf expansion in leaves 3, 4 and5 were checked by TMV 5–7 days after inoculation but notin leaf 2. In the period 7–25 days after inoculation therelative growth rates of whole plant and leaves 3 and 6 andnet assimilation rate were not affected by TMV. Stem heightand dry weight were not affected by TMV but ‘root’dry weight was reduced from days 5–15. Virus was presentin the stem and in leaves 2 and 6 by days 3, 5 and 15 respectivelyso that infection per se did not always check growth. Chlorophyllcontent of systemically-infected leaves was reduced 10 daysafter inoculation. Total N and ammonia contents were not affectedby TMV but infected leaves contained less nitrate. At the two lower levels of incident radiation the initial effectof TMV was to reduce the content of total free protein aminoacids and amides, which were minimal 5–7 days after inoculation.In the glasshouse experiment a reduction could be measured only1 day after inoculation or before virus was present in the youngerleaves. With high incident radiation there was no initial reductionbut an increase at day 13 when mottling symptoms were visible.Total non-protein amino acids, of which amino butyric acidwas the major constituent, were increased by TMV in all threeexperiments for up to 13 days after inoculation. It is suggested that inoculation of a leaf with TMV temporarilyinterferes with export of photosynthates and import of root-synthesisedamino acids and that the results reported above can be interpretedin this context. Evidence in support of this is adduced froman experiment in which 13 foliar sprays of gibberellic acid(2·5 ppm) were combined with TMV inoculation and thechanges in free amino compounds followed. It is concluded that analyses of the changes in individual freeamino compounds are unlikely to provide useful information concerningthe sources of virus coat protein.     

Response of Membrane-bound Mg-activated ATPase of Tobacco Leaves to Tobacco Mosaic Virus

Infectious material was formed at an early stage, and migrated into the mesophyll from the epidermis of tobacco leaves (Nicotiana tabacum cv. Samsun NN) during the period of 1 to 3 hours after inoculation with tobacco mosaic virus (TMV). The activity of membrane-bound Mg²⁺-activated ATPase from the mesophyll was stimulated two to four times within 30 minutes after inoculation with 1.0 microgram per milliliter of TMV. Maximum TMV stimulation of membrane-bound Mg²⁺-activated ATPase activity in epidermis and mesophyll was observed at 0.5 and 3.0 hours after inoculation, respectively. This stimulation was also observed with ultraviolet irradiated TMV (only RNA was destroyed), whereas, the stimulation was not observed with heat-irradiated TMV (both coat and RNA were destroyed). Stimulation equal to that of TMV was observed by inoculation with cucumber green mottle mosaic virus and to a lesser extent with cucumber mosaic virus.

These results illustrate that the stimulus resulting from inoculation with TMV transfers to underlying cells faster than the migration of TMV particles. This stimulus might be closely correlated to the structure of virus, but not to the infectivity of virus.

     

Early trans-plasma membrane responses to Tobacco mosaic virus infection

Early trans-plasma membrane behavior after in vivo mechanical inoculation of Nicotiana tabacum with Tobacco mosaic virus (TMV) was investigated and compared to virus quantification in leaf tissues. To identify early events related to virus/host interaction, the systemic virus TMV was used to infect lower leaves and tests were carried out on upper leaves which were not directly infected. Non-invasive microelectrodes were used to estimate trans-plasma membrane electron transport and membrane potential after artificial inoculum of virus, monitoring the plant for the following 15 days. Virus infection was assessed by ELISA and quantified by quantitative RT-PCR. Collected data showed that after 2-day post-inoculation (dpi), TMV was able to modify membrane parameters: transient hyperpolarization of trans-membrane potential was observed until 10 dpi, while redox activity in infected samples was higher compared to control until end of tests. Conversely, ELISA diagnostic test was not able to reveal the virus presence in tobacco leaves until 6 dpi, while leaf symptoms were manifested after 13 dpi.     

Glucose-6-Phosphate Dehydrogenase, Ribonucleases and Esterases upon Tobacco Mosaic Virus Infection and Benzothiodiazole Treatment in Tobacco

Effect of the benzothiodiazole (BTH) pre-treatment was monitored during the acute infection stage in the susceptible and the hypersensitive tobacco plants infected with the tobacco mosaic virus (TMV). Dynamic changes in the contents of chlorophyll, the total proteins, and the pathogenesis related proteins (PR-proteins), and activities of ribonucleases (RNase), phosphomonoesterase (PME), phosphodiesterase (PDE), and glucose-6-phosphate dehydrogenase (G6P DH) were studied. Neither the protein nor the chlorophyll contents were significantly changed by the TMV infection and/or the BTH treatment. The BTH pre-treatment caused a substantial reduction in the multiplication of TMV in the locally-infected leaves of the hypersensitive cultivar Xanthi-nc (to 15.1%). A lesser decrease (to 50.3%) was observed in the locally-infected leaves of susceptible cultivar Samsun. But in the systemically-infected leaves of this cultivar, only a 4-d delay in the multiplication of TMV was found. In the locally-infected leaves of both cultivars, the activities of the RNase, PME, PDE and G6P DH were sharply increased during the acute phase of TMV multiplication (when compared with the healthy plants) and the curves of these activities correlated with the multiplication curves of TMV. The BTH alone also strongly enhanced the activities of these enzymes early after application. Only low additional increases in some enzymes and even slight declines in the others were observed when the inoculation of leaves of cultivar Xanthi-nc followed the pre-treatment with the BTH. No inhibition of the enzymes was observed when the direct effect of different concentration of the BTH (1 – 1000 M) was examined in vitro during a measurement of the activity. The analysis of intercellular proteins by PAGE under native conditions shows the similar spectrum of the proteins extracted from either the BTH-treated or the TMV-infected tobacco cv. Xanthi-nc.     

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves

Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g-1 fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-Gtase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7-27.0 [mu]g g-1 fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]-GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity.     

Tobacco proteinase inhibitor I genes are locally,but not systemically induced by stress

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.     

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.