Relationships between tobacco mosaic virus and the non-virus proteins

1. The non-virus proteins, A4, B3, and B6, characteristically found in tobacco leaf infected with TMV exhibit specific immunochemical cross-reactions with serum prepared against the virus. The close immunochemical relations which occur among these proteins do not extend to any normal tobacco leaf proteins. 2. The rate of appearance of the non-virus proteins in newly infected cultured leaf tissue at various times after inoculation has been determined by immunochemical techniques and by direct isolation of the proteins. Both methods give comparable results. The non-virus proteins appear abruptly at about 220 hours after inoculation, when the TMV content is about one-third its final value. The amount of A4 rises rapidly and then levels off. The B6 content rises rapidly and continuously over the course of the experiment. B3 appears last, and increases in amount considerably more slowly than A4 and B6. 3. The isotope contents of TMV, B3, and B6 which appear in given intervals after inoculation in newly infected leaf cultured in nutrient containing N¹⁵H₄ have been compared. The isotope levels of concurrent TMV, B3, and B6 are identical within the experimental error. The isotope conditions employed in this experiment lead to the conclusion that this coincidence of N¹⁵ levels means that the virus and non-virus proteins are probably synthesized at about the same time from the same non-protein source of nitrogen. 4. Possible interpretations of the available data on the non-virus proteins are discussed. It is likely that one or more of these proteins represents small protein units which occur in the TMV nucleoprotein.As they exist in the infected leaf, the non-virus proteins are probably no longer available to the biochemical processes which lead to TMV synthesis. They are probably not precursors of TMV protein in a temporal sense.     

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.     

Azocoll-digesting Proteinases in Soybean Leaves: Characteristics and Changes during Leaf Maturation and Senescence

Two different endopeptidases which digest the chromogenic substrate Azocoll were found in soybean leaves. Azocollase A has a molecular weight of 17,500 and a pI of 6.0. Azocollase B has a molecular weight of 52,000 and a pI of 9.0. Both digest Azocoll optimally at pH 9.0. Azocollase A is inhibited by 3 millimolar ethylenediamine tetraacetate (EDTA) and azocollase B by 100 micromolar parachloromercuribenzoate. Studies on whole plants grown in the greenhouse and in the field show that total azocollase activity gradually increased during leaf maturation when leaf protein and chlorophyll increased, and then declined again during leaf senescence. Young leaves which are still expanding contain mostly azocollase B and little azocollase A. Leaf maturation was associated with a dramatic increase in azocollase A (40- to 50-fold), while azocollase B activity increased more slowly. This increase in azocollase A occurred in the 2- to 3-week period following leaf expansion. Azocollase A, separated from other proteinases by gel filtration on Sephadex G-100, digested denatured leaf protein and casein, resulting in the release of free α-amino groups. Break-down of leaf proteins by autodigestion of extracts at pH 9.0 resulted in the release of free α-amino groups and endopeptidic cleavage of polypeptides. However, polypeptide cleavage was not inhibited by parachloromercuribenzoate or EDTA indicating that the azocollases do not play a major role in the hydrolysis of leaf proteins in crude extracts.     

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.     

Growth, Sporulation and Spore Germination of Colletotrichum capsici as Influenced by Tobacco Mosaic Virus Infection in Capsicum annuum L.

Growth and sporulation of C. capsici were lesser in different solid and liquid media containing of virus infected leaves than in media containing extracts of healthy leaves. Spore germination and formation of appressoria in different dilutions of the extract were poor as compared to healthy leaf extracts The antimetabolite/inhibitor in TMV infected plants is water soluble.     

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.     

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.     

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.     

Characterization of the proteins of guinea-pig hair and hair-follicle tissue

This work forms a part of a study of the mechanism and control of protein synthesis in the hair follicle and concerns the characterization of the proteins of hair-follicle tissue and for comparative reasons those of the hair itself. 1. Five different groups of reduced carboxymethylated proteins were delineated from both tissues; these were: group 1A proteins, which appeared to be aggregates of the group 2 proteins; group 1B proteins, soluble at pH4.4, which were thought to originate from the medulla and inner-rootsheath layers; group 2 proteins, which were defined as the main low-sulphur keratin proteins insoluble at pH4.4; group 3 proteins, the precise origin of which is not known; and the group 4 proteins, which were defined as the main high-sulphur keratin proteins soluble at pH4.4. 2. With the single exception of the group 1B proteins, the types and properties of all hair and hair-follicle proteins were identical as far as could be determined by use of such criteria as multiplicity of components, molecular charge, molecular weight and amino acid composition. 3. Two significant quantitative differences were noted: in follicle extracts there were more group 2 proteins but less group 3 and group 4 proteins than in hair extracts; and secondly, in the follicle group 4 proteins, there were more proteins of lowest molecular weight and S-carboxymethylcysteine content, but fewer proteins of the highest molecular weight and S-carboxymethylcysteine conent than in the hair group 4 proteins. 4. These quantitative differences are discussed in terms of the mechanism of synthesis of the keratin proteins. 5. Follicle group 1B proteins are postulated to have arisen from the trichohyalin droplets of the developing medulla and inner-root-sheath layers of the follicle and may be precursors of the proteins of the mature medulla and inner root sheath.     

Proteomic analysis of tobacco mosaic virus-infected tomato (Lycopersicon esculentum M.) fruits and detection of viral coat protein

Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects tobacco and tomato plants causing a pathology characterised by cell breakage and disorganisation in plant leaves and fruits. In this study we undertook a proteomic approach to investigate the molecular and biochemical mechanisms potentially involved in tomato fruit defence against the viral infection. The comparison of 2-D gels from control and TMV-infected but asymptomatic tomato fruits revealed changes in several proteins. The differential expression of peptidases, endoglucanase, chitinase and proteins participating in the ascorbate-glutathione cycle in infected fruits suggests that pathogenesis-related proteins and antioxidant enzymes may play a role in the protection against TMV infection. TMV coat protein appeared as a prominent spot in 2-D gels from TMV-infected asymptomatic fruits. A Triton X-114 phase-partitioning step of tomato protein extracts favoured the solubilisation of TMV coat protein and the enrichment of two aminopeptidases not present in control fruits. PMF and MS/MS data of the 2-D gel-isolated TMV coat protein is proposed as a powerful analysis method for the simultaneous tobamovirus detection, species determination and strain differentiation in virus-infected fruit commodities.     

Mutants of tobacco mosaic virus with temperature-sensitive coat proteins induce heat shock response in tobacco leaves.

We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30 degrees C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30 degrees C. After 4 to 6 days, HSP70 was also induced at 23 degrees C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.     

The effect of virus infection on morphology and protein components of pollen grains

Morphology of pollen grains collected from healthy and virus infected plants ofChenopodium quinoa L.,Chenopodium album L. andNicotiana tabacum L. cv. Samsun was investigated using scanning electron microscopy (SEM). Pollen grains from tobacco plans infected with tobacco mosaic virus (TMV) were smaller, with rounded shape and conspicuous deformation of aperture unlike oval and smooth pollen grains from healthy plants. No morphological alterations were observed inC. quinoa andC. album plants infected with TMV and cucumber mosaic virus (CMV). Polyacrylamide gel electrophoresis of pollen proteins revealed substantial quantitative and qualitative differences in protein components of pollen grains collected from healthy and virus infected plants     

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 – 16 kDa (group 1), 27 – 28 kDa (group 3: chitinases) and 36 – 40 kDa (group 2a: -1,3-glucanases).     

TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector

Transient expression is a rapid, useful approach for producing proteins of interest in plants. Tobacco mosaic virus (TMV)-based transient expression vectors can express very high levels of foreign proteins in plants. However, TMV vectors are, in general, not efficiently delivered to plant cells by agroinfection. It was determined that agroinfection was very efficient with a 35S promoter-driven TMV replicon that lacked the TMV coat protein gene sequence. This coat protein deletion vector had several useful features as a transient expression system, including improved ease of use, higher protein expression rates, and improved biocontainment. Using this TMV expression vector, some foreign proteins were expressed at levels of 3 to 5 mg/g fresh weight of plant tissue. It is proposed that this new transient expression vector will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.     

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea.

The ery A region of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previously been shown to contain three large open reading frames (ORFs) that encode the components of 6-deoxyerythronolide B synthase (DEBS). Polyclonal antibodies were raised against recombinant proteins obtained by overexpression of 3' regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different high molecular weight protein in extracts of erythromycin-producing S. erythraea cells. These putative DEBS 2 and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences of ORFs 2 and 3. A third high molecular weight protein co-purified with DEBS 2 and DEBS 3 and had an N-terminal sequence that matched a protein sequence translated from the DNA sequence some 155 base pairs upstream from the previously proposed start codon of ORF1.     

Estimation of population bottlenecks during systemic movement of tobacco mosaic virus in tobacco plants

More often than not, analyses of virus evolution have considered that virus populations are so large that evolution can be explained by purely deterministic models. However, virus populations could have much smaller effective numbers than the huge reported census numbers, and random genetic drift could be important in virus evolution. A reason for this would be population bottlenecks during the virus life cycle. Here we report a quantitative estimate of population bottlenecks during the systemic colonization of tobacco leaves by Tobacco mosaic virus (TMV). Our analysis is based on the experimental estimation of the frequency of different genotypes of TMV in the inoculated leaf, and in systemically infected leaves, of tobacco plants coinoculated with two TMV genotypes. A simple model, based on the probability that a leaf in coinoculated plants is infected by just one genotype and on the frequency of each genotype in the source, was used to estimate the effective number of founders for the populations in each leaf. Results from the analysis of three leaves per plant in plants inoculated with different combinations of three TMV genotypes yielded highly consistent estimates. Founder numbers for each leaf were small, in the order of units. This would result in effective population numbers much smaller than the census numbers and indicates that random effects due to genetic drift should be considered for understanding virus evolution within an infected plant.