Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

THE RESPIRATION OF TOBACCO LEAVES AFTER SYSTEMIC INFECTION WITH TOBACCO MOSAIC VIRUS

The rate of CO, production per g. dry matter of the younger leaves of tobacco plants systemically infected with tobacco mosaic virus was about 10 yo less than that of comparable healthy leaves. Older infected leaves, showing well-developed mosaic symptoms, had the same respiration rate as comparable healthy leaves. These results were independent of seasonal change in light conditions during the growth of the plants. Older leaves, but not younger leaves, of infected plants had a lower initial water content, and both absorbed less water during the experimental period, than leaves from healthy plants. The effects of TMV infection on water content were so great that the rate of CO, production per g. fresh weight was sometimes significantly increased by infection. This reversal of the apparent effect of infection on respiration rate, depending on the basis of reference may partly account for contradictory results reported previously by other workers. Other causes for contradictory results are discussed.     

SOME EFFECTS OF TANNIC ACID AND OF LEAF EXTRACTS WHICH CONTAIN TANNINS ON THE INFECTIVITY OF TOBACCO MOSAIC AND TOBACCO NECROSIS VIRUSES

The inhibition of infection by tobacco necrosis and tobacco mosaic viruses by tannic acid, and by extracts of raspberry and strawberry leaves, was associated with the precipitation of the viruses. Precipitation and inhibition were reversible, and infective virus was obtained from the precipitate formed between the viruses and tannins. Infectivity was fully restored by diluting mixtures of virus and tannin adequately and partially restored by adding alumina or nicotine sulphate.
Viruses and tannins are thought to form non-infective complexes, in which the virus and tannin components are held together by co-ordinate linkages or hydrogen bonds.
Macerating tobacco leaves infected with tobacco mosaic virus together with raspberry leaves greatly decreased the infectivity of the extracts; adding nicotine sulphate to the mixture of leaves before it was ground increased the infectivity, even though nicotine sulphate alone decreases the infectivity of tobacco mosaic virus. Even in the presence of nicotine sulphate, much of the virus was precipitated by substances from the raspberry leaves.
Extracts of roots of Fragaria vesca plants, infected with a tobacco necrosis virus, were more infective when made by macerating the roots with four times their weight of buffer at pH 8 than when made without buffer. Various methods are suggested for facilitating the transmission of viruses from plants that contain tannin.     

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.

     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

Studies on Induced Resistance to Tobacco Mosaic Virus, in Samsun NN Tobacco and Changes in Ribosomal Fractions

Resistance to tobacco mosaic virus (TMV) was activated by various forms of induction in Samsun NN tobacco leaves, and the intensity of the different forms was compared. Induced resistance was highest in leaf tissue between TMV inoculated stripes parallel to the mid-vein and after injection of ethylene maleic anhydride copolymer (EMA), followed by that induced in distal half leaves after inoculating the basal halves with TMV. Resistance in upper leaves following inoculation of the lower leaves with TMV was relatively low, while induction due to lesions caused by ethrel gave an intermediate degree of resistance. Estimation of resistance by size and number of local lesions was correlated with the amount of extractable virus as measured by enzyme-linked immunosorbent assay (ELISA), thus indicating that in the resistant tissue virus replication, and not only the development of necrotic local lesions, is suppressed. An increase in a specific ribosomal fraction (R₂), recovered by a two-step procedure, was observed in tissues where resistance was most intense, i.e., between TMV stripes and after EMA injection. It may be that this specific ribosomal fraction participates in maintaining the resistant state.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter

An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.     

检测烟草中烟草花叶病毒的RNA斑点杂交法

用普通烟草花叶病毒OM株3′-端约2kb的cDNA为探针,探索了用RNA斑点杂交法对烟草组织中烟草花叶病毒RNA进行检测的条件。这些条件包括用分子杂交法观察云南烟区和上海烟草上分到的烟草花叶病毒与OM株的同源性,从烟草组织中提取烟草花叶病毒的几种方法的比较,使RNA有效地固定在硝酸纤维素滤膜上的方法,烟草组织中是否有干扰RNA固定和杂交的物质,斑点杂交方法检测烟草花叶病毒的特异性、灵敏度等。     

Systemic PCD occurs in TMV-tomato interaction

In hypersensitive response (HR), programmed cell death (PCD) is reported as a powerful defense mechanism in plant immune responses to pathogen. However, little is known about the PCD in sys-temic acquired resistance (SAR). Using tobacco mosaic virus (TMV) to infect the tomato (Lycopersicon esculentum cv. Jiafen 16) we found that localized TMV-infection could induce cell death in the uninoculated parts of the tomatoes, where the enzyme-linked immunosorbent assay (ELISA) showed no spreading virus. The biological and molecular characterization of this cell death was shown as fol-lowing: chromatin condensed and formed peripheral conglomeration in nuclei; cell nucleus were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; mitochondria and chloroplast were disrupted; tonoplast and plasma membrane were shrunk and degradated. These re-sults suggested that with an absence of TMV spread, the local TMV-infection on certain tomato leaves could induce systemic PCD in the root-tips, stem-apices and uninoculated leaves. The systemic PCD has various initiation and synchronization in such tissues and is distinct in inducement and exhibition from HR-PCD and SAR.     

CYTOLOGICAL AND CYTOCHEMICAL OBSERVATIONS ON THE EARLY STAGE OF INFECTION OF TOMATO HAIR CELLS BY TOBACCO MOSAIC VIRUS

Strips of epidermal tissue bearing hair cells were removed fromtomato stems. The lower surface exposed by the stripping wasinoculated with tobacco mosaic virus and the progress of infectionof the hair cells was followed microscopically and by extractionof infectivity. Shortly before infectivity could be extractedfrom the hair cells, the nuclei were observed to have migratedtowards the tran sverse wall of the basal hair cell nearestthe epidermis, apparently the site at which infection of thecell commences. After infection was apparent, the staining characteristicsof the nuclei, before and after RNase and DNase treatment, suggestedan accumulation of RNA. A granular body, different from X-bodiesmaintained a close association with the nucleus of TMV infectedhair cells, and was found to be rich in RNA, suggesting thatviral RNA of the nucleus may have been entering the cytoplasmvia the granular body. A possible mechanism of X-body formationarising from the granular body is suggested. ¹ Supported in part by a grant E-536 (C9) from the U. S. PublicHealth Service. ² Present address: Plant Pathology Laboratory, Faculty of Agriculture,Nagoya University, Anjo, Aichi (Japan). (Received March 4, 1963; )     

Model System for the Adsorption of Tobacco Mosaic Virus

Materials which can adsorb tobacco mosaic virus (TMV) were isolated from tobacco leaves and studied for applicability as a model system for TMV adsorption. Leaves were homogenized and fractionated by sucrose density gradient centrifugation. One fraction adsorbed TMV in the presence of polyornithine. Deduced from its sensitivity to trypsin and detergent as well as from its manner of isolation, the material responsible for adsorption of TMV seemed to be cytoplasmic membrane. Membrane derived from light particulate, as well as cytoplasmic membrane, seemed to be capable of adsorbing TMV. Shorter rods obtained by sodium dodecyl sulfate or sonic treatment of TMV could adsorb to membrane as efficiently as TMV. Viral protein subunit could not adsorb whereas helical rods made of viral protein aggregates could. A two-step nature of the adsorption of TMV was suggested: a salt-sensitive and a subsequent salt-resistant steps. In the first step, ionic bonding plays a main role in the combination between TMV and membrane. Adsorption of ¹⁴C-labeled TMV was inhibited by an excess amount of non-labeled TMV or cucumber green mottle mosaic virus but not by potato virus X or rice dwarf virus, suggesting the specific nature of adsorption. In contrast to the observed specificity on the part of virus, a membrane fraction isolated from various plants, including non-hosts for TMV, could adsorb TMV. This may imply that adsorption and injection are not the determinant of host specificity in plant viral infection.     

THE STRUCTURE OF CELLS DURING TOBACCO MOSAIC VIRUS REPRODUCTION : Mesophyll Cells Containing Virus Crystals

The submicroscopic organization of mesophyll cells from tobacco leaves systemically infected with tobacco mosaic virus (TMV) is described. After fixation with glutaraldehyde and osmium tetroxide the arrangement of the TMV particles within the crystalline inclusions is well preserved. Only the ribonucleic acid-containing core of the virus particles is visible in the micrographs. Besides the hexagonal virus crystals, several characteristic types of "inclusion bodies" are definable in the cytoplasm: The so-called fluid crystals seem to correspond to single layers of oriented TMV particles between a network of the endoplasmic reticulum and ribosomes. Unordered groups or well oriented masses of tubes with the diameter of the TMV capsid are found in certain areas of the cytoplasm. A complicated inclusion body is characterized by an extensively branched and folded part of the endoplasmic reticulum, containing in its folds long aggregates of flexible rods. Certain parts of the cytoplasm are filled with large, strongly electron-scattering globules, probably of lipid composition. These various cytoplasmic differentiations and the different forms of presumed virus material are discussed in relation to late stages of TMV reproduction and virus crystal formation.     

RELATION OF TOBACCO MOSAIC VIRUS TO THE HOST CELLS

The relation of tobacco mosaic virus (TMV) to host cells was studied in leaves of Nicotiana tabacum L. systemically infected with the virus. The typical TMV inclusions, striate or crystalline material and ameboid or X-bodies, which are discernible with the light microscope, and/or particles of virus, which are identifiable with the electron microscope, were observed in epidermal cells, mesophyll cells, parenchyma cells of the vascular bundles, differentiating and mature tracheary elements, and immature and mature sieve elements. Virus particles were observed in the nuclei and the chloroplasts of parenchyma cells as well as in the ground cytoplasm, the vacuole, and between the plasma membrane and the cell wall. The nature of the conformations of the particle aggregates in the chloroplasts was compatible with the concept that some virus particles may be assembled in these organelles. The virus particles in the nuclei appeared to be complete particles. Under the electron microscope the X-body constitutes a membraneless assemblage of endoplasmic reticulum, ribosomes, virus particles, and of virus-related material in the form of wide filaments indistinctly resolvable as bundles of tubules. Some parenchyma cells contained aggregates of discrete tubules in parallel arrangement. These groups of tubules were relatively free from components of host protoplasts.     

Effect of abscissic acid on tobacco mosaic virus

Abscisic acid (ABA) did not affect the infectivity of tobacco mosaic virus (TMV) in vitro. The same dilutions of ABA when applied on the leaves of Chenopodium amaranticolor Coste and Reyn. at different intervals before inoculation affected development of local lesions variably at different dilutions. The inhibition of local lesion formation was reduced at other intervals leading to stimulation at thirty minutes and six hours intervals. Post-inoculation treatments with 2 mg/l of ABA gave stimulation of local lesion formation, though other dilutions gave inhibition. Viral concentration was stimulated in the tomato seedlings root dipped in 0.2 mg/l of ABA for 6 hours and inoculated 24 hours after transplantation. Incorporation of different concentrations of ABA into tissue culture medium reduced the growth of the TMV infected tobacco callus tissue and stimulated the infectivity of the tissue grown over it assayed after three weeks.     

Systemic PCD occurs in TMV-tomato interaction

In hypersensitive response (HR), programmed cell death (PCD) is reported as a powerful defense mechanism in plant immune responses to pathogen. However, little is known about the PCD in systemic acquired resistance (SAR). Using tobacco mosaic virus (TMV) to infect the tomato (Lycopersicon esculentum cv. Jiafen 16) we found that localized TMV-infection could induce cell death in the uninoculated parts of the tomatoes, where the enzyme-linked immunosorbent assay (ELISA) showed no spreading virus. The biological and molecular characterization of this cell death was shown as following: chromatin condensed and formed peripheral conglomeration in nuclei; cell nucleus were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; mitochondria and chloroplast were disrupted; tonoplast and plasma membrane were shrunk and degradated. These results suggested that with an absence of TMV spread, the local TMV-infection on certain tomato leaves could induce systemic PCD in the root-tips, stem-apices and uninoculated leaves. The systemic PCD has various initiation and synchronization in such tissues and is distinct in inducement and exhibition from HR-PCD and SAR.     

5'-dephosphorylated 2',5'-adenylate trimer and its analogs. Inhibition of tobacco mosaic virus replication in tobacco mosaic virus-infected leaf discs, protoplasts, and intact tobacco plants

The effect of the 5'-dephosphorylated 2',5'-adenylate trimer and its 2',5'-trimer core analogs on the inhibition of tobacco mosaic virus (TMV) replication was determined in tobacco leaf discs, protoplasts, and whole tobacco plants, using infectivity tests and enzyme-linked immunosorbent assays. A structure-activity-metabolic stability-toxicity analysis of the 2',5'-adenylate trimer core molecule in TMV-infected Nicotiana glutinosa was determined. Modification at either the 6-amino position of the adenylate residues (i.e. inosinate trimer core) or at the 2' terminus (i.e. A-A-ara-A or A-A-Tu) inhibited replication of TMV. Modification of the 3'-hydroxyl group of the adenylate residues to 3-deoxyribose (i.e. the 2',5'-cordycepin trimer core) inhibited TMV replication better than the 2',5'-adenylate trimer core molecule. With enzyme-linked immunosorbent assays, there was complete inhibition of TMV replication by 200 nM 2',5'-adenylate trimer core for 60 h and by 200 nM 2',5'-cordycepin trimer core for 96 h. The amount of 2',5'-oligonucleotides associated with the leaves was determined using 2',5'-[3H]cordycepin trimer core; 1 X 10(-12) mol/cm2 of plant leaves inhibited TMV replication by 99%. No 2',5'-phosphodiesterase activity was detected in TMV-infected and noninfected leaf extracts. Therefore, the 2',5'-trimer cores were potent inhibitors of TMV replication at nanomolar concentrations, i.e. at 1000-fold lower concentration than that required in mammalian systems.     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.     

Infectivity suppressing and virus-binding activities of a membrane material isolated from tobacco leaves.

TMV binding substance (R) was isolated from a tobacco leaf membrane fraction and was purified by extraction with organic solvents and by column chromatography. Experimental results suggest that the binding of R with TMV results in inactivation of TMV. When tobacco leaves were inoculated with the R-TMV complex, it was found that the formation of polysome containing infecting viral RNA was inhibited. Model experiments showed that the mode of R-TMV adsorption to the membrane is different from that of TMV adsorption and that stripping of coat protein from TMV by SDS was inhibited by R. A possible explanation for the mechanism of this inhibition by R is that the R-TMV complex follows a pathway which does not lead to establishment of infection. Although less efficient, R was still active when it was applied after virus inoculation. Due to its affinity to coat protein, R might also interfere with a later process of viral multiplication.