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Glucan preparations were obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-β-1 .3-glucanase from marine mollusks. These preparations, like those of other sources, have an inhibitory effect on tobacco mosaic virus (TMV) infection of detached leaves of local and systemic host tobacco plants. [ABSTRACT FROM AUTHOR]

It was shown that tobacco leaf treatment with 100 mM H₂0₂ increased their content ofendogenous H₂0₂ and activities of catalase and hydrolases (acid phosphatase, proteases, and RNase) and also caused'various chang- es in the cell structure. In this case, programmed cell death (PCD) occurred in some cells, which was ob- served as chromatin condensation, cytoplasm collapse, etc. In the meantime, many cells displayed organelle activation rather than PCD. It is suggested that cells that undergo H₂0₂-dependent PCD release signaling molecules inducing protective mechanisms against oxidative stress in neighboring cells not exhibiting PCD.

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.

Effect of chitosan on the mesophyll cell ultrastucture and activity of hydrolases in leaves of tobacco cv. Samsun was studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased) and, at the same time, caused some activation of lytic compartment expressed in the stimulation of the formation of dictyosomes, smooth ER elements and cytoplasmic vacuoles, which are all prominent constituents of this compartment. In biochemical experiments, it was established that chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) in the leaves as compared to untreated leaves. In some cells chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm and so on) that can be classified as a result of programmed cell death development.

Ultrastructural examination of tobacco mosaic virus-induced local lesions developing in leaves of Datura stramonium plants demonstrated that, in the central area of the lesions, the cell response to viral invasion was not uniform. Most cells exhibited an acute hypersensitive reaction and underwent rapid and complete necrosis. However, some cells, despite considerable virus accumulation and immediate contact with completely collapsed cells, maintained a certain degree of structural integrity. Analysis performed showed that the proportion of collapsed and uncollapsed cells in the lesion centre 3 to 5 days after infection did not change essentially. These data suggest that the absence of hypersensitive response in some cells in the lesion centre is not due to an early stage of infection but is likely caused by cell tolerance of the virus.

Enzymatic (the action of lysozyme) and chemical (hydrogen peroxide) hydrolysis of chitosans with various degree ofacetylation (DA)--25, 17, and 1.5%--was performed. Purification and fractioning of the hydrolysis products were performed using dialysis, ultrafiltration, and gel-penetrating chromatography Low-molecular (LM) derivatives of the polysaccharide with molecular masses from 17 to 2 kDa were obtained. The study of their antiviral activity against the tobacco mosaic virus (TMV) showed that these samples inhibited the formation of local necroses induced by the virus for 50-90%. The antiviral activity of the LM chitosans significantly increased with the lowering of their polymerization degree. Furthermore, the products of the enzymatic hydrolysis possessed higher activity than the chitosan samples obtained as a result of chemical hydrolysis. It was revealed that the exhibition of the antiviral activity weakly depended on the degree of acetylation of the samples.

Influence of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in the mesophyll cells of Nicotiana tabacum L. var Samsun leaves in early period of infection development (3 days after infection of leaves) has been studied. The virus accumulated in the cells of the leaves treated for 24 h before infection with chitosan to a lesser degree than in the control cells. The chitosan affected the formation of TMV-specific granular and tubular inclusions which are known to consist of the viral replicase components. Three days after infection of the leaves treated with the chitosan, a typical sign of the infection development was the predominant formation of granular inclusions which are known to appear at the early stages of TMV replication. The infected cells of the leaves untreated with chitosan contained mainly tubular inclusions which had been shown previously to be formed from granular ones at the last stages of the infection process. This indicates that chitosan treatment of the leaves leads to a delay of the development of infection. In phosphotungstic acid-stained suspensions obtained from the infected leaves, abnormal (swollen and "thin") TMV particles were observed along with normal ones. The appearance of abnormal virus particles seems to be caused by virus-induced activation of intracellular lytic processes. The most lytic activity in the infected cells as well as the highest number of abnormal viral particles was observed under the chitosan action. Therefore, it appears that chitosan-mediated stimulation of lytic processes causing destruction of TMV particles may be one of the protective mechanisms limiting virus accumulation in cells.

The effect of kappa/beta-carrageenan from red alda Tichocarpus crinitus on the development of a potato virus X (PVX) infection in the leaves of Datura stramonium L. has been studied. The treatment of leaves with carrageenan stimulates a protein synthesis in the cells, causing an increase in the size of nucleoli and in the number of mitochondria and membranes of the granular endoplasmic reticulum. At the same time, such treatment slightly stimulates lytic processes, causing an increase in the number of agranular endoplasmic reticulum cisterns, dictyosomes, and cytoplasmic vacuoles and the formation of cytoplasmic zones, transparent to electron microscopy. The carrageenan-induced stimulation of lytic processes results in the destruction of viral particles and can be considered as one of the defense mechanisms, preventing the intracellular accumulation of viruses. The carrageenan-stimulated formation of PVX-specific laminar structures, able to bind viral particles and, therefore, prevent their intracellular translocation and reproduction, represents another carrageenan-induced mechanism of the antiviral defense in plant cells.

The effect of disodium salt of 2-acetyl-4-hydroxycarbonylmethylthio-5-chlorocyclopent-4-en-1,3-dione on development of the infection caused by tobacco mosaic virus in the leaves of two Nicotiana tabacum L. cultivars--an oversensitive cultivar Xanthi-nc and systemically affected Samsun--was studied. The results suggest that this compound interferes with reproduction of the virus. This antiviral displayed the highest activity when applied in a mixture with the virus, presumably due to its action on both the plant and the virus.

Influence of fucoidan from brown alga Fucus evanescens C. Ag. on the development of infection induced by potato virus X (PVX) in Datura stramonium leaves was studied. It as been shown that 24 h after the treatment of the leaves with fucoidan and following infection of them with PVX the accumulation of virus particles in infected cells during early infection period was substantially less than that in untreated control. Using ultrastructure-morphometric analysis, it has been established that fucoidan treatment increases at protein-synthesizing capability of cells (nucleolus dimension, amount of mitochondria and rough endoplasmic reticulum membranes become increased). At the same time, the fucoidan treatment causes some activation of lytic compartment which leads to destruction of virus particles and, therefore, might be considered as one of fucoidan-dependent protective mechanisms limiting virus accumulation in cells. Fucoidan stimulation of the formation of PVX-specific laminated structures capable of virus particles binding is possibly another induced antiviral cell mechanism, preventing from virus reproduction and transposition.