Marguerite Kopp

Structural features of fungal β-d-glucans for the efficient inhibition of the initiation of virus infection on Nicotiana tabacum

Glucans of fungal origin have been shown to inhibit the early stages of infection of Nicotiana by numerous viruses of different taxonomic groups. Several glucans were isolated from the cell walls of Phytophthora parasitica, Phytophthora megasperma f. sp. glycinea (Pmg) and Fusarium oxysporum, and their antiviral activity compared on tobacco leaves inoculated with tobacco mosaic virus. These polysaccharides consist of a mixture of (1-->3)(1-->6)-beta-D-glucans with M(r) varying from 1.1 x 10(3) to 2 x 10(6). Requirements for a prominent antiviral activity of the fungal polysaccharides are a beta-(1-->3)(1-->6)-D-glucan structure with mono-, di-, tri- or tetra-glucosidic side branches attached to a linear main chain of beta-(1-->3)-linked-D-glucose residues. Very high activity is correlated with a high degree of branching at position 6 and with the size and glycosidic nature of the side chains. The molecular masses and the organized structure of fungal beta-D-glucans are not essential for their antiviral activity. The structural motif for antiviral activity in Nicotiana is distinct from that required for elicitation of phytoalexins in soybean cotyledons.

Virus-induced glycanhydrolases and effects of oligosaccharide signals on plant-virus interactions

The hypersensitive response is one of the most efficient mechanisms of defence that is induced by the infection itself. Even though the triggering of the hypersensitivity reaction results from a very specific gene-for-gene recognition between plant and pathogen, what the plant does thereafter to defend itself appears to be specific only to the host. The alterations of host metabolism believed to participate in active defence include cell wall thickening resulting from production and deposition of various macromolecules, and the production of defence enzymes and proteins. Among the cell wall macromolecules are lignins and other phenolic polymers, polysaccharides such as callose, and proteins such as the hydroxyproline rich glycoproteins. Defence enzymes fall into two classes: enzymes that catalyze the production of various metabolites participating in resistance (ethylene, phytoalexins, aromatic compounds, oxidized metabolites), and direct defence enzymes (hydrolases such as chitinases and glucanases). The defence proteins include inhibitors of microbial proteases or of polygalacturonases and “pathogenesis-related” (PR) proteins.

Phenylalanine Ammonia-Lyase Levels in Protoplasts Isolated from Hypersensitive Tobacco Pre-infected with Tobacco Mosaic Virus

Leaves of tobacco varieties carrying the N gene for hypersensitiviy react to tobacco mosaic virus (TMV) infection by forming necrotic lesions and by localizing the virus in the vicinity of these lesions. These changes are accompanied in the host by an increased metabolic activity, in particular by an increased production of phenolic compounds derived from phenylalanine. Necrogenesis apparently destroys cells which have become heavily infected despite this strong defense reaction. However, it has been demonstrated previously (Otsuki et al., 1972) that protoplasts derived from leaves which normally respond in vivo to virus inoculation by forming necrotic local lesions, show no such response when inoculated in vitro. In the present study we have investigated the effect of pre-infecting hypersensitive leaves with TMV on the production or the non-production of the factor(s) of necrosis at the level of either protoplasts or mesophyll cells isolated from these preinfected leaves. Phenylalanine ammonia-lyase (PAL), whose rate of synthesis has been shown (Duchesne et al., 1977) to increase in stimulated cells of infected leaves, was used as a biochemical marker in the search for the stimulus preceding necrogenesis. We found that this stimulus concerning PAL activity was never elicited in either protoplasts or mesophyll cells which were prepared just before the appearance of necrotic local lesions. This result did not depend on the conditions of pre-infection or on the methods used to isolate the protoplasts or mesophyll cells. We also assayed samples derived from pre-infected leaves that were already carrying local lesions, i.e., in which the stimulus and necrogenesis were already operating: not only did the isolated protoplasts and mesophyll cells not sustain the stimulus concerning PAL activity, but the stimulated enzyme activity decreased abruptly and, in most of the experiments, had disappeared within the time necessary for maceration. Evidence is presented showing that the non-elicitation or the abrupt decrease of stimulated PAL activity could not result from a selection of unstimulated cells or from a preferential destruction of stimulated cells during maceration of the leaves.Our results support the view that hypertonic osmotic pressure is responsible for the non-occurence of the hypersensitive response by acting according to one or both of the following processes: it suppresses the contacts through plasmodesmata between neighboring cells and, hence, it also suppresses the cell-to-cell diffusion of the factor(s) eliciting the stimulus; and/or since hypertonic osmotic pressure causes striking differences between leaf cells and protoplasts in total RNA and protein synthesis, these differences might include the suppression of synthesis of the elicitor of hypersensitivity.

Defence Proteins, Glycanhydrolases and Oligosaccharide Signals in Plant-Virus Interactions

Most defence proteins and enzymes that are typical of incompatible plant-fungi interactions are also induced or stimulated in hypersensitive plant-virus interactions. Examples include “pathogenesis-related” (PR) proteins, proteases, inhibitors of proteases, hydrolases (chitinases and 1,3-s-glucanases), enzymes of ethylene biosynthesis, of aromatic metabolism, of phytoalexin biosynthesis and oxidative enzymes. At least 10 PR proteins are glycanhydrolases (4 chitinases and 6 glucanases) with differential specific activity. They are direct anti-microbial defence enzymes and are supposed to cause other defence responses through the release of oligosaccharidic elicitors of pathogen and/or plant origin. Oligosaccharides are shown to protect plants from infection by viruses by a host-mediated process that may not be a classical defence mechanism.

Plant disease and the regulation of enzymes involved in lignification Effects of osmotic pressure on phenylpropanoid enzymes and on the hypersensitive response of tobacco to tobacco mosaic virus

Tobacco varieties carrying the N gene from Nicotiana glutinosa respond to infection by Tobacco Mosaic Virus (TMV) by forming necrotic local lesions (hypersensitive reaction), thereby localizing the infection. In this study, infected mesophyll leaf tissue of N. tabacum Samsun NN was treated with the non-permeating, non-metabolizable carbohydrate mannitol. The local lesions developed under iso-osmotic conditions (0.28 M mannitol), though with a slight delay and with a reduced rate of growth, as compared to those on attached leaves. At increasing plasmolysing concentrations of mannitol, necrotization was progressively inhibited, but not completely suppressed. The leaf tissue produced tiny translucent zones, with a delay that increased between the virus inoculation and application of the plasmolytica. Activities of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and O-methyltransferase (OMT, EC 2.1.1.6) are strongly stimulated in hypersensitively reacting tobacco and were used as biochemical markers in the present study. This study was done to determine whether the inhibitory effect of plasmolysis on the elicitation of the hypersensitive response is due to a decrease in virus spread, resulting from the rupture of plasmodesmata or, at least in part, to metabolic alterations of the host cell exposed to osmotic stress. Since necrotization is normally preceded by intense virus multiplication, the inhibitory effects found for early applications (i.e., before local lesion appearance) of plasmolytica could easily be related to an inhibition of virus spread which also occurred in similarly treated leaf tissue of the systemically reacting variety Samsun. The most meaningful data were obtained from mannitol treatments performed on leaf tissue already carrying local lesions, i.e., in which the elicitor(s) and/or the factor(s) of necrotization were already operating. Under iso-osmotic conditions, we found the stimulated PAL and OMT activities characteristic of the hypersensitive response. At plasmolysing concentrations of mannitol, we observed the counteracting effects of two different mechanisms controlling the phenylpropanoid enzymes. Floating the leaf material on the liquid medium induced an ageing-like effect with a continuous increase in enzyme activities that was independent on osmotic pressure and sensitive to cycloheximide. At the same time, the stimulated enzyme activities related to hypersensitivity decreased at a rate related to osmotic pressure. Since PAL and OMT of tobacco leaves are long-lived enzymes, it is likely that the increased de novo synthesis of the enzymes was suppressed by plasmolysis while their degradation and/or inactivation was maintained or even increased. From these results it is concluded that the apparent inhibition of the hypersensitive response by plasmolysis is due to both a decrease in virus spead (artificially caused by the rupture of connections between cells) and to drastic metabolic alterations of the host cell exposed to high osmotic pressure.

Phenylalanine ammonia-lyase activity of protoplasts: In vitro inhibition by mannitol and sorbitol

Abstract Phenylalanine ammonia-lyase (PAL) activity was strongly inhibited in vitro by D -mannitol and D -sorbitol at concentrations exceeding 50 mM,

Plant Genes Involved in Resistance to Viruses

The model system that will be considered here is the hypersensitive response (HR) since it is one of the most efficient natural mechanisms of defence of plants against pathogens. This is also true in the case of virus infections. In hypersensitivity to viruses the same characteristics are found as in incompatible interactions between plants and fungi or bacteria: i) necrosis at and around each point at which the leaf tissue was infected; ii) localization of the pathogen to the region of each initiated infection; iii) induction of marked metabolic changes in the cells surrounding the necrotic area, these changes being believed to cause, or at least to contribute to, the resistance observed. Furthermore, the hypersensitive response to viruses involves a cascade of events and signals (Fritig et. al., 1987) similar to that proposed for active defence against fungi and bacteria (Lamb et al., 1989): the response would be initiated by a specific gene-for-gene recognition between the plant and the virus that would lead to cell damage (death of a number of cells) and the release of intermediary signals which in turn would enter a reception-transduction pathway leading to major changes in gene expression (usually gene activation) responsible for the resistance observed.