The hypersensitive reaction to a pathogen is one of the most efficient defense mechanisms in nature and leads to the induction of numerous plant genes encoding defense proteins. These proteins include: 1) structural proteins that are incorporated into the extracellular matrix and participate in the confinement of the pathogen; 2) enzymes of secondary metabolism, for instance those of the biosynthesis of plant antibiotics; 3) pathogenesis-related (PR) proteins which represent major quantitative changes in soluble protein during the defense response. The PRs have typical physicochemical properties that enable them to resist to acidic pH and proteolytic cleavage and thus survive in the harsh environments where they occur: vacuolar compartment or cell wall or intercellular spaces. Since the discovery of the first PRs in tobacco many other similar proteins have been isolated from tobacco but also from other plant species, including dicots and monocots, the widest range being characterized from hypersensitively reacting tobacco. Based first on serological properties and later on sequence data, the tobacco PRs have been classified in five major groups. Group PR-1 contains the first discovered PRs of 15-17 kDa molecular mass, whose biological activity is still unknown, but some members have been shown recently to have antifungal activity. Group PR-2 contains three structurally distinct classes of 1,3-beta-glucanases, with acidic and basic counterparts, with dramatically different specific activity towards linear 1,3-beta-glucans and with different substrate specificity. Group PR-3 consists of various chitinases-lysozymes that belong to three distinct classes, are vacuolar or extracellular, and exhibit differential chitinase and lysozyme activities. Some of them, either alone or in combination with 1,3-beta-glucanases, have been shown to be antifungal in vitro and in vivo (transgenic plants), probably by hydrolysing their substrates as structural components in the fungal cell wall. Group PR-4 is the less studied, and in tobacco contains four members of 13-14.5 kDa of unknown activity and function. Group PR-5 contains acidic-neutral and very basic members with extracellular and vacuolar localization, respectively, and all members show sequence similarity to the sweet-tasting protein thaumatin. Several members of the PR-5 group from tobacco and other plant species were shown to display significant in vitro activity of inhibiting hyphal growth or spore germination of various fungi probably by a membrane permeabilizing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Laminarin, a linear ␤-1,3 glucan (mean degree of polymerization of 33) was extracted and purified from the brown alga Laminaria digitata. Its elicitor activity on tobacco (Nicotiana tabacum) was compared to that of oligogalacturonides with a mean degree of polymerization of 10. The two oligosaccharides were perceived by suspension-cultured cells as distinct chemical stimuli but triggered a similar and broad spectrum of defense responses. A dose of 200 g mL Ϫ1 laminarin or oligogalacturonides induced within a few minutes a 1.9-pH-units alkalinization of the extracellular medium and a transient release of H 2 O 2 . After a few hours, a strong stimulation of Phe ammonia-lyase, caffeic acid O-methyltransferase, and lipoxygenase activities occurred, as well as accumulation of salicylic acid. Neither of the two oligosaccharides induced tissue damage or cell death nor did they induce accumulation of the typical tobacco phytoalexin capsidiol, in contrast with the effects of the proteinaceous elicitor ␤-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight ␤-1,3-1,6 glucans from fungal cell walls, and the ␤-1,6-1,3 heptaglucan showed that the elicitor effects observed in tobacco with ␤-glucans are specific to linear ␤-1,3 linkages, with laminaripentaose being the smallest elicitor-active structure. In accordance with its strong stimulating effect on defense responses in tobacco cells, infiltration of 200 g mL Ϫ1 laminarin in tobacco leaves triggered accumulation within 48 h of the four families of antimicrobial pathogenesis-related proteins investigated. Challenge of the laminarin-infiltrated leaves 5 d after treatment with the soft rot pathogen Erwinia carotovora subsp. carotovora resulted in a strong reduction of the infection when compared with water-treated leaves.

Four endochitinases (poly [1,4-(N-acetyl-j8-D-glu samlnide)J glycanohydrolase, EC 3.2.1.14) have been purified from leaves of Niotiana tabacum cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Two of them are acidic proteins of molecular weights 27,500 and 28,500 and have been identified as 2 ofthe 10 pathogenesis-related proteins that are known to accumulate in tobacco in response to stress or pathogen attack. These two pathogenesis-related proteins, named "P9 and "Q" when their biological function was unknown, account for one-third of tobacco mosaic virusinduced chitinase activity of tobacco leaves. They are serologIcally closely related to the two other chitinases, which can be considered as new basic pathogenesis-related proteins. These two basic chitinass exhibit higher molecular weights (32,000 and 34,000) and higher specific enzyme activity than the two acidic isoforms. treatment with salicylic acid (27) or TMV infection (28), in agreement with the rapid de novo synthesis of PR-proteins, which has been demonstrated in TMV-infected tobacco leaves (29). Similarly, treatment ofcell suspension cultures of parsley with a fungal elicitor resulted in a rapid increase in the transcription rate of two .In spite of the great wealth of data available on PRproteins, thus far no biological function has been demonstrated for these proteins. Here we report on the catalytic activity of two of them that are acidic chitinases (namely, proteins PR-P and PR-Q). We have purified and characterized two other basic chitinases from tobacco leaves reacting hypersensitively to TMV. These two basic isoforms can be considered as new PR-proteins of tobacco. Serological relationships between all four chitinases of tobacco have been demonstrated.Upon infection of plants with viruses (1-6), viroids (4, 7), fungi (4, 8), or bacteria (9), the development of symptoms is accompanied by the accumulation of soluble host-encoded proteins. Such proteins were first detected in tobacco cultivars reacting hypersensitively to tobacco mosaic virus (TMV) (1, 2) but now have been found in 16 plant species (10) under various circumstances. Since their appearance at first could only be related to pathological conditions, they were named "pathogenesis-related" proteins (PR-proteins) (11). However, several PR-proteins appear spontaneously in healthy tobacco plants when they start to flower (12,13) and in tomato leaves during natural aging (4), and they are expressed constitutively in interspecific hybrids of tobacco (14). They can also be induced by plasmolysis (15), by the application of a variety of chemicals (16,17), or by high concentrations of plant hormones (18).PR-proteins have characteristic properties that aid in their detection: they are selectively extractable at low pH (19,20), highly resistant to proteolytic enzymes (21, 22), localized predominantly in the intercellular spaces (23), and easily resolved by electrophoresis in polyacrylamide gels under native conditions. In tobacco, 10 major PR-proteins can be detected (13,...

Three of the ten acidic ‘pathogenesis‐related’ (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely −O, −N and −2, have been shown to have 1,3‐β‐glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross‐reacted with a basic protein which is also a 1,3‐β‐glucanase induced by virus infection and which can be considered as a new basic pathogenesis‐related protein of tobacco. Protein PR‐O and the basic 1,3‐β‐glucanase display about the same specific enzymatic activity, i.e. 50‐fold and 250‐fold higher than specific activities of proteins PR‐N and −2 respectively.