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.

H 2 O 2 from the oxidative burst, cell death, and defense responses such as the production of phenylalanine ammonia lyase (PAL), salicylic acid (SA), and scopoletin were analyzed in cultured tobacco (Nicotiana tabacum) cells treated with three proteinaceous elicitors: two elicitins (␣-megaspermin and ␤-megaspermin) and one glycoprotein. These three proteins have been isolated from Phytophthora megasperma H20 and have been previously shown to be equally efficient in inducing a hypersensitive response (HR) upon infiltration into tobacco leaves. However, in cultured tobacco cells these elicitors exhibited strikingly different biological activities. ␤-Megaspermin was the only elicitor that caused cell death and induced a strong, biphasic H 2 O 2 burst. Both elicitins stimulated PAL activity similarly and strongly, while the glycoprotein caused only a slight increase. Only elicitins induced SA accumulation and scopoletin consumption, and ␤-megaspermin was more efficient. To assess the role of H 2 O 2 in HR cell death and defense response expression in elicitin-treated cells, a gain and loss of function strategy was used. Our results indicated that H 2 O 2 was neither necessary nor sufficient for HR cell death, PAL activation, or SA accumulation, and that extracellular H 2 O 2 was not a direct cause of intracellular scopoletin consumption.

SummaryA 32 kDa glycoprotein whose effects in tobacco and other Nicotianae mimic a typical hypersensitive response, was isolated from Phytophthora megasperma. Infiltration of a few nanograms of the protein into leaves caused the formation of lesions that closely resemble hypersensitive response lesions. Transcripts of genes encoding enzymes of the phenylpropenoid and cesquiterpenoid pathways accumulated rapidly after elicitor application followed by salicylic acid production. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of PR protein genes was activated. After several days systemic acquired resistance was also induced. Thus, tobacco plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in • Nicotianae and possibly into crop plant species.

A glucan preparation obtained from the mycelial walls of the fungus Phytophthora megasperma f.sp. glycinea and known as an elicitor of phytoalexins in soybean was shown to be a very efficient inducer of resistance against viruses in tobacco. The glucan preparation protected against mechanically transmitted viral infections on the upper and lower leaf surfaces. Whether the glucan preparation was applied by injection, inoculation, or spraying, it protected the plants if applied before, at the same time as, or not later than 8 hours after virus inoculation. At concentrations ranging from 0.1 to 10 micrograms per milliliter, the glucan preparation induced protection ranging from 50 to 100% against both symptom production (necrotic local lesions, necrotic rings, or systemic mosaic) and virus accumulation in all Nicotiana-virus combinations examined. However, no significant protection against some of the same viruses was observed in bean or turnip. The host plants successfully protected included N. tabacum (9 different cultivars), N. sylvestris, N. glutlnosa, and N. clevelandii.The viruses belonged to several taxonomic groups including tobacco mosaic virus, alfalfa mosaic virus, and tomato black ring virus. The glucan preparation did not act directly on the virus and did not interfere with virus disassembly; rather, it appeared to induce changes in the host plant that prevented infections from being initiated or recently established infections from enlarging. The induced resistance does not depend on induction of pathogenesis-related proteins, the phenylpropanoid pathway, ligninlike substances, or callose-like materials. We believe the induced resistance results from a mechanism that has yet to be described. the observed resistance (for recent reviews, see 8, 26). The metabolic alterations that occur during a viral-induced hypersensitive reaction appear to be similar to those that occur in the same host during incompatible interactions with pathogenic bacteria and fungi (7). The most commonly observed metabolic changes are the production of phytoalexins and phenylpropanoid metabolites. In previous reports, it was shown that the phenylpropanoid pathway is strongly activated ( 15) in tobacco leaves reacting hypersensitively to TMV2 and that the virus localizing mechanism was weakened (larger lesions) if infected leaves were treated with competitive inhibitors of phenylalanine ammonia-lyase, a key enzyme in the pathway (19,20). This suggested an involvement of the activated phenylpropanoid pathway in the localizing mechanism.Treatment of various plant materials with elicitors of microbial origin have been shown to mimic defense responses and to stimulate the pathways leading to phenylpropanoids and flavonoids (for reviews, see 1, 4). Among these elicitors were cell wall fragments and substances present in the culture filtrates ofthe fungus Phytophthora megasperma f.sp. glycinea (Pmg) (1, 2). In particular, poly-and oligosaccharides extracted and purified from cell walls of this fungus were shown to exhibit very h...