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Extracellular polysaccharides /EPS/ produced by bacterial phytopathogens are known to take part in triggering or suppressing plant defense reactions. However, the functions of these compounds synthesized by ring rot pathogen - bacteria Clavibacter miсhiganensis subsp. sepedonicus /Cms/, are obviously studied insufficiently.

Isolation of protoplasts. Leaves of two-week-old in vitro plants of two cultivars were used for protoplasts extraction: Lugovskoi cultivar - resistant to ring rot pathogen and Luk’yanovskii - susceptible cultivar. Test-tube plants were grown in factor-state conditions on the Murashige-Skoog solid medium with the addition of hormones and vitamins /13/. To facilitate the isolation of protoplasts, the plants were first kept in darkness for 24 h. Then leaves separated from the plants were cut into fragments of 4-6 mm² in Murashige-Scoog medium on 0.06 М К–Na phosphate buffer at рН 5.8 and saturated via creating reverse pressure by enzymatic solution of the following composition with the final concentrations: 1% macerozyme R-10 (Serva, Germany-USA); 1% cellulase; 0,5% hemicellulase (Sigma, USА); 0,3 М sorbitol; 3 мМ CaCl₂; 1% BSA (Reakhim, Russia); 0,06 М K–Na phosphate buffer, рН 5.8 : Murashige-Skoog medium (1:1). Total volume of the medium amounted to 3 ml. For preventing the hydrolysis of the receptors’ protein part with proteases which could be present in the preparations of pectolytic enzymes used, as well as for blocking non-specific EPS binding with surface plasma membrane determinants 1% BSA was added to the medium /9,14/. Solutions with leaf fragments were placed in the bottles and put in the shaker at 28⁰ С for 3 h. Protoplasts were separated from damaged cells by means of filtration through a kapron fabric, and then, twice, 5 min each time, were washed by isolation medium without enzymes.

EPS conjugation with rhodamine isothiocyanate (RITC). EPS were isolated and purified from the cultivation medium of Cms strain 5369 virulent for Luk’yanovskii cultivar by the method described earlier /11/. 0.2 g of EPS were dissolved in 10 ml of 0.1 М of carbonate-bicarbonate buffer, рН 9.3. 0.02 g of RITC were dissolved in the same buffer and volume. The solutions were mixed and placed in thermostat (27⁰ С) for 3 h. The conjugate obtained was purified from both salt and non-bound dye on the column (2.2 х 40 cm) with sefadex G-25 (Sigma, USA) by eluating with distilled water. Eluate going out was controlled with the wave length of 206 nm using chromatographic equipment Uvicord (LKB, Sweden). The conjugate was evaporated to dry condition on the vacuum rotor vaporizer at 35⁰ С and stored in the refrigerator.

Protoplasts incubation with EPS-RITC conjugate, luminescent microscopy. 1 ml of 0.05% EPS-RITC conjugate dissolved in protoplasts extraction medium without enzymes was added to 1 ml of the obtained protoplasts suspension (10⁴ cells/ml), then the samples were placed in the shaker and incubated for 1 h in the darkness. To remove non-bound conjugate the protoplasts were twice washed (10 minutes each time) in the isolation medium without enzymes and precipitated by centrifuging at 100 g for 3 min. The cells were watched in a luminescent microscope ML-2 (LOMO, Russia). Mercury lamp DRSH-250 was used as a light source. In order to observe proper and induced fluorescence a pervious light filter DBLF-15 (maximum permeation at λ = 400 nm) and a locking light filter YLF-18 (permeation at λ ³ 490 nm) was used. Non-colored protoplasts and RITC-treated protoplasts (0.05% solution in the isolation medium without enzymes) were used as control of induced fluorescence specificity. The objects were photographed using photo-nozzle FMN-10 (LOMO, Russia) and photo-film Kodak Academy-200 (KODAK, England). Two independent tests were done, with no less than 50 protoplasts analyzed for each variant.

Results and Discussion

With the light filter DBLF-15 pervious for blue-violet and ultra-violet parts of spectrum (range 340-480 nm), protoplasts treated with EPS-RITC complex fluoresced with blue light. The intensity of this fluorescence differed significantly in protoplasts of two cultivar cells. Thus, the surface of protoplasts isolated from the leaves of resistant cultivar plants (protoplasts-I) practically did not fluoresce (Fig. 1 a), whereas protoplasts isolated from the analogous plant tissues of susceptible cultivar (protoplasts-II) fluoresced fairly brightly (Fig. 1 b). The surface of control samples (protoplasts without incubation with EPS-RITC, and those treated with RITC only) isolated from both cultivars did not possess proper fluorescence (Fig.1 c, data shown only for protoplasts treated with RITC only). It is to be noted that chloroplasts present in significant amounts in cytoplasm (Fig. 1 d,f) fluoresced with intense red light (Fig.1 a,c). The results obtained allow a conclusion to be made that the receptors to EPS Сms are present in the plasma membrane of potato cells. This can be judged by the sorbtion of EPS-RITC complex on the surface of protoplasts and by its binding possibly with the receptors given. The intensive EPS-RITC fluorescence on protoplasts-II (Fig. 1 b) proves the great number of receptors supposed to be on the plasma membrane of susceptible cultivar cells. In contrast, nearly complete fluorescence absence of this compex on the surface of protoplasts-I (Fig. 1 a) points to the essentially less number of such receptors in resistant cultivar cells. The similar results have been obtained for the plasma membrane fractions isolated from potato suspension cells the same cultivars /12/, and for the receptors of cotton plant cells to phytotoxin producing Verticillium dahliae in the work by Dubery and Meyer /15/.

The role of EPS in bacteria virulence has been widely discussed in literature /10, 16-17/. According to our data, EPS of Cms virulent strain demonstrated activating and suppressing action on the peroxidase activity of potato suspension cells of resistant and susceptible cultivars, respectively /18/. Thus, these compounds may act both as elicitors and suppressors of plants defence responses. This is probably due to heterogenous nature of EPS, i.e.: they consist of four-six components with molecular weights ranging from more than 700 to less than 1 kDa /10-11, 16/. According to the data published, depending on the degree of polymerization, EPS may show elicitor or suppressor properties /19/. The following facts can prove the possible role of EPS Cms in triggering elicitor-induced defensive responses of potato plants. We have shown that under high infection load by Cms unusual symptoms – local necroses on the leaves characteristic of hypersensitive response (HR) typical of incompatible interactions under pathogenesis in resistant cultivar take place /18/. Free oxygen radicals and hydrogen peroxide are known to be capable of initiating HR reaction /20/ in which realization of lipoxygenase and superoxidsynthase signal systems take part /21/.

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8. Pertl H., Himly M., Gehwolf R., Kriechbaumer R., Strasser D., Michalke W., Richter K., Ferreira F., Obermeyer G. // Planta, 2001, v. 213, p.132-141.

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19. Ozeretskovskaya O.L., Romenskaya I.G. // Rus. J. Plant Physiol., 1996, v. 43, p. 743-752 /in Russian/.

21. Tarchevsky I.A. // Rus. J. Plant Physiol., 2000, v. 47, p. 321-331 /in Russian/.

Fig. 1. Binding of extracellular polysaccharides (EPS) of C. michiganensis ssp. sepedonicus (Cms), strain 5369, on protoplasts isolated from potato leaf tissue cells.

b, c, e, f – Lukyanovskii cv (susceptible); a, b, d, e – treatment by EPS-RITC; c, f - control (treatment with RITC only); а-c – fluorescent microscopy; d-f – light microcscopy. Long arrows show chloroplasts; short arrows show vacuoles. The line is 20 m m.

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