Plant Molecular Biology 51: 109–118, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin Sylvain Cordelier, Patrice de Ruffray, Bernard Fritig and Serge Kauffmann∗
Institut de Biologie Mol´eculaire des Plantes du C.N.R.S., Universit´e Louis Pasteur. 12, rue du G´en´eral Zimmer, 67084 Strasbourg, France (∗ author for correspondence; e-mail:
[email protected]) Received 14 February 2002; accepted in final form 15 May 2002
Key words: acquired resistance, defense response, elicitor, ethylene, Nicotiana tabacum, salicylic acid, signalling pathway Abstract We have compared localized (LAR) and systemic (SAR) acquired resistance induced in tobacco by a hypersensitive response (HR) inducing Phytophthora megasperma glycoprotein elicitin. Three different zones were taken into account: LAR, SART and SARS . The LAR zone was 5–10 mm wide and surrounded the HR lesion. SART was the tissue of the elicitor-treated leaf immediately beyond the LAR zone. The systemic leaf was called SARS . Glycoprotein-treated plants showed enhanced resistance to challenge infection by tobacco mosaic virus (TMV). Disease resistance was similar in SART and SARS , and higher in LAR. The expression pattern, in glycoproteintreated plants, of acidic and basic PR1, PR2, PR3 and PR5 proteins and of O-methyltransferases (OMT), enzymes of the phenylpropanoid pathway, was similar to that in TMV-infected plants. OMT was stimulated in LAR but not in SART and SARS . The four classes of acidic and basic PR proteins accumulated strongly in LAR. Reduced amounts of acidic PR1, PR2, PR3 and only minute amounts of basic PR2 and PR3 accumulated in SART and SARS . In glycoprotein-treated plants, expression of the acidic and basic PR proteins in LAR and SAR of transgenic NahG and ETR tobacco plants and in LAR of plants treated with inhibitors of salicylic acid accumulation and of ethylene biosynthesis indicated a salicylic acid-dependent signalling pathway for acidic isoform activation and an ethylenedependent signalling pathway for basic isoform activation. Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; AOPP, α-aminooxy-ß-phenylpropionate; HR, hypersensitive response; LAR, localized acquired resistance; OMT, O-methyltransferase; PR, pathogenesis-related; SA, salicylic acid; SAR, systemic acquired resistance; TMV, tobacco mosaic virus. Introduction Plants have evolved elaborated strategies to defend themselves against pathogens. They include restriction of the pathogen at its site of penetration and development of systemic acquired resistance (SAR). Restriction can be achieved through the combined effects of the hypersensitive response (HR) and localized acquired resistance (LAR) (Dorey et al. 1997; Heath 1998). The HR is a commonly activated resistance process induced upon recognition of pathogen-derived
factors, and characterized by the rapid induction of localized host cell death. LAR is induced by plant signals diffusing out of the HR cells (Dorey et al. 1997). In tobacco mosaic virus (TMV)-infected tobacco plants containing the N gene, LAR was described as a 2 mm wide region, fluorescent under UV light, surrounding the virus lesions and showing a high level of resistance to challenge virus inoculation (Ross 1961a). A strong expression of a broad range of defense responses occurs in LAR. In tobacco reacting hypersensitively to TMV, they include accumulation
110 of antimicrobial proteins such as the pathogenesisrelated (PR) proteins (Fritig et al. 1998), and stimulation of enzymes of the phenylpropanoid pathway such as O-methyltransferases (OMT) (Legrand et al. 1978). This pathway provides antibiotics such as scopoletin which is fluorescent under UV light. Hence, LAR provides a locally and highly inhospitable environment for the invading pathogen (Heath 1998). LAR is clearly different from SAR. SAR provides a low, though significant, level of resistance against a subsequent infection (Ryals et al. 1996; Sticher et al. 1997). SAR develops beyond tissues exhibiting LAR (Ross 1961a; Ross 1961b). Thus, SAR occurs in the primary infected parts of the plant and throughout the host. For instance, in TMV-infected Samsun NN tobacco plants, the level of enhanced resistance to a challenge TMV infection was similar in the primary inoculated leaves and in the systemic leaves (Ross 1961b; Ross 1966). Messenger RNA coding for acidic and basic PR1, PR2, PR3, and PR5 proteins were shown to accumulate in high amounts in TMVinfected N-gene containing tobacco leaves (Brederode et al. 1991; Ward et al. 1991). In SAR leaves, expression of the acidic PR genes was relatively high while that of the basic PR2, PR3 and PR5 genes was very low and detected only at early times (Brederode et al. 1991; Ward et al. 1991). Concerning the basic PR1 gene expression in SAR leaves, Brederode et al. (1991) reported no response while Ward et al. (1991) found high steady state levels of the mRNA. Salicylic acid (SA) and ethylene are two plant signals playing crucial roles in resistance to pathogens (Boller 1991; Delaney et al. 1994). While expression of acidic PR genes was shown to be induced by SA (Ward et al. 1991; Yalpani et al. 1991), ethylene regulates that of the basic PR genes (Brederode et al. 1991; Knoester et al. 1998; Ohtsubo et al. 1999). Few reports have shown that proteinaceous elicitors of the HR also displayed SAR-inducing activity. Phytophthora elicitins induce the HR and SAR in tobacco, but SAR is a consequence of the systemic movement of the elicitins (Bonnet et al. 1996; Keller et al. 1996). Harpins, bacterial proteins, induce the HR in several plants and SAR in cucumber and Arabidopsis (Dong et al. 1999; Strobel et al. 1996). The localization of the harpin-induced HR to the treated tissue suggests that harpins do not diffuse. A P. megasperma glycoprotein induces the HR (Baillieul et al. 1995) and LAR (Dorey et al. 1997) when infiltrated into tobacco leaves. The glycoprotein does not diffuse out of the treated tissue, which un-
Figure 1. Scheme of the different tissues considered in the study. One, two or three leaves from a tobacco plant (treated leaf) were infiltrated with 50 nM glycoprotein to induce the HR, which remained strictly limited to the infiltration site, and LAR, which surrounded the HR lesion. LAR was defined as the tissue showing a bright blue fluorescence under UV light and was about 5–10 mm wide. The region immediately beyond LAR was called SART . The upper non-treated leaf, or systemic leaf, was called SARS .
dergoes the HR, demonstrating that a plant signal(s) is released to trigger LAR (Dorey et al. 1997). In this system, before cell death occurs, SA accumulates, OMT expression is stimulated, catalases are repressed and a strong oxidative burst occurs (Dorey et al. 1997; Dorey et al. 1998). LAR takes place in a ring of living cells, 5-10 mm wide and surrounding the HR lesion, exhibiting phenylpropanoid, sesquiterpenoid, catalase and PR gene activation, accumulation of SA and scopoletin (Costet et al. 2002). Here we have characterized and compared the glycoprotein induced LAR and SAR in tobacco at both the biological and molecular levels. We analyzed expression of the acidic and basic isoforms of PR1, PR2, PR3 and PR5 proteins, as well as OMT activity. Using genetical and pharmacological approaches, we investigated the signalling pathways involved in PR protein activation, focussing on SA and ethylene. Materials and methods Plant material and treatments Nicotiana tabacum L cv. Samsun NN, N. tabacum L cv. Samsun NN transgenic for the etr gene, and N. tabacum L cv. Xanthi nc transgenic for the nahg bacterial gene were grown in a greenhouse under controlled conditions. Three-months-old plants were placed 2–3 days before treatments in a growth chamber at 22±1 ◦ C with a photoperiod of 16 h. The glycoprotein elicitor was purified from the culture medium of the fungus Phytophthora megasperma H20 as described previously (Baillieul et al. 1995). Plant
111 treatments were realized by infiltration of aqueous solutions of the glycoprotein with a syringe into the mesophyll of 1 to 3 fully developed leaves. The infiltrated tissue (4 to 5 cm2 ) was immediately delineated with a felt-tip marker when necessary. For some experiments the glycoprotein was also co-infiltrated with cobalt chloride (CoCl2 , Sigma) or with αaminooxy-ß-phenylpropionate (AOPP). For virus infection, plants were inoculated with the common strain of TMV by rubbing the upper leaf surface with a glass pad and a suspension of purified virus in the presence of abrasive celite. The virus concentration was adjusted in order to induce about 150 local lesions per leaf of control noninfiltrated plants. Diameter of TMV lesions was determined 6 days after inoculation using an ocular micrometer. Protein and enzyme analysis Protein extraction was performed from 100 to 200 mg fresh weight tissue collected from the different zones, LAR, SART and SARS as defined in Figure 1. Tissues were ground in 20 mM Na-phosphate buffer (400 µL for 100 mg of fresh tissue), pH 7.5, containing 15 mM ß-mercaptoethanol, charcoal and quartz. The crude extract was centrifuged at 13000 rpm for 30 min and the supernatant was used to perform OMT activity and acidic and basic PR protein analysis. OMT activity was assayed as described elsewhere (Pellegrini et al. 1993). PR proteins were analyzed by western blotting as described in Baillieul et al. (1995). Protein extracts corresponding to 4 mg fresh weight were loaded onto the gels. The specificity of the different rabbit antisera used in this study to probe acidic and basic PR proteins was as follows. The anti-PR1b serum crossreacted with the 3 acidic PR1a, PR1b and PR1c, but not with the basic PR1g. The anti-PR1g serum recognized only PR1g. The anti-PR2a serum crossreacted with the 3 acidic PR2a (PR-2), PR2b (PR-N) and PR2c (PRO), but not with the basic PR2e (glub). The anti-PR2e serum recognized only the basic PR2e. The anti-PR3a serum crossreacted with the 2 acidic PR3a (PR-P) and PR3b (PR-Q) and the 2 basic PR3d (Chi32) and PR3e (Chi34). The anti-PR3d serum recognized only the 2 basic PR3d and PR3e. The anti-PR5a serum crossreacted with the 2 acidic PR5a (PR-R), PR5b (PR-S), but not with the basic PR5c (osmotin). The anti-PR5c serum recognized the basic PR5c and the 2 acidic PR5a and PR5b. Immunodetection was performed with the immun-star chemiluminescent kit of Bio-Rad.
Results The glycoprotein induces both LAR and SAR resistance to TMV infection in tobacco The experimental protocol designed to analyze TMV resistance in elicitor-treated Samsun NN tobacco plants was similar to that commonly used to analyze TMV resistance induced after a primary TMV infection (Ross 1961b; Ward et al. 1991). Basically, 2 or 3 leaves of 4 plants were infiltrated at 4 to 10 spots per leaf with 50 nM glycoprotein or water as a control of the treatment. Five days later, infiltrated and upper nontreated (systemic) leaves were inoculated with TMV. Symptoms and diameter of TMV lesions were analyzed after a further 6 days period. Three different zones in elicitor-treated plants were taken into account: the LAR, SART and SARS zones as shown in Figure 1. The LAR zone was the tissue showing the bright blue fluorescence under UV light, observed 2 days after elicitor treatments, and delineated with a felt-tip marker. SART corresponded to the tissue immediately beyond the LAR zone that surrounded the HR lesion. The tissue of the systemic non-treated leaf was called SARS . Controls were plants kept nontreated (control of the plant batch) before the challenge virus inoculation, plants infiltrated with water (negative control of the treatment) and plants virus-inoculated as the first treatment (positive control). Figure 2A shows the typical HR symptoms induced by the infiltration at 6 spots of the glycoprotein into a tobacco leaf while infiltration of water remained the leaf symptomless. The glycoprotein treatment induced SAR to TMV infection, and the glycoprotein was as efficient as a primary TMV inoculation to induce SAR. Figure 2B shows smaller TMV lesions in SARS leaves of glycoprotein-treated plants versus control water-treated plants. The magnitude of enhanced TMV resistance, as measured by the lesion size, was similar to that obtained after a primary TMV infection (Table 1). Lesion size analysis (table 1) revealed i) that resistance in LAR was higher than that in SART and SARS and ii) that resistance in SART was not significantly different from that in SARS . In 2 out of 3 experiments, the mean of lesion size in SART and SARS was different, lesion size mean in SART being lower than that in SARS . However, applying the Student’s t test to SART and SARS lesion size means indicated that there was no statistical difference between the 2 means. To apply Student’s
112 Table 1. Reduction of TMV lesion size in LAR, SART and SARS zones of glycroprotein-treated Samsun NN tobacco plants. Plants were infiltrated with 50 nM glycoprotein, with water, or were inoculated with TMV. The LAR zone was delineated with a felt type marker 2 days after elicitor application. It was defined as the tissue surrounding the HR lesion and showing a bright blue fluorescence under UV light. Five days after the treatments, plants were challenge inoculated with TMV. Virus inoculation was performed on elicitor- and water-treated leaves and systemic leaves of elicitor- and water-treated plants and of TMV infected plants. Lesion size was determined 6 days later. Results were expressed as the mean diameter (in mm) of the lesions with the standard deviation, and also as a percentage, indicated between brackets, versus controls which were plants kept nontreated before the challenge virus inoculation. Mean and standard deviation were calculated from a minimum of 300 lesions for LAR tissues and 900 lesions for SART and SARS tissues. Treatmentsa
Treated leaves Waterb
Systemic leaves Water
Glycoprotein LAR
SART
4 (2)
nd
nd
nd
4 (2)
2.39 ± 0.40 (96%) 2.29 ± 0.56 (100%) nd
1.45 ± 0.44 (60.6%) 0.92 ± 0.47 (40%) nd
1.75 ± 0.43 (73.2%) 1.19 ± 0.46 (52%) nd
2.29 ± 0.56 (100%)
0.82 ± 0.29 (36%)
1.06 ± 0.42 (46%)
6 (3) 8 (3) 10(3)
3.32 ± 0.38 (100%) 2.49 ± 0.36 100%) 2.29 ± 0.55 100%) 3.27 ± 0.46 100%) 2.29 ± 0.55 100%)
Glycoprotein SARS
TMVc
2.54 ± 0.31 (76.5%) 2.12 ± 0.5 (85%) 1.46 ± 0.53 (64%) 1.84 ± 0.52 (56.3%) 1.07 ± 0.44 (47%)
2.55 ± 0.44 (76.8%) nd 1.05 ± 0.49 (46%) 2.06 ± 0.52 (63.1%) 1.05 ± 0.49 (46%)
a: number of elicitor or water infiltration sites per leaf and, between bracketts, number of treated leaves per plant b: there was no difference in lesion size between those lesions found close to the infiltration site (equivalent to the LAR zone of elicitorinfiltrated leaves) and those found beyond (equivalent to the SART zone of elicitor-infiltrated leaves). Numbers indicate the lesion size mean of the pool of the 2 populations. c: plants were inoculated with TMV 5 days prior challenge virus inoculation nd: not determined
t-test to compare means, we need to check that the lesion distribution follows a normal distribution (Vu and Gros 1970). Lesion size in control plants, SART and SARS gave a near normal distribution curve, whereas lesion distribution in LAR was markedly skewed as we did not record the very small lesions. Most probably, several lesions had not progressed enough to result in a visible lesion, thus escaping counting. This phenomenon is particularly pronounced when lesion size tends towards small values, suggesting that the mean of lesion size in LAR should be even lower. A similar observation was reported during SAR to TMV in N-gene containing tobacco (Ross 1966). We also observed that the larger number of elicitor-infiltration sites the greater reduction in lesion size whatever the analyzed zone, LAR, SART or SARS (Table 1). OMT activity in LAR, SART and SARS OMT activity was monitored in LAR, SART and SARS as well as in tobacco leaves reacting hypersensitively to TMV infection and in the corresponding systemic leaves developing SAR. In TMV-infected tobacco plants, OMT activity was strongly induced in infected leaves but remained at basal levels in systemic
non-infected leaves (Figure 3A). A similar pattern was observed after treatment with the elicitor. OMT activity was induced in LAR but not in SARS (Figure 3B). Interestingly, no significant increase in OMT activity was monitored in SART , whether activity was expressed as pkat/mg protein (Figure 3B) or as pkat/g fresh weight (Figure 3C). Figure 3B and 3C present the results of 2 independent experiments realized at several months interval. In the experiment presented in Figure 3B, three leaves per plant were infiltrated with 50 nM glycoprotein or water at six different spots per leaf. In the Figure 3C experiment, one leaf was treated at 2 spots. In both experiments SART samples were collected at a distance between 1.5 to 2 cm from the external limit of the HR zone, corresponding to a distance between 0.5 to 1 cm beyond the LAR zone showing the bright blue fluorescence under UV light. Thus, a sharp drop in OMT activity occurred in close vicinity of tissues undergoing LAR. This result is further illustrated in Figure 3D. Samples S1 to S4 were collected radially with a 5mm diameter cork borer starting from the external limit of the HR zone, S1 being proximal to the HR lesion. In this experiment, most of the blue fluorescence under UV light
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Figure 2. Systemic acquired resistance to tobacco mosaic virus infection in glycoprotein-treated tobacco plants. Three leaves per plant were infiltrated with the glycoprotein or water at 6 spots per leaf and systemic leaves (SARS ) were inoculated with TMV 5 days later. A. Symptoms induced by the infiltration of water (left leaf) or glycoprotein (right leaf), and photographed 5 days after treatments. B. SARS leaves photographed 6 days after inoculation of TMV. Left leaf: SARS leaf from a water-treated plant. Right leaf: SARS leaf from a glycoprotein-treated plant.
was in S1, only residual fluorescence was in S2 and no fluorescence was in S3 and S4. OMT activity was stimulated in S1, a sharp decrease occurred in S2, and only basal levels compared to the control were measured in S3 and S4. OMT activity could thus be used as an internal control indicating that collected SART tissues were not contaminated with LAR tissues. Expression of acidic and basic PR proteins in LAR, SART and SARS We monitored by western blotting the expression of acidic and basic PR1, PR2, PR3, PR5 in LAR, SART and SARS of glycoprotein–treated plants (Figure 4) and of TMV inoculated plants (Figure 5). For virusinfected plants, LAR corresponded to the tissue centered to the HR lesion and the surrounding tissue showing the bright blue fluorescence under UV light, SART to the tissue between the lesions (taking care not
to collect tissue showing the fluorescence) and SARS to the tissue of the systemic non-inoculated leaf. As a control of tissue sampling, especially for SART samples from glycoprotein-treated plants, OMT activity was also measured in the same protein extracts. In all SART samples, OMT activity remained to basal levels. In LAR of elicitor-treated plants accumulation of acidic PR1, PR2 and PR3 occurred earlier than that of the basic counterpart. Quantification of the blots showed similar signals for acidic and basic counterparts at days 7 and 9 post-treatments. When PR proteins, acidic or basic, were detected in the corresponding SAR samples, there was no clear difference in protein accumulation between SART and SARS , their accumulation was decreased compared to that in LAR, and acidic isoforms accumulated in higher levels compared to basic isoforms. For instance, acidic PR1, PR2 and PR3, and basic PR2 and PR3 were detected in
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Figure 3. OMT activity in TMV-infected tobacco plants and in LAR, SART and SARS of glycoprotein-treated tobacco plants. A. OMT activity in TMV-infected plants. Three leaves per plant were inoculated with the virus, and enzyme activity was determined in infected leaves (closed squares) and in the systemic non-inoculated leaf (open squares). B. OMT activity in LAR, SART and SARS of glycoprotein-treated plants and expressed as pkat/mg protein. Three leaves per plant were infiltrated with 50 nM glycoprotein or water at 6 spots per leaf, and enzyme activity was determined in LAR (closed diamonds), SART (closed circles), SARS (closed triangles) and in water-infiltrated leaves (open circles). C. OMT activity in LAR and SART of glycoprotein-treated plants and expressed as pkat/g fresh weight. One leaf per plant was infiltrated with 50 nM glycoprotein at 2 spots located on the same half leaf, and enzyme activity was determined in LAR (closed diamonds) and SART (closed circles). SART samples were collected from tissues located between the two elicitor-infiltrated spots. D. OMT activity in tissues proximal and distal from the glycoprotein-infiltration site. One leaf per plant was infiltrated with 50 nM glycoprotein at 2 spots located on each half leaf. Samples (S1 to S4) were collected radially starting from the external limit of the glycoprotein-induced HR lesion. Leaf disks were punched out with a cork borer of diameter 5 mm, sample S1 being the most proximal to the HR lesion and S4 the most distal. OMT activity from non-treated tissues (lane C) was included as control.
Figure 4. Expression of acidic and basic PR1, PR2, PR3 and PR5 proteins in LAR, SART and SARS of glycoprotein-treated tobacco plants. Three leaves per plant were infiltrated with 50 nM glycoprotein at 6 spots per leaf. LAR, SART and SARS samples were collected at different times in days after treatments (dat) and PR protein accumulation was monitored by western blotting.
Figure 5. Expression of acidic and basic PR1, PR2, PR3 and PR5 proteins in LAR, SART and SARS of TMV-infected tobacco plants. Three leaves per plant were inoculated with TMV. LAR, SART and SARS samples were collected 6 days after inoculation and PR protein accumulation was monitored by western blotting. LAR was the tissue centered to the HR lesion, SART the tissue between the lesions and SARS to the tissue of the systemic non-inoculated leaf.
Figure 6. Expression of acidic and basic PR1, PR2, PR3 and PR5 proteins in LAR and SAR of glycoprotein-treated NahG and ETR transgenic tobacco plants. Three leaves per plant were infiltrated with 50 nM glycoprotein or water at 6 spots per leaf. LAR and SARS (labelled SAR in the figure) samples were collected 5 days after treatments and PR protein accumulation was monitored by western blotting. Control samples (lanes C) were the water-infiltrated tissues.
SART and SARS , their level of expression was much lower to that found in LAR, and levels of acidic isoforms were higher than levels of basic isoforms. Some PRs remained barely detectable in SAR samples such as acidic and basic PR5 and basic PR1. Overall, there was a good correlation between PR protein expression and resistance to TMV in LAR, SART and SARS . Furthermore the pattern of PR protein expression in elicitor-treated plants was similar to that occurring in TMV-infected plants. In the latter plants, there was a strong accumulation of acidic and basic PR proteins in LAR, a lower accumulation in SART and SARS , and no difference between SART and SARS . We further analyzed expression of acidic and basic PR proteins in LAR and SAR tissues of NahG
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Figure 7. Expression of acidic and basic PR1, PR2, PR3 and PR5 proteins in LAR of tobacco leaves co-infiltrated with the glycoprotein and AOPP or CoCl2 . One leaf per plant was infiltrated at 4 spots (2 spots per half leaf) with water (lane C), 50 nM glycoprotein (GP), 50 nM glycoprotein + 250 µM AOPP (GP+AOPP), and 50 nM glycoprotein + 0.1 mM CoCl2 (GP + CoCl2 ). LAR samples were collected 3 days after treatments and PR protein accumulation was monitored by western blotting.
and ETR tobacco plants (Figure 6). Transgenic NahG plants catabolize the SA signal into the inactive catechol (Delaney et al. 1994). Transgenic ETR plants are ethylene-insensitive (Knoester et al. 1998). Three leaves per plant were infiltrated with 50 nM glycoprotein or water at six different spots per leaf. LAR and SAR samples were collected 5 days after elicitor application. Since there was no clear difference in PR protein accumulation in SART and SARS , and since there was no clear difference in TMV resistance in SART and SARS , analysis was performed on SARS samples. As controls, water-infiltrated tissues were collected. The HR induced in NahG plants was similar to that induced in the Samsun NN non transgenic control plants: the kinetics of necrotic symptom appearance was similar and necrosis covered all the infiltrated tissue. Elicitor-treated ETR plants showed slightly delayed HR symptoms and necrosis almost never covered the entire infiltrated tissue. In LAR of NahG plants, expression of acidic PR1 and PR2 was clearly suppressed, that of acidic PR5 was reduced, and that of acidic PR3 remained unchanged. The experiment was repeated with similar results, particularly accumulation of acidic PR3 was never suppressed or decreased. Expression of the basic counterparts remained unchanged in NahG plants. In LAR of ETR plants, expression of acidic PR1, PR2 and PR5 was slightly decreased compared to non-transgenic plants. Expression of acidic PR3 was not affected. Ex-
pression of the four basic isoforms was reduced, but never completely suppressed. Expression of the acidic and basic isoforms was suppressed in SAR of NahG and ETR plants. In an other series of experiments, the glycoprotein elicitor was co-infiltrated with 250 µM α-aminooxyß-phenylpropionate (AOPP) or with 0.1 mM CoCl2 , and expression of acidic and basic PR proteins was analyzed in LAR (Figure 7). AOPP is a specific inhibitor of phenylalanine ammonia lyase (Massala et al. 1987) which is involved in SA biosynthesis (Chong et al. 2001). CoCl2 is an inhibitor of the ACC oxydase the enzyme converting 1aminocyclopropane-1-carboxylic acid (ACC) into ethylene (Ohtsubo et al. 1999). To better compare the results, all four treatments (water, elicitor alone, elicitor+AOPP, elicitor+CoCl2 ) were performed on the same leaf. Co-infiltration of the elicitor with the chemicals did not affect the development of the HR. Expression of the acidic and basic isoforms remained unchanged in LAR surrounding the tissue infiltrated with the elicitor+AOPP. In LAR of the treatment elicitor+CoCl2 , accumulation of the basic isoforms was suppressed, that of the acidic PR1, PR2 and PR5 was reduced, and that of acidic PR3 remained unchanged. Discussion We have compared in tobacco the glycoproteininduced LAR and SAR in terms of resistance against TMV infection and of expression pattern of OMT and of acidic and basic PR1, PR2, PR3, and PR5. The glycoprotein belongs to the elicitin family: it has the typical elicitin domain (amino-acid 1 to 98) with the 6 conserved cysteines (Kamoun et al. 1997), and it shares common epitopes with alpha-elicitins (Baillieul et al. 1996). Elicitins have been shown to function as avirulence factors inducing the HR in Nicotianae species (Kamoun et al. 1999) as P. infestans deficient in the elicitin INF1 induces disease lesion on N. benthamiana (Kamoun et al. 1998). One major characteristic of the glycoprotein is its strict localization to its infiltration site (Dorey et al. 1997): it does not diffuse in LAR tissues and beyond. Thus, the responses induced in LAR and SAR tissues result from the diffusion of host-released signals. The glycoprotein-induced HR and LAR in tobacco have been characterized at the molecular level (Baillieul et al. 1995; Dorey et al. 1997). Here, we further
116 show that the glycoprotein-treated Samsun NN tobacco plants exhibited the features characteristic of an authentic SAR. First, treated and systemic leaves showed enhanced resistance against challenge TMV inoculation. The level of resistance in systemic leaves (SARS ), as measured by the reduction in virus lesion size, was similar to that induced after a primary TMV infection. It indicated that the elicitor treatment was as efficient as a primary TMV inoculation to induce SAR. Second, expression of typical SAR molecular marker, the acidic PR proteins (Brederode et al. 1991; Ward et al. 1991), was induced in tissues exhibiting SAR to TMV. SART disclosed SAR activity similar to that observed for SARS . In 2 out of 3 experiments, when comparing the mean size, lesions found in SART were smaller than those found in SARS . However, that difference was not of statistical significance. Studying SAR in TMV-infected Samsun NN tobacco plants, Ross (1961b) reported also that no gradient in the size of TMV lesions was evident when the challenge virus inoculation was performed on the primary infected leaves or on systemic leaves. Thus, a similar magnitude of SAR was occurring in SART and SARS of virus inoculated or glycoprotein-treated tobacco plants. SART and SARS disclosed also similar molecular characteristics. We found similar PR levels in SART and SARS of virus-inoculated or glycoproteintreated plants. Furthermore, these levels were much lower than those measured in LAR. The pattern of PR proteins found in SAR samples correlated well with that of mRNA analyzed independently by Brederode et al. (1991) in TMV-infected Samsun NN tobacco plants. They found relatively high amounts of acidic PR1, PR2, PR3 mRNA, and very low amounts of acidic PR5 and basic PR3 and PR5 mRNA. LAR clearly distinguishes from SART and SARS . The levels of resistance to challenge virus infection and of PR protein expression were found significantly higher in LAR than in SART and SARS . One remarkable feature, however, was the strong stimulation of OMT activity in LAR while no stimulation occurred during SAR. This was particularly noteworthy in SART tissues, which were collected in close vicinity of LAR tissues. It should be noted that OMT activity was measured following a very sensitive assay using a radioactively labeled substrate, S-adenosylL-methyl-[3H]-methionine, allowing measurements of pmoles of enzyme product (Legrand et al. 1976). It suggests that the signal(s) triggering OMT expression in LAR is not diffusing out of LAR tissues. It also further supports the view that SART and SARS
cannot be distinghuished, in other terms that SAR in glycoprotein-treated plants develops already in the vicinity of the elicitor application. This feature of SAR was also reported in TMV-infected tobacco plants (Ross 1961b). The genetical and pharmacological approaches indicate that expression of acidic PR1, PR2, PR3 and PR5 proteins during the elicitor-induced LAR and SAR follows a SA-dependent signalling pathway. Amounts of acidic PR1, PR2, and PR5 were clearly reduced in LAR, and suppressed in SAR of NahG plants (Figure 6). Expression of acidic PR3 remained unchanged in LAR, however, but was suppressed in SAR of elicitor-treated NahG plants. It suggests that different acidic PR protein gene may display different sensitivity to SA. Residual SA levels have also been shown to occur in the NahG plants (Friedrich et al. 1995). Thus, low SA levels combined with a high sensitivity to SA may explain that PR3 expression remained unaffected in LAR but was suppressed in SAR of NahG plants. Expression of basic PR1, PR2, PR3 and PR5 proteins during the elicitor-induced LAR follows an ethylene-dependent signalling pathway. In ETR plants, reduced levels of the basic PR proteins (Figure 6) and of OMT activity (data not shown) were found. The results with the PR proteins correlated well with the results of Knoester et al. (1998) showing a decrease in the basic PR1 and PR5 mRNA levels in TMV-infected leaves of the same ETR plants. The co-infiltration of the glycoprotein and an inhibtor of ethylene synthesis resulted in the suppression of basic PR1, PR2, PR3 and PR5 protein expression and in a strong decrease (50 to 80%) in OMT activity (data not shown) in LAR. It also resulted in reduced levels of the acidic PR protein counterparts. This latter result fits with previous observations that ethylene enhances the sensitivity of plants to SA (Lawton et al. 1994). Overall, our results indicate that the glycoprotein treatment triggers similar signalling pathways to those found in a typical incompatible interaction. Whereas the AOPP treatment did not change PR protein expression in LAR, the CoCl2 treatment did. This could be explained by a diffusion of CoCl2 to LAR tissues and not of AOPP. Diffusion of CoCl2 may not occur, however. We observed that infiltration of an aqueous solution into leaves results in the rapid taken up of the water by the cells. This is thought to create a driving force counteracting the diffusion of small molecules. We have infiltrated different chemicals into tobacco leaves either alone or in combination with the
117 glycoprotein. We never observed diffusion to the LAR tissues of N-acetylcystein (Costet et al. 2002), AOPP (Dorey et al. 1997), H2 O2 (Dorey et al. 1998), or other reactive oxygen species produced from rose bengal or from the oxidative burst occurring in HR cells (Costet et al. 2002). Elicitins applied to decapitated tobacco plants are able to move troughout the plant (Keller et al. 1996; Zanetti et al. 1992). However, when elicitins are infiltrated into the leaves, they remain strickly restricted to the infiltrated site (Baillieul et al. 1996; Keller et al. 1996). Thus, if CoCl2 remains strictly localized to its the infiltration site, this would suggest that ethylene is diffusing out of the HR cells to activate a subset of defense genes in LAR tissues. Ethylene production is often correlated with necrotizing tissues (Boller, 1991). Consequently, at least two chemically different signals would be released by cells undergoing the HR cell death: ethylene activating basic PR genes in LAR and another signal activating acidic PR genes in LAR via a SA-dependent signalling pathway. SA does not appear to be the second signal as AOPP treatment was shown to strongly decrease SA levels in HR cells but not in LAR cells (Dorey et al. 1997) and there is no reports indicating that low SA levels can induced SA synthesis. Recently, we have provided some clues that, in our system, reactive oxygen species produced in HR cells during the oxidative burst do not act as a diffusible LAR signal (Costet et al. 2002). These and previous results (Baillieul et al. 1995; Dorey et al. 1997; Dorey et al. 1998) show that, in tobacco, the glycoprotein induces the HR, LAR and SAR with similar biological, molecular, and signalling pathway features to the typical HR, LAR and SAR induced in TMV-infected N-gene containing tobacco plants. The gene-for-gene model governing HR induction in a race-cultivar specificity was recognized sufficient to explain non-host (or species specific) resistance of Nicotianae species to Phytophthora, the non-host determinants being elicitins (Kamoun et al. 1999). So far, we could not find in tobacco a feature which differentiates the HR, LAR, and SAR induced by the glycoprotein elicitin, from the HR, LAR, and SAR induced by a race-cultivar specific avirulence factor. Acknowledgements Sylvain Cordelier was supported by a BDI fellowship granted by both Aventis Crop Science and the CNRS. Authors are thankful to Prof. J. Bol (Leiden
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