JXB Advance Access originally published online on February 24, 2007
Journal of Experimental Botany 2007 58(6):1463-1472; doi:10.1093/jxb/erm008
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RESEARCH PAPER |
Elicitor and resistance-inducing activities of ß-1,4 cellodextrins in grapevine, comparison with ß-1,3 glucans and 
-1,4 oligogalacturonides
1URVVC-EA 2069, Stress, Défenses et Reproduction des Plantes, Université de Reims Champagne-Ardenne, BP 1039, F-51687 Reims cedex 2, France
2UMR Plante–Microbe–Environnement, INRA 1088, CNRS 5184, Université de Bourgogne, 17 rue Sully, BP 86510, 21065 Dijon cedex, France
3Laboratoires Goëmar, Avenue du Général Patton, F-35400 Saint Malo cedex, France
4Centre de Recherche sur les Macromolécules Végétales, CNRS, BP 53 X, F-38041 Grenoble, France
* Present address and to whom correspondence should be sent: Plantes, Pesticides, Développement Durable, URVVC-EA 2069, Université de Reims, BP 1039, F-51687 Reims cedex 2, France. E-mail: aziz.aziz{at}univ-reims.fr
Received 20 September 2006; Revised 18 December 2006 Accepted 5 January 2007
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Abstract |
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Cellodextrins (CD), water-soluble derivatives of cellulose composed of ß-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and ß-1,3 glucanase activities. Most of these defence reactions were also induced by linear ß-1,3 glucans (ßGlu) and
-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with ßGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, ßGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than ßGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response. Key words: Cellodextrins, defence responses, grapevine, induced resistance
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plants rely on an innate immune system to defend themselves against potentially pathogenic micro-organisms. This system is based on the capacity to perceive micro-organisms as ‘non-self’ or to recognize ‘pathogen-induced modified self’. Current models mention the perception of micro-organisms through receptors of pathogen-associated molecular patterns (PAMPs) during the induced non-host resistance (Gomez-Gomez and Boller, 2002; Nürnberger and Brunner, 2002; Jones and Takemoto, 2004). The PAMPs are in fact a new term for the ‘old’ so-called general elicitors. These include ß-heptaglucan structures or a glycoprotein (GP42) from Phytophthora cell walls, elicitins secreted by various Phytophthora and Pythium species, fungal cell wall chitin fragments, and N-terminal fragments of bacterial flagellin (Kamoun, 2001; Nürnberger and Brunner, 2002; Brunner et al., 2002; Gomez-Gomez and Boller, 2002). The ‘pathogen-induced modified self’ perception has been mentioned for the ‘guard model’ concept occurring during most gene-for-gene relationships (Jones and Dangl, 2006). During host–pathogen interaction many pathogens secrete cell-wall-degrading enzymes, including endopolygalacturonases and xylanases (Vidal et al., 1998; Furman-Matarasso et al., 1999; Boudart et al., 2003; Poinssot et al., 2003). These enzymes can themselves function as elicitors (Rotblat et al., 2002; Poinssot et al., 2003), but their enzymatic products are also known to be general elicitors of plant defence responses (Shibuya and Minami, 2001).
With regard to the concept mentioned above, plant cell-wall degradation products can be considered as ‘microbe-induced molecular patterns’ (MIMPs) recognized through receptors as ‘pathogen-induced modified self’ (Mackey and McFall, 2006). So cell-wall oligosaccharides originating from plant or pathogenic micro-organisms, can play an important role in the perception of the invading pathogen by the plant. It is also thought that mimicking pathogen attack with such non-specific elicitors might prove useful in the development of alternative strategies for crop protection, even if the activation of plant defence responses in a non-cultivar-specific manner may not necessarily mediate resistance (Brunner et al., 2002).
The oligosaccharides that have been frequently investigated are ß-1,3 (1,6) glucans and -1,4 oligogalacturonides (OGA) (Côté et al., 1998; Ridley et al., 2001; Aziz et al., 2004). The ß-1,3 glucans were shown to induce a variety of defence reactions in tobacco (Klarzynski et al., 2000), Arabidopsis (Ménard et al., 2004), rice (Inui et al., 1997), alfalfa (Kobayashi et al., 1993), and grapevine (Aziz et al., 2003), conferring resistance to viral, bacterial, and fungal pathogens. The biological activity of ß-1,3 glucans was dependent on the degree of polymerization (DP) and decorations carried by the sugar backbone (Darvill et al., 1992; Inui et al., 1997; Ménard et al., 2004). The activity of -1,4-linked OGA depends on plant species (Côté et al., 1998) and DP (Darvill et al., 1992; Simpson et al., 1998). For many responses, OGA oligomers with a DP higher than 9 have been shown to be active in several plants, including Arabidospis (Moscatiello et al., 2006) and grapevine (Poinssot et al., 2003; Aziz et al., 2004). This has generally been attributed to the ability of the OGA of DP >9 to form multioligomer complexes with Ca2+ (Liners et al., 1992). However, in some reports small-sized oligomers have also been shown to induce the accumulation of protease inhibitor proteins and ethylene synthesis in tomato, while larger oligomers were ineffective (Simpson et al., 1998).
The possibility that cellulose-derived oligosaccharides may be actors in defence mechanisms could be supported by their possible occurrence through cellulase activity during plant–microbe interaction (Vidal et al., 1998). Cellodextrins, consisting of a linear ß-(1,4)-linked glucose backbone, are the predominant end-products from cellulose degradation in plant cell walls and are also produced by fungi and bacteria (Scheible and Pauly, 2004; Matthysse et al., 2005). They have frequently been reported to participate in the regulation of cellulose synthesis and bacterial cell growth (Scheible and Pauly, 2004; Matthysse et al., 2005). In animal systems, hemicellulose derived from soybean hull, containing ß-(1,3)-(1,4) glucan, has been reported to stimulate macrophages to produce nitric oxide and interleukin-1ß (Nagata et al., 2001). Similarly, recent studies have shown that soluble ß-1,4 glucans derived from Acetobacter sp. exert an immunostimulatory activity in the mouse macrophage cells and prevent infectious diseases with pathogenic bacteria (Saito et al., 2003; Li et al., 2004). The authors suggested that a backbone of ß-1,4-glucan is crucial for the biological activity. However, the role of these oligosaccharides in triggering plant defence reactions has not yet been established.
In this study, the elicitor activity of ß-1,4 cellodextrins (CD) in grapevine cells was investigated with respect to the oxidative burst, free cytosolic calcium concentration, expression of nine defence-related genes, and the stimulation of chitinase and ß-1,3 glucanase activities. The defence-inducing activities of CD fragments were compared to those of linear ß-1,3 glucans (ßGlu) and -1,4 oligogalacturonides (OGA) to evaluate the importance of their structural motif and DP. Furthermore, the ability of CD oligomers, compared with ßGlu and OGA oligomers, to protect grapevine leaves against B. cinerea has been examined.
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Materials and methods |
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Biological materials
Grapevine plantlets (Vitis vinifera cv. Chardonnay) were obtained by multiplication through in vitro micro-cuttings on pH 5.9 modified Murashige and Skoog (1962) medium, as described by Aziz et al. (2003). Grapevine cells (V. vinifera cv. Gamay) were cultivated in the medium of Nitsch and Nitsch (1969) without hormones (80 ml in 250 ml Erlenmeyer flasks) and propagated at 130 rpm on a rotary shaker at 25 °C in continuous light. Cells were maintained in the exponential phase and subcultured 24 h prior to utilization. Transformed grapevine cells (V. vinifera cv. Gamay) expressing apoaequorin were used to generate cell suspensions which subcultured in Nitsch–Nitsch medium as described in Vandelle et al. (2006). Botrytis cinerea strain 630 (gift of Dr Y Brygoo, INRA, Versailles, France) was grown on potato dextrose agar at 22 °C.
Preparation of oligosaccharides
Cellodextrins (CD, ß-1,4 glucose oligomers) were obtained from CERMAV (Grenoble, France) (Fig.1). They were prepared by precipitation of oligomers with 1-propanol and ethanol after acetolysis of cellulose (Avicel from the cotton linters). CD oligomers (CD 2 to CD 9) can be isolated by high-resolution size-exclusion chromatography on Bio-Gel P 2 using water as eluent as described by Schmid et al. (1988). Linear ß-1,3 Glucans (ßGlu) were obtained from Goëmar (Saint Malo, France). They were extracted and purified from the marine brown algae Laminaria digitata as described by Klarzynski et al. (2000). The average degree of polymerization (DP) was estimated by molecular size chromatography coupled with a refractometric detector. -1,4 Oligogalacturonides (OGA) were obtained from CERMAV (Grenoble, France). They were generated by partial digestion of polygalacturonic acid (sodium salt) (Sigma) and size-homogeneous OGA was prepared by high-performance anion-exchange chromatography on Bio-Gel P 6, as described by Gouvion et al. (1994). All oligosaccharides were dissolved in the culture medium and sterilized by filtration through membranes (pore size 0.45 µm, Millipore).
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Treatments
Cells were collected during the exponential growth phase and washed by filtration in a suspension buffer containing 175 mM mannitol, 0.5 mM K2SO4, 0.5 mM CaCl2, and 2 mM MES, pH 5.5. Cells were resuspended at 0.1 g FW ml–1 with suspension buffer and equilibrated for 2 h on a rotary shaker (130 rpm, 24 °C). Grapevine cells were then used for measurements of H2O2 production and cell death after treatment with oligosaccharides. Control cells were incubated under the same conditions without elicitor. For gene expression, chitinase and ß-1,3 glucanase activities, cells were collected and treated as above except that they were resuspended in a freshly prepared Nitsch medium before the addition of elicitor. For all elicitors, experiments were performed and replicated on the same batches of cell suspensions.
Measurement of H2O2 production
H2O2 production was determined using chemiluminescence of luminol as described previously (Poinssot et al., 2003). Chemiluminescence, measured with luminometer (Lumat LB 9507, Berthold), was integrated and expressed in nmol H2O2 g–1 FW, using a standard calibration curve by H2O2 added in cell suspension aliquots.
Free cytosolic calcium analysis
Measurements of aequorin luminescence were carried out on transformed grapevine cells as described by Vandelle et al. (2006). The bioluminescence of 250 µl aliquots of reconstituted cells, transferred to a luminometer glass, was recorded at 1 s intervals using a digital luminometer (Lumat LB9507, Berthold). The luminescence counts were recorded and exported simultaneously (using Win Term software; Berthold) into an Excel spreadsheet (Microsoft, Redmond, WA) on a computer. Residual functional aequorin was quantified by adding 300 µl of lysis buffer (10 mM CaCl2; 2% Nonidet P40 v/v; 20% ethanol v/v) and the luminescence data were transformed into Ca2+ concentrations, as described by Vandelle et al. (2006).
RNA isolation and real-time quantitative RT-PCR
Aliquots of 0.5 g FW were collected by filtration from the culture medium and frozen in liquid nitrogen. Total RNA was extracted by 4 ml of sodium perchlorate buffer as described by Davies and Robinson (1996). Residual genomic DNA was removed by incubating the RNA solution with 15 units of RNase-free DNase I (Promega) for 30 min at 37 °C. The DNase reaction was stopped with a mix of phenol/chloroform/isoamyl alcohol (25/24/1, by vol.). 2 µg of DNase-treated RNA were reverse transcribed with 5 µM oligo(dT) (Life Technologies/Gibco-BRL) following the manufacturer's instructions.
The transcript levels were determined by real-time quantitative PCR using the GeneAmp 5700 Sequence Detector (Applied Biosystems) and the SYBR Green Master Mix PCR kit as recommended by the manufacturer (Applied Biosystems). PCR reactions were carried out in triplicates in 96-well plates (25 µl per well) in a buffer containing 1x SYBR Green I mix (including Taq polymerase, dNTPs, SYBR Green dye), 300 nM forward and reverse primers and 1:250 dilution of reverse transcript RNA. After denaturation at 95 °C for 10 min, amplification occurred in a two-step procedure: 15 s of denaturation at 95 °C and 1 min of annealing/extension at 60 °C, with a total of 40 cycles. Identical thermal cycling conditions were used for all targets. The gene-specific primers were designed based on sequences present in databases (Aziz et al., 2003). Transcript level was calculated using the standard curve method and normalized against grapevine actin gene as an internal control and non-treated cells as a reference sample (Bézier et al., 2002a). For each gene the reference sample was defined as the 1x expression level, and results were expressed as the fold increase of mRNA level over the reference sample as previously described (Aziz et al., 2003).
Determination of chitinase and ß-1,3 glucanase activity
Chitinase activity was measured according to the procedure described by Wirth and Wolf (1992) using carboxymethyl-chitin-Remazol-brilliant violet (Loewe Biochemica, Germany) as a substrate. For ß-1,3 glucanase assays, proteins from the crude extract (0.5 ml) were precipitated with 80% ammonium sulphate and redissolved in 0.5 ml of 50 mM sodium acetate buffer, pH 5.0. ß-1,3 Glucanase activity was assayed according to Derckel et al. (1998), using laminarin (Fluka) as a substrate.
Protection assays
Conidia of Botrytis cinerea strain 630 (gifted by Dr Y Brygoo, INRA Versailles, France) were collected from 10-d-old potato dextrose agar culture with 10 ml sterile water, filtered to remove mycelial debris and concentration was adjusted to 106 conidia ml–1. For each treatment, 30 leaves were excised from 10-week-old in vitro grapevine plantlets and preincubated in 2 mM MES buffer, pH 5.9 containing 0.5 mM CaCl2 and 0.5 mM K2SO4, in the presence of each oligosaccharide. After 24 h, the leaves were placed on wet filter paper in plastic Petri dishes. One needle-prick wound was applied to each leaf, and the fresh wounds were covered with 5 µl drops of conidial suspension of B. cinerea. Quantification of disease development in grapevine leaves after inoculation with B. cinerea was measured as average diameter of lesions formed during infection.
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Results |
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Induction of oxidative burst and cytosolic calcium variation
The rapid release of H2O2 is generally assumed to be a key event in the orchestration of various cellular defence responses (Alvarez et al., 1998) and is a well-described response of plant cell suspensions to molecules with eliciting activities. Here grapevine cells were elicited with cellodextrin (CD) oligomers (Fig. 1) spanning the whole range of DP 3 to 9. As shown in Fig. 2, grapevine cells responded to CD with a rapid release of H2O2. The production of H2O2 increased with increasing concentrations of CD and became saturated between 0.3–0.9 mM, which corresponded to 0.5 mg ml–1 for each oligomer (data not shown). The level of oxidative burst response was directly dependent on the DP of the CD (Fig. 2a). For all oligomers, the production of H2O2 showed similar profiles, with a maximum level at 20 min after elicitation. Thereafter, H2O2 level declined progressively. Oligomers of DP 3 and 4 induced a slight production of H2O2, those of DP 5 and 6 were unable to induce any significant response, while CD of DP >6 were active. CD 7 oligomer was 2-fold more active than CD 8 and 9. These effects were further compared with those of linear ß-1,3 glucans and
-1,4 OGA of different DP, at the optimal concentration of 0.5 mg ml–1 (Fig. 2b). ß-1,3 glucans of DP 6 (ßGlu 6) and DP 10 (ßGlu 10) induced similar responses to CD 8 and 9 (Fig. 2b, b), while ßGlu 3 had no effect (Fig. 2b). The OGA oligomers displayed the strongest H2O2 production. CD 7, ßGlu 10, and OGA 7 remained the most active molecules in each category.
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Free cytosolic [Ca2+] increase has been reported in plants in response to various stimuli, including elicitors (Lecourieux et al., 2002; Moscatiello et al., 2006; Vandelle et al., 2006). In this study, variations of [Ca2+]cyt, have been investigated using grapevine cells transformed with the gene encoding apoaequorin addressed to the cytosol. In control cells, the resting [Ca2+]cyt is 0.15 µM during the assay period. When cells were challenged with CD 7 at 0.5 mg ml–1, a rapid and transient increase of [Ca2+]cyt was observed (Fig. 2c). This increase started within 1 min, peaked at 0.6 µM after 2.5 min, and decreased to 0.32 µM after 8 min. Cytosolic [Ca2+] was then maintained at this level for about 4 min, and then decreased slowly to the background level. The Ca2+ signature was different in peak time or intensity after treatment with other oligosaccharides. OGA (0.5 mg ml–1 cell suspension) induced a [Ca2+]cyt increase comparable in a peak time with CD 7 response, but with a maximum reaching 0.5 µM. With ßGlu 10 at the same concentration, the cells responded weakly with a maximum [Ca2+]cyt of 0.4 µM at 3.7 min.
As CD 7 and OGA 7 show the same calcium signatures, except in intensity, it was analysed further whether successive addition of CD 7 and OGA 7 would result in a refractory state (i.e. the inability of the cells to react to a second application of the same elicitor) by measuring H2O2 production (Fig. 2d). Grape cells pretreated with CD 7 at 0.5 mg ml–1 were shown to be refractory to a second application of CD 7. However, they were not refractory to an application of OGA 7. This indicates a different mode of perception of CD and OGA by grapevine cells.
Differential up-regulation of defence-related genes
It was investigated whether CD oligomers, compared with known oligosaccharides with different structures and DP, can induce various defence-related genes in grapevine cells. The expression of genes was analysed using real-time quantitative RT-PCR (qRT-PCR) and specific primers with an actin gene as the internal standard (Bézier et al., 2002a). In control cells, the transcript level of the different genes was low during the period of treatment.
Expression of genes encoding phenylalanine ammonia lyase (PAL) was induced by all CD fragments even with low DP (Fig. 3a), while stilbene synthase (STS) expression was significantly induced only by CD of DP 
6 (Fig. 3b). PAL transcript was greatly accumulated in response to CD 3 and to oligomers of DP >6 (45–70-fold induction), the intermediary sizes remained less active. Maximal expression was achieved with CD 7 for PAL and with CD 7 and CD 8 for STS. A strong expression of PAL and STS was also recorded in response to OGA 7 and OGA 10 (Fig. 3c, d). Transcript level of PAL increased by 90-fold and 75-fold and that of STS increased by about 700-fold and 350-fold, respectively. However, ßGlu 6 induced PAL expression by only 20-fold and STS by about 170-fold. ßGlu 3 and ßGlu 10 had a lower effect on PAL expression (about 5-fold), but efficiently up-regulated STS expression by about 80-fold and 300-fold, respectively. The highest expression was obtained with OGA and CD for PAL, and with OGA for STS.
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Induction of PR genes encoding basic (Chit1b) and acidic (Chit3, Chit4c) chitinases was also dependent on the DP of CD and significantly up-regulated with oligomers of DP
6 (Fig. 3e, f, g). Maximal expression was achieved with DP 9 for Chit1b (400-fold), with DP 7 for Chit3 (20-fold) and with DP 8 for Chit4c (1500-fold). For the three genes, ßGlu 6 and ßGlu 10 (Fig. 3h, i, j) displayed an elicitor activity similar to that of CD 7 and CD 8. ßGlu 3 remained less active than CD 6. OGA were also potent inducers of all three PR-genes. They had similar effects than ßGlu of DP 6 and 10, but remained less active than CD of DP >7 in inducing Chit1b (Fig. 3h) and Chit3 (Fig. 3i) expression. Nevertheless, the highest expression of Chit4c occurred with both CD of higher DP (Fig. 3g) and OGA (Fig. 3j). In all cases, ßGlu 3 oligomer remained less active as CD 6.
Like STS and chitinase transcripts, in CD-treated cells the expression of genes encoding ß-1,3 glucanase (ß-Glu1), polygalacturonase inhibiting protein (PGIP), and serine-protease inhibitor (PIN) increased with increasing DP (Fig. 3k, l, m). CD of a DP 6 induced a greater mRNA accumulation, compared with smaller oligomers. A maximal induction was achieved with CD 8 for ß-Glu1 (200-fold) and with CD 9 for PGIP (24-fold) and PIN (140-fold). A similar pattern was observed in cells treated with ß-1,3 glucans (Fig. 3n, o, p). ßGlu 3 treatment was always less effective than ßGlu of higher DP. Highest transcript levels of ß-Glu1 (280-fold), PGIP (40-fold), and PIN (250-fold) were induced by ßGlu 6 and ßGlu 10. Steady-state levels of ß-Glu1, PGIP, and PIN mRNA were also induced by OGA. The effect of OGA 10 was weakly higher than the OGA 7 one (Fig. 3n, o, p).
Chitinase and ß-1,3 glucanase activities
In addition, chitinase and ß-1,3 glucanase activities in elicited grapevine cells were analysed. Consistent with their elicitor activity in terms of gene induction, the CD oligomers stimulated chitinase and ß-1,3 glucanase activities in a size-dependent manner (Fig. 4a, b). Overall, the high values were obtained with CD of DP 7 to 9. Linear ß-1,3 Glu of DP 6 also stimulated activity of both enzymes in grapevine cells. As for CD of DP 6, ßGlu 3 remained less active. OGA also proved to be effective stimulators of chitinase and ß-1,3 glucanase activities (Fig. 4c, d). The highest activity of chitinase was obtained with OGA and CD of DP >6, while ß-1,3 glucanase was comparable in response to CD of DP 7, ßGlu of DP 6 and OGA 7.
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Protection of grapevine leaves against Botrytis cinerea
To evaluate the resistance of grapevine to B. cinerea, the causal agent of gray mould disease, detached leaves from in vitro-cultivated plantlets were first preincubated for 25 h with each oligosaccharide at a concentration of 0.5 mg ml–1 before inoculation with a B. cinerea conidial suspension. Under these conditions, the average diameter of the necrotic lesion measured 5 d post-inoculation was reduced by CD with higher DP (Fig. 5a). The diameter of the necrotic lesions was significantly reduced with oligomers of DP
7 (by about 45%). Oligomers with smaller DP had no significant effect. ßGlu with DP 6 partially protected grapevine leaves against B. cinerea (by about 30–38%) (Fig. 5b). OGA were more effective and triggered a high protection against gray mould disease reaching 63% (Fig. 5b).
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Discussion |
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The data reported in this study show that cellodextrins (CD) consisting of linear ß-1,4 glucoside units derived from cellulose (Fig. 1), induce multiple defence and resistance responses in grapevine cells. CD triggered induction of oxidative burst, transient elevation of [Ca2+]cyt, expression of defence-related genes, and stimulation of chitinase and ß-1,3 glucanase activities as well as protection against Botrytis cinerea. The rapid and transient production of H2O2 increased in a CD size-dependent manner. The heptasaccharide exhibits the greatest oxidative burst-inducing activity. This response was also dose-dependent and saturated at 0.3–0.4 mM of oligomers of DP 7 to DP 9, suggesting the existence of a receptor mediating CD effects. This is consistent with studies in animal systems, showing that cellulose or hemicellulose-derived ß-1,4 glucans are strong inducers of nitric oxide burst (Nagata et al., 2001) and innate immunity mediated by Toll-like receptor (TLR4) in macrophages (Saito et al., 2003).
In common with other elicitors (Klarzynski et al., 2000; Aziz et al., 2003; Ménard et al., 2004), CD of DP 7 induced an oxidative burst with a similar level to that triggered by linear ß-1,3 glucans of DP 6. -1,4 OGA 7 was more effective in inducing the H2O2 burst than the other oligomers and oligosaccharides, suggesting a distinct mode of perception of the CD, ßGlu, and OGA fragments by grapevine cells. CD also triggered a Ca2+ signature in apoaequorin-transformed grapevine cells characterized by a first transient peak and a second sustained phase. The OGA 7 and ßGlu 10 also induced signatures resembling the CD response, but differ in peak time and intensities. CD was slightly more efficient in inducing the [Ca2+]cyt increase, whereas OGA were better inducers of oxidative burst. Oligosaccharide elicitors have been reported to induce [Ca2+]cyt variations resulting from both the extracellular medium and intracellular Ca2+ pools (Lecourieux et al., 2002). As OGA 7 and CD 7 show similar calcium signatures (except in intensity), the establishment of a refractory state has been tested. Grapevine cells first treated with CD 7 at 0.5 mg ml–1 failed to respond to a second treatment with the same CD concentration, but highly responsive to a second treatment with OGA 7 at 0.5 mg ml–1. Loss of elicitor responsiveness is therefore assumed to indicate desensitization of the CD perception system. Such desensitization behaviour has also been reported for cultured tobacco cells treated with laminarin (a ß-1,3 glucan of DP 25 with up to 3% of ß-1,6 glucose) or OGA, with respect to medium alkalinization (Klarzynski et al., 2000).
A strong oxidative burst as well as a sustained increase in [Ca2+]cyt have been described as events triggering a diverse set of subsequent defence responses in plants, frequently related to hypersensitive reaction (HR) and systemic acquired resistance (SAR) (Alvarez et al., 1998; Lecourieux et al., 2002). It is shown that CD, OGA, as well as ßGlu, induced both oxidative burst and [Ca2+]cyt increase to moderate levels. Moreover, none of them induced the HSR gene (data not shown), which is considered as an HR-like marker (Pontier et al., 1998; Bézier et al., 2002b), nor did they cause significant cell death (data not shown), suggesting that resistance to pathogen invasion is not necessarily associated with cell death, but depends on other defence responses. Furthermore, with a necrotrophic pathogen like B. cinerea, the HR could facilitate the plant infection (Govrin and Levine, 2000) instead of confining the pathogen.
CD treated-cells exhibit a differential induction of a set of defence-related genes encoding PAL and STS which are involved in lignin, SA, and phytoalexin synthesis; acidic (Chit3, Chit4c) and basic (Chit1b) chitinases, and ß-1,3 glucanase, PR proteins that hydrolyse chitin and ß-1,3 glucans of fungal cell walls; PIN and PGIP which inhibit, respectively, proteases and polygalacturonases produced by some pathogenic fungi (Farmer et al., 1991; De Lorenzo and Ferrari, 2002). Most of these genes were induced by CD in a size-dependent manner, excepted for PAL which was up-regulated independently. In response to ßGlu, the PAL expression was also much less pronounced, while STS was efficiently up-regulated, indicating that PAL and STS pathways are not co-ordinately activated by these oligosaccharides. Many of these genes have also been differentially up-regulated in grapevine leaves and berries during interaction with fungal or bacterial pathogens (Busam et al., 1997; Bézier et al., 2002a; Robert et al., 2002) and in response to elicitors (Aziz et al., 2003, 2004; Bonomelli et al., 2004). As for CD 7, ßGlu of DP 6 and DP 10 showed similar effects in the defence gene expression (except for PAL, Chit4c, and PGIP) and PR protein activities. This suggests that some defence responses could be induced independently on acetylene linkages of glucopyranose units. CD and ßGlu are composed of ß-1,4- and ß-1,3-linked glucose residues, respectively (Fig. 1). To our knowledge, this is the first report showing CD as potent elicitors of plant defence reactions. This is consistent with an earlier report indicating that cellulases from Trichoderma viride can induce grapevine defence mechanisms (Calderon et al., 1993), probably in part by releasing CD fragments from cell wall cellulose. On the other hand, the importance of the cellulose-derived ß-1,4 glucan backbone in inducing innate immunity in animals has been emphasized (Saito et al., 2003). This activity involves a specific receptor allowing the recognition of pathogen-associated molecular patterns (Saito et al., 2003; Li et al., 2004). In addition, the molecular size of ß-1,4 glucan seemed to be important for exerting this biological activity, because fragments with low DP were less effective (Saito et al., 2003).
Although they were perceived as distinct oligosaccharide signals, larger oligomers of CD, ßGlu, and OGA had globally similar elicitor effects in inducing certain defence responses. CD with higher DP, however, were slightly more efficient in inducing the expression of genes encoding PAL and acidic chitinases (Chit3 and Chit4c), whereas ßGlu were better inducers of genes encoding basic ß-1,3 glucanase (ßGlu1) and PGIP. OGA can be thought of as efficient elicitors of STS and PIN gene expressions. Nevertheless, these data strengthen the notion that CD could operate via similar reaction cascades than ßGlu and OGA or that convergence of distinct signalling pathways must occur downstream.
It should also be noted that transcript accumulation of PR genes is correlated to the activity of chitinase and ß-1,3 glucanase in grapevine cells. Intensity of both PR activities was associated with disease resistance of different grapevine cultivars to fungal pathogens (Giannakis et al., 1998; Aziz et al., 2004; Bonomelli et al., 2004). Based on their hydrolytic activities they exert an antimicrobial effect against different pathogens (Fritig et al., 1998), and might also play an important role in the amplification of defence reactions through the release of chitin and glucan oligomers from the pathogen and host cell walls (Fritig et al., 1998).
The majority of induced responses at a cellular level correlated with an enhanced protection of grapevine leaves against B. cinerea. This protection did not apparently result from any direct effect on fungal pathogen growth (data not shown) but rather from a higher inducing efficiency of plant resistance pathways. The infection was significantly reduced by larger oligomers of CD (DP 7) and ßGlu (DP 6). Oligomers with lower DP had no significant effect. Recent reports indicated that cellulose-derived ß-1,4 glucans have ben shown to prevent infectious processes by pathogenic bacteria in animals through the activation of innate immunity (Li et al., 2004). Data with ßGlu and OGA are consistent with other findings on Arabidopsis and tobacco (Klarzynski et al., 2000; Ménard et al., 2004). OGA 7 was more effective and triggered a high protection against B. cinerea (55–60%).
In conclusion, these results suggest that CD are important elicitors of various defence reactions in grapevine, conferring resistance to B. cinerea. Most of these responses are generally concentration and DP-dependent. CD can be thought a member of oligosaccharide elicitors, with an activity in grapevine globally comparable (with some exceptions) to ß-1,3 glucans or OGA. It should also be noted that ß-1,4-CD are structural analogues of the linear ß-1,3-glucans as major components of fungal cell walls that play an active part in host resistance. This highlights the importance of cellulose-derived ß-1,4-glucans as potent MIMPs in plant defence mechanisms needed for disease control.
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Acknowledgements |
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We thank C Hachet for her technical assistance. This work was supported by funds from Europôl'Agro and Fondation du Site Paris-Reims. AG was supported by a grant from the European social fund, the Conseil Régional de Bourgogne, and the BIVB.
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Abbreviations |
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AOS, active oxygen species; CD, ß-1,4 cellodextrins; Chit, chitinase; DP, degree of polymerization; ßGlu, ß-1,3 glucan; HSR, hypersensitive-related; OGA,
-1,4 oligogalacturonides; PAL, phenylalanine ammonia-lyase; PGIP, polygalacturonase inhibiting protein; PIN, proteinase inhibitor; PR, pathogenesis-related; STS, stilbene synthase. |
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