O-glycans from Fusarium sp. M7-1 �

So far only little data have been available concerning the eliciting capacity of well defined glycan molecules isolated from plant pathogens. This study brings new information about changes in plant cells caused by fungal pathogens. Sugar fractions derived from glycoproteins isolated from the fungus Fusarium sp. M7-1 have been tested here as signaling molecules. The ability of three O-glycan fractions (named in this work inducer I, II, III) to trigger responses in Rubus protoplasts has been examined. It was found that inducer III was the most efficient as it elicited changes in the levels of phenylpropanoid pathway intermediates in relation to phenylalanine-ammonia lyase (PAL) activation.

Plants have developed efficient defense mechanisms to cope with pathogens.Plant cells have the ability to rapidly perceive the pathogenic organisms, and to trigger metabolic changes resulting in the activation of transcription of specific genes.Many of the genes encoding proteins or enzymes involved in plant defense reactions are those encoding peroxidases (Chittoor et al., 1997), pathogenesis-related proteins, e.g.glucanases, chitinases, proteinase inhibitors (Chang et al., 1995;Pinto & Ricardo, 1995) or those encoding phenolic biosynthetic enzymes.The enzymes of phenolic pathways generate prod-ucts with numerous functions in plant defense, such as lignins which reinforce cell-walls, phytoalexins with antimicrobial properties (Bell et al., 1986), or salicylic acid with a signaling function (Ryals et al., 1996).
The fungus Fusarium causes many physiological or genetic disorders, nutrient deficiencies, and environmental stress both in plants and in animals, including humans.It can produce lytic enzymes, e.g.pectinases, glucanases, xylanases (Benhamou et al., 1990;Alconada & Martinez, 1995;Christakopoulos et al., 1996), and large amounts of biologically active metabolites such as carotenoids, bikaverins, mycotoxins, phytotropins, gibberellins and oestrogens (Bruckner et al., 1989).Plants react to penetration by Fusarium by accumulation of callose or plant cell-wall components (Rodriguez-Galvez & Mendgen, 1995), by an increase of the steady-state mRNA level of phenylpropanoid pathway enzymes (Ni et al., 1996) or pathogenesis-related like proteins (Casacuberta et al., 1992).Plants infested by the fungus also elicit chitinases (Koga et al., 1992) and accumulate phytoalexins (Stevenson et al., 1997).Little is known, though, about the Fusarium component(s) which act(s) as the natural elicitors of the above responses.

Materials.
Suspensions cultures of Rubus fruticosus L. (raspberry) originally derived from cambial explants from twigs, were grown as described by Hustache et al. (1975).
The inducers I, II and III were prepared and identified according to Jikibara et al. (1992a;1992b), Iwahara et al. (1992;1995).Briefly, glycoproteins were isolated from the mycelium of Fusarium sp.M7-1, and the O-glycosidic fractions released by alkaline borohydride treatment were purified by gel filtration chromatography and high performance liquid chromatography (HPLC).The primary structure of the oligosaccharides was resolved by nuclear magnetic resonance spec- Man-ol-P.In each experimental set, the concentration of inducers II and III is expressed as molarity, and the molar concentration of inducer I is reported by reference to its monomer unit.
Preparation of protoplasts.Cells of Rubus fruticosus L. (40 g fresh mass) were collected in the exponential growth phase (15 to 18 days after subculturing).They were incubated overnight at room temperature in 300 ml of Heller's medium (pH 5.9) supplemented with 0.56 M mannitol, 0.25% (w/v) caylase 345 and 0.01% (w/v) caylase M3.The released protoplasts were filtered through a 100 mm nylon mesh, washed twice with the incubation medium with no cell-wall degrading enzymes, recovered by centrifugation (500 ´g, 5 min) and then suspended in 25 mM Tris/HCl buffer, pH 4.8, containing 0.56 M mannitol, 0.06 M sucrose, 1 mM KCl, 1 mM CaCl 2 , and 6% (w/v) Ficoll 400.The purified protoplasts were isolated by centrifugation at 100 ´g for 8 min at 4°C.Finally, before use, they were suspended in the above buffer without Ficoll.Protoplast yields ranged from 70 to 85% of the initial number of treated cells.The viability of protoplasts was controlled by using Evan's blue indicator.
Elicitation experiments for PAL assays.Rubus protoplasts (2´10 6 ) or cells (4´10 6 ) were suspended for 0 to 30 h in 25 ml buffer, pH 4.8 (25 mM Bis-Tris/HCl containing 1 mM KCl, 1 mM CaCl 2 , 0.06 M sucrose, 0.56 M mannitol supplemented or not with cycloheximide (1 mM)) on a roller mixer in the presence or not (in controls) of 100 nM fungal inducer I, II or III.When appropriate, the elicitation medium was removed by centrifugation (500 ´g, 5 min, 4°C), and the protoplasts were subjected to PAL analysis.At least, four replicates were monitored from three independent elicitation sets.PAL assays.The protoplasts (cells) were subjected to PAL assays carried out according to the modified procedure of Hagendoorn et al. (1991).Briefly, the protoplasts (cells) were sonicated on ice (70 W, 20 s) with a Vibra cell (Bioblock) in 0.1 M sodium borate buffer (pH 8.8) containing 2 mM dithiothreitol.The homogenate was centrifuged (5000 ´g, 15 min), and the supernatant and then mixed with 100 mg of  for 10 min at 4°C.The mixture was centrifuged for 5 min at 5000 ´g, and the supernatant was added to an equal volume of 0.05% polyvinylpyrrolidone (w/v).The mixture was kept for 10 min at 4°C and then centrifuged for 5 min at 5000 ´g, and the supernatant was dialyzed and concentrated with Ultrafree units equipped with a 10 kDa molecular-mass cut-off membrane (Ultrafree Millipore, Bedford, MA, U.S.A.).The crude enzymes were subjected to PAL assays at 40°C as in Zucker (1965).Kinetics were developed at 290 nm using a Beckman DU 640 spectrophotometer: changes in A 290 of the reaction mixture containing enzyme extract (6 mg protein/ml according to the method of Bradford ( 1976)), L-phenylalanine substrate (up to 3 mM) in 0.1 M sodium borate, pH 8.8, supplemented with 2 mM dithiothreitol were followed up to 30 min.At least four kinetic curves per experimental condition were developed, and experiments were monitored from three independent protoplast preparations.The data between 2 and 10 min were fitted with secondorder polynomial regressions giving an equation, and a coefficient r 2 value of 0.82-0.88.The slope (DA 290 /min) gave the PAL activity expressed as mkat/kg protein, 1 mkat being 1 mmol product formed per sec.The enzyme activation is expressed by R value (the slopes of fitted curves obtained from induced protoplasts versus slopes of fitted curves obtained from controls).
Extraction of PAL reaction products.PAL was assayed using 2.25 mM L-phenyl- alanine substrate, at 37°C for up to 4 h as detailed above.The enzyme reaction was stopped by adding 30 ml of 2 M HCl, and the PAL products extracted with 500 ml ethyl acetate according to Khan and Vaidyanathan (1986) were dried by evaporation.The residue was then dissolved in a known volume of the starting solvent used for HPLC, and an aliquot of 100 ml was injected into the chromatograph.HPLC-PAD analysis.HPLC analyses were carried out as described in Chevolot et al.
(1997) but using a Kontron Liquid Chromatograph (Kontron Instruments, Milano, Italy) instead of a Waters one.The system encompassed a manual Rheodyne model 9125 injector, an HPLC 325 pump system, and a 440 photodiode array detector (PAD).All these modules were controlled by a microcomputer equipped with KromaSystem 2000 software (Kontron Instruments, Milano, Italy) which also stored and processed the data.Guard column (LichroCART 4-4, RP-18) and analytical column (Lichrospher 100 RP-18; 125 ´4 mm; 5 mm particle size) were from Merck (Darmstadt, Germany).The conditions of elution are given in the legend to Fig. 3.The detection wavelength range was 230-350 nm and wavelength resolution was 4 nm.
The phenolics were identified by comparison of their retention time (R t values) and their UV spectra with those of authentic compounds using the comparison software available in KromaSystem 2000.Chromatograms were recorded at selected wavelengths (see Figs. legends).Quantification was performed at the most favorable wavelength (310 nm) by external calibration, and the data were expressed in nmole.The R t values were the mean values of at least three runs from two biological samples prepared from two independent elicitation sets.
Controls.Kinetic curves were developed from elicited samples and from non-induced ones assayed in parallel.The controls consisted of protoplasts (cells) incubated in buffer without inducer, in the presence or absence of an effector, e.g.cycloheximide.The viability of protoplasts (cells) was controlled during each experimental set.Blanks for enzyme assays were performed with boiled enzyme or without L-phenylalanine substrate.

Activation of PAL by inducers I, II and III
Rubus protoplasts treated or not with 100 nM inducer I, II or III for 30 min or up to 30 h were subjected to PAL extraction.It was verified that the viability of protoplasts (cells) was not affected by the inducer since it remained as high as in controls (85 or 95%).Briefly, the response to the inducer was then followed as changes in absorbance at 290 nm of reaction mixtures containing the substrate L-phenylalanine used at the optimal concentration of 2.25 mM, and the enzyme extract prepared from treated or untreated protoplasts.Data in Fig. 1 clearly show that inducer III is the most efficient with an activity of 900 mkat/kg protein while inducer I exhibits an activity of 101 mkat/kg protein only, and inducer II is not active.During 30 h of treatment, the response to inducer III was biphasic with respect to the kinetics (Fig. 2, curve b), and PAL activity in controls did not change over 20 h (Fig. 2, curve a).Initially, the treatment with inducer III resulted in a rapid and transient increase of PAL activity which peaked at 30 min approximately with the activity of 900 mkat/kg protein, giving the R value of 108.Incubation for a longer time resulted in a second initiation of PAL activity with the level of 775 mkat/kg protein, equivalent to the R value of 90 at 30 h.In the presence of 1 mM cycloheximide, the early response was maintained at R value of approximately 88 whereas the long-term induction was markedly attenuated since it decreased to the value of 20 at 30 h (Fig. 2, curve c).After 30 h the slope was increasing slowly (only for elicited protoplasts) and after 35 h the presence of contaminants was observed.The data revealed that the treatment for up to 5 h affects the specific enzyme activity since it did not significantly change the total amount of extractable proteins.In a marked contrast, the long term-treatment promoted a large increase of protein content, thus suggesting involvement of de novo synthesized proteins in the delayed response (not shown).It was observed that when suspended cells were monitored instead of protoplasts, the detected responses from inducer III were largely attenuated, by about 35-40%.

Changes in the phenylpropanoid pathway elicited by inducer III
HPLC-PAD investigations of PAL extracts prepared from elicited protoplasts or from controls were run.Typical HPLC profiles of standards with detection at 256 and 310 nm were firstly achieved as in Chevolot et al. (1997).
The chromatograms at 256 nm for the samples obtained from inducer treatment (Fig. 3A, curve a) showed the presence of major peaks F, X 1 , X 2 , X 3 , X 4 , X 5 with the R t values of 13.45, 31.07, 33.1, 37.60, 40.74 and 42.37 min, respectively.The profiles from control protoplasts (Fig. 3 A, curve b) did not exhibit the same pattern since the peak F (R t value of 13.45) is absent and lesser amounts of X 5 and higher amounts of X 1 , X 2 , X 3 and X 4 were detected.The phenolic compound F was identified as ferulic acid by comparison of its UV spectrum (l max at 234 nm, shoulder at 325 nm) and especially by the identity of its R t value with the authentic compound.The chromatograms at 310 nm indicated also that the peak F can be identified as ferulic acid (Fig. 3B, curve a), and that it is not detected in controls (Fig. 3B,  curve b).Quantification of the phenolics at 310 nm revealed that the treatment of 2´10 6 protoplasts for 30 min with 100 nM inducer III promoted the accumulation of 1.3 nmols of ferulic acid.Up to now, we failed to establish the identity of the other eluted products.For unknown compounds, comparison of peak areas at 256 nm indicated that X 5 increased by a factor of l.7 but X 1 , X 2 , X 3 and X 4 decreased by a factor of 0.4, 0.45, 0.42 and 0.37, respectively.

DISCUSSION
O-Glycan fractions obtained from Fusarium glycoproteins were used here as potential signaling molecules in raspberry protoplast suspensions.The biochemical study focused on the characterization of PAL activation generated by inducer III.This inducer, an acidic oligosaccharide fraction derived from uronic acid-containing glycoproteins from Fusarium sp.M7-1, has a chemical structure similar to that of the glycan part of cell-wall glycoproteins from Gibberella (Takegawa et al., 1991;1997).Our data indicate that inducer III elicits early events such as the induction of a PAL complex.Following the inducer treatment, the PAL response exhibited a maximal intensity after 30 min.Detection of oligosaccharides by protoplasts has also been previously reported in a number of plant systems (Dangl et al., 1987;Kauss et al., 1989;Patier et al., 1995), and the fact that the protoplasts responded here better than intact cells indicated that the signal of Fusarium origin is attenuated by plant cell walls.
Following the application of inducer III, very high levels of PAL activity were rapidly detected in protoplasts.The response is transient and it is maintained in the presence of cycloheximide.This suggested involvement of a signal cascade associated with plasma-membranes as in Nurnberger et al. (1997), and brought evidence for a post-translational regulation of the enzyme.A delayed PAL response was detected: it decreased in the presence of cycloheximide, and therefore involved late changes in PAL mRNA translation levels.An elevation of PAL response, favored as an indicator of plant resistance, has often been reported to occur at the transcriptional level.But it is worth noting that the levels of PAL mRNA did not always increase proportionally to the enzyme activity measured (Lee et al., 1992), thus suggesting that PAL can also be affected by post-transcriptional modifications (Shaw et al., 1990).Extraction of phenolics from PAL reaction mixtures by suitable organic phase followed by HPLC-PAD revealed the presence of ferulic acid, thus suggesting the activation of a PAL complex including PAL, cinnamate 4-hydroxylase, 4-coumarate CoA ligase and caffeoyl 3-O-methyltransferase, directly channeling t-cinnamic acid to ferulic acid.The presence of a PAL complex directing the flow of secondary compounds after hormone or elicitor treatment has been reported previously.The enzymes of branch pathways for phenylpropanoids, benzoates, or for flavonoids are known to be differentially expressed in response to environmental factors (Habereder et al., 1989;Funk & Brodelius, 1992;Chevolot et al., 1997).Likewise, specific enzymes, such as 3-O-methyltransferases acting at the terminal step in the biosynthesis of phenylpropanoids or of isoflavonoids, increased in response to elicitor or to pathogen (Busam et al., 1997;Grimmig & Matern, 1997;Preisig et al., 1991).O-Methyltransferases are thought to be a target of modulation in the systemic acquired resistance predisposition of tissues for en-hanced lignification (Busam et al., 1997).Caffeic acid, ferulic acid or its derivative curcumin exhibited inhibitory effect on ornithine decarboxylase (Huang et al., 1988).
In conclusion, the results revealed that a carbohydrate fraction of Fusarium origin can be a signaling molecule that causes, at a post-translational level, changes in phenolic metabolism.Based on the elicitation of ferulic acid, it can be proposed that the pathogen Fusarium affected the oxidant system of the host.Up to now, enzymes of the phenolic pathway had been often described as playing a central role in the orchestration of hypersensitive responses in plants, but here they are shown for the first time to be dependent on the application of a well defined elicitor from Fusarium.

R E F E R E N C E S
Alconada TM, Martinez MJ. ( 1995 trometry ( 1 H) in combination with methylation mass spectrometry and fast-atom bombardment mass spectrometry.Inducer I is an acidic polysaccharide with a M r of about 50000.The chain backbone is a galactan linked through b(1®6) galactofuranoside linkages, with Man b(1®4) GlcA, Glc, and Man b(1®2) Man b(1®4) GlcAa1 as main side chains.Inducer II (with an average M r of 1500) consists of neutral oligosaccharides with Man-ol, Man b(1®2) Man-ol as the main sugars.Inducer III, a Dowex 1-X2-bound fraction, is a mixture of acidic oligosaccharides, having the average M r of 1000, with the following structures: (a) Man b(1®4) GlcA a(1®2) Galf b(1®6) Man a(1®2) Man-ol and Man b

Figure 1 .
Figure 1.PAL response in Rubus protoplasts.PAL activity of 2´10 6 protoplasts challenged for 30 min with 100 nM inducer I, II or III or in non-induced protoplasts (C) was determined at 290 nm.The values are expressed as relative rates of reaction products (mkat/kg protein).All experiments were carried out in triplicate.

Figure 2 .
Figure 2. Time-course for PAL activity (PAL activation) in protoplasts challenged with 100 nM inducer III only (curve b) or 100 nM inducer III plus 1 mM cycloheximide (curve c); curve a shows the response in non-induced protoplasts.2´106protoplasts were elicited for up to 30 h, and PAL extracts were then assayed at 290 nm.The slope of kinetic curves obtained by linear-regression fits of the data from four assays performed in three independent sets gave the relative rate of reaction products expressed in mkat/ kg protein or by the R value (the activity in treated protoplasts vs controls).

Figure 3 .
Figure 3. HPLC chromatograms of phenolics in PAL reaction mixtures derived from Rubus protoplasts elicited for 30 min with 100 nM inducer III (curve b) or from controls (curve a).The phenolics in the reaction mixtures were eluted on RP-18 column, and the mobile phase was solvent A (5% (v/v) acetic acid in Milli-Q water) and solvent B (MeOH).Elution programme was: 0-5 min, isocratic 15% B in A; 5-35 min, linear gradient 15% B in A to 80% B in A; 35-45 min, isocratic 80% B in A. The flow rate was 1 ml/min.The detection and quantification were carried out with PAD at 256 nm in (A) or at 310 nm in (B) from 100 ml of injected sample.Peak F was identified as ferulic acid.