Ste ´phane

Maury • A Delaunay

F Mesnard

D Cro

B Chabbert

P Geoffroy

M Legrand

S Maury

Á M Legrand

Á A Delaunay

Á B Chabbert

O-methyltransferase(s)-suppressed plants produce lower amounts of phenolic vir inducers and are less susceptible to Agrobacterium tumefaciens infection

Keywords: Acetosyringone, Agrobacterium, Caffeic acid O-methyltransferase, Caffeoyl-coenzyme A O-methyltransferase, HPLC

The first step of Agrobacterium tumefaciens/ plant interaction corresponds to the activation of a transduction pathway of the bacterium by plant exudate. Phenolic compounds rapidly secreted by wounded plant cells induce the expression of bacterial virulence (vir) genes; however, little is known about their biosynthesis in plant. Here we show that inoculation of an Agrobacterium tumefaciens virulent strain on orthodiphenol-O-methyltransferases-suppressed tobacco plants leads to significantly smaller tumors compared to control plants. These transgenic plants are inhibited for caffeic acid O-methyltransferase class I or II (OMT; EC 2.1.1.6) and/or caffeoyl-coenzyme A O-methyltransferase (CCoAOMT; EC 2.1.1.104) that are involved in monolignol biosynthesis. The significant decrease of tumor size could be suppressed by the pre-activation of bacterial virulence, before inoculation, using acetosyringone a known vir inducer. Total soluble phenolic amounts and cell wall composition analyzed by FT-IR analysis did not show significant differences between transgenic and control plants. The potential of phenolic extracts from control and OMT-suppressed plants to induce virulence was evaluated using an Agrobacterium tumefaciens reporter strain carrying a vir::LacZ gene fusion plasmid. Lower virinducing activities were recorded for plants that show inhibition to caffeic acid O-methyltransferase activity. HPLC analysis confirmed that the levels of several phenolic compounds were differently affected by wounding and/or by bacterial inoculation. Statistical correlations were established between tumor sizes, vir-inducing activities, O-methyltransferases proteins accumulations and the levels of various soluble phenolic compounds such as acetosyringone. These results demonstrate the role of the O-methyltransferases of the phenylpropanoid pathway in the early production of soluble Agrobacterium tumefaciens vir inducers.

1

Introduction

The phenylpropanoid pathway that provides many phenolic compounds to the plant cell, such as salicylic acid, flavonoids, coumarins and lignin-building units, is strongly activated upon infection by pathogens or treatment with elicitors [START_REF] Dixon | Stress-induced phenylpropanoid metabolism[END_REF][START_REF] Dixon | The phenylpropanoid pathway and plant defense-a genomics perspective[END_REF][START_REF] Ferrer | Structure and function of enzymes involved in the biosynthesis of phenylpropanoids[END_REF]. Plant phenolics play important roles in the outcome of plant/microbe interactions. On the one hand, in infected plants, the deposition of phenylpropanoid compounds participates in cell wall reinforcement that restricts pathogen invasion [START_REF] Vance | Lignification as a mechanism of disease resistance[END_REF][START_REF] Nicholson | Phenolic compounds and their role in disease resistance[END_REF][START_REF] Dixon | Stress-induced phenylpropanoid metabolism[END_REF] by cross-linking with cell wall constituents [START_REF] Iiyama | Covalent cross-links in the cell wall[END_REF]). On the other hand, phenolic compounds act as primary signals for several bacteria such as Rhizobium and Agrobacterium [START_REF] Gelvin | Agrobacterium virulence gene induction[END_REF][START_REF] Mccullen | Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer[END_REF] to activate an early transduction pathway controlling the expression of bacterial genes involved in symbiosis or pathogenesis.

Wounded plant cells secrete exudate with high concentration of various phenolics (Walker et al. 2003a, b) that induce Agrobacterium tumefaciens virulence (vir) genes expression. Agrobacterium tumefaciens is a Gram-negative soil bacterium that causes crown gall disease. It affects many woody and herbaceous plants belonging to 140 genera of more than 60 families and particularly fruit trees of economic interest. This bacterium has the capacity to transfer genetic material (T-DNA) into the plant cell genome after vir genes induction by plant phenolics [START_REF] Gelvin | Agrobacterium virulence gene induction[END_REF][START_REF] Mccullen | Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer[END_REF][START_REF] Dafny-Yelin | The ongoing saga of Agrobacterium-host interactions[END_REF]. The specific composition of phenolic compounds in plant exudates may influence the host specificity of Agrobacterium tumefaciens.

The best characterized and most effective vir inducers are monocyclic phenolics such as acetosyringone (Stachel et al. 1985a), coniferyl and sinapyl alcohol [START_REF] Melchers | Specificity of signal molecules in the activation of Agrobacterium virulence gene expression[END_REF]; diphenolic compounds such as dehydrodiconiferyl alcohol aglycone and glucosides [START_REF] Lynn | The structural characterization of endogenous factor from Vinca rosea crow, gall tumors that promote cell division of tobacco cells[END_REF][START_REF] Orr | Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth[END_REF][START_REF] Teutonico | Activity and accumulation of cell division-promoting phenolics in tobacco tissue cultures[END_REF]; hydroxycinnamic acid amides [START_REF] Berthelot | Vir-gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens[END_REF]; sugars or acidic pH [START_REF] Shimoda | Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides[END_REF][START_REF] Zhu | The bases of crown gall tumorigenesis[END_REF] and GABA [START_REF] Chevrot | GABA controls the level of quorum-sensing signal in Agrobacterium tumefaciens[END_REF]. The vir regulon is coordinately induced in response to host phenolic compounds in combination with monosaccharides and extracellular acidity in the range of pH 5-5.5. Vir inducers are not detected or detected at very low levels in uninjured plants, but their amounts increase rapidly in wounded plant cells (Stachel et al. 1985a;[START_REF] Teutonico | Activity and accumulation of cell division-promoting phenolics in tobacco tissue cultures[END_REF][START_REF] Tamagnone | Inhibition of phenolic acid metabolism results in precocious cell death and altered cell morphology in leaves of transgenic tobacco plants[END_REF][START_REF] Chevrot | GABA controls the level of quorum-sensing signal in Agrobacterium tumefaciens[END_REF]. These chemical stimuli are detected by the transmembrane twocomponent sensor kinase VirA [START_REF] Lee | Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens[END_REF][START_REF] Gelvin | Agrobacterium virulence gene induction[END_REF]. This sensor contains four functional domains (periplasmic, linker, kinase and receiver). Its periplasmic domain is required for detection of a sugar binding protein named ChvE, while the linker domain allows the detection of phenolic compounds. Then, VirA can phosphorylate the response regulator VirG that positively regulates all vir promoters. It was recently shown that salicylic acid could attenuate the function of the VirA kinase domain [START_REF] Yuan | The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium[END_REF].

Most of the phenolic vir inducers are mono-or di-methoxylated compounds, the biosynthesis and relative importance for the establishment of tumors of which are poorly understood. Two S-adenosyl-L-methionine (SAM)dependent O-methyltransferases (OMTs) methylate the phenyl ring of monolignol precursors: caffeic acid OMTs (COMTs) and caffeoyl-CoA OMTs (CCoAOMTs). COMT methylates caffeoyl-and 5-hydroxyferuloyl-containing acids, aldehydes and alcohols in vitro but displays a kinetic preference for the alcohols and aldehydes over the free acids, while CCoAOMT accepts only the CoA-ester as a substrate [START_REF] Maury | Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different CCoAOMT and COMT classes have distinct substrate specificities and expression patterns[END_REF][START_REF] Hoffmann | Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis[END_REF][START_REF] Do | Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid 3-O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis[END_REF][START_REF] Ferrer | Structure and function of enzymes involved in the biosynthesis of phenylpropanoids[END_REF]. Furthermore, transgenic tobacco plants suppressed for the different orthodiphenol-O-methyltransferases (OMTs) involved in the phenylpropanoid pathway were produced and the impact on lignin biosynthesis and plant growth was studied [START_REF] Atanassova | Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation[END_REF]Pinc ¸on et al. 2001a). These plants show inhibition to caffeic acid orthodiphenol-O-methyltransferase (COMT) of class I or II and/or caffeoyl-coenzyme A orthodiphenol-O-methyltransferase (CCoAOMT) activities by antisense RNA expression. The activation of the OMTs during plant/pathogen interaction such as tobacco/TMV has been reported [START_REF] Maury | Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different CCoAOMT and COMT classes have distinct substrate specificities and expression patterns[END_REF][START_REF] Toquin | Structure of the tobacco caffeic acid O-methyltransferase (COMT) II gene: identification of promoter sequences involved in gene inductibility by various stimuli[END_REF], but no data were available about their possible role in the production of vir inducers during infection of tobacco by Agrobacterium tumefaciens. Here, we show that OMTsuppressed transgenic plants inoculated with a virulent Agrobacterium tumefaciens strain developed smaller tumors than those of control plants. This phenotype could be suppressed by the pre-activation of bacterial virulence with acetosyringone before inoculation. Lower vir gene induction activities were measured using an Agrobacterium tumefaciens reporter strain carrying a vir::LacZ gene fusion in plants expressing COMTI, COMTII and COMTI:: CCoAOMT. Finally, HPLC analysis demonstrated that a few phenolic compounds such as acetosyringone are major vir inducers that are produced in lower amounts in OMTsuppressed tobacco plants compared to controls upon infection. These results demonstrate the dual role of OMTs of the phenylpropanoid pathway in monolignol synthesis and in the early production of soluble Agrobacterium tumefaciens vir inducers.

Materials and methods

Bacterial strain and culture conditions

Agrobacterium tumefaciens strain A348 harboring the octopine type Ti plasmid (pTiA6) was used for plant inoculation [START_REF] Anand | Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing[END_REF]). We used an A348 strain harboring the pSM219 plasmid that carries lacZ under control of the virH promoter in trans to the wild-type pTiA6 plasmid for virulence induction measurements (Stachel et al. 1985b). Bacteria were cultured in Luria-Bertani medium supplemented with kanamycin (50 lgm l -1 ) and rifampicin (10 lgm l -1 )a t2 8 °C [START_REF] Anand | Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing[END_REF]). Overnight cultures were centrifuged and resuspended in culture medium to obtain 0.6 absorbance unit ml -1 at 660 nm before use. [START_REF] Atanassova | Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation[END_REF][START_REF] Martz | Modification de l'activite ´O-me ´thyltransfe ´rase dans des tabacs transge ´niques: conse ´quences sur la lignine et la re ´sistance au Virus de la Mosaı ¨que du Tabac[END_REF]Pinc ¸on et al. 2001a) has been published previously. Plants were grown in the greenhouse under controlled conditions at 22°C with a 16-h/8-h photoperiod.

Stems of 6-week-old plants were wounded at the level of the same internode using a needle and inoculated with 10 ll of a culture at 10 9 CFU ml -1 of virulent Agrobacterium tumefaciens strain A348 induced or not with commercial acetosyringone (Sigma-Aldrich, Saint-Quentin Fallavier, France) according to [START_REF] Anand | Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing[END_REF]. Briefly, overnight cultures were centrifuged, washed and resuspended in agroinduction medium supplemented or not with acetosyringone (150 lgm l -1 )at24°C for 15 h. The cultures were washed with sterile distilled water and resuspended in 0.9% NaCl at 10 9 CFU ml -1 for in planta tumor assay. Two types of control plants were used: wounded plants inoculated with 10 ll of bacterial media alone and unwounded plants. At 6 h after infection, treated and unwounded stem tissues of half of the plants were harvested for further analyses. Thus, previously published data on agro-inoculated tobacco BY-2 cell suspensions suggest a model in which tobacco undergoes a response in the first 6 h [START_REF] Veena | Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defence gene expression[END_REF]. The other half of the plants were grown in the greenhouse for 1 more month to obtain tumors. Tumors were pictured with a numeric camera, separated from stem tissues using a scalpel, weighed and frozen in liquid nitrogen. The numeric pictures were analyzed using imaging software (ImageTool for Windows version 3.00) to determine the area of each tumor. Two independent experiments with at least 20 individuals of the same clone by genotype and treatment were done.

Callus induction and proliferation

Callus induction and proliferation were carried out to check if the response to growth regulators (similar to those produced in crown galls) of transgenic tissues was affected or not compared to control plants. Callus induction was realized according to a standard protocol [START_REF] Atanassova | Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation[END_REF]. Briefly, leaves and shoots from transgenic and control plants from 2-week-old in vitro seedling were cultivated separately on MS medium supplemented with 2 mg l -1 of 6-benzylaminopurine and 0.05 mg l -1 of naphthalene acetic acid (Sigma-Aldrich). Subcultures were performed every 2 weeks during 6 weeks. Calli obtained for each type of plant were initiated with the same number of starting explants (size homogenized between plants). At the end of the 6 weeks, the total fresh mass of callus was determined by weighing each Petridish. Eight replicates and two independent experiments were done for each type of plant and explant.

SDS-PAGE and immunoblotting

The basic procedures for electrophoresis under denaturing conditions, immunoblotting and OMT antibodies dilutions have been described previously [START_REF] Maury | Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different CCoAOMT and COMT classes have distinct substrate specificities and expression patterns[END_REF]). The blots were scanned and the protein amounts were quantified using imaging software (ImageTool for Windows version 3.00).

Extraction and quantification of phenolic compounds and vir gene induction assay

Tobacco stems (60 mg) were ground in liquid nitrogen and extracted twice with 70% methanol (1/20) (v/v). After centrifugation at 3,000g for 15 min, supernatants were collected, evaporated and resuspended in 1 ml of 70% methanol. Spectrophotometric quantification of total phenolic compounds was done using Folin-Ciocalteu's reagent (Carlo Erba Reagenti, Rodano, Italy) with an adapted method from [START_REF] Causevic | DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines[END_REF]. Catechin (0-10 lg ll -1 ) was used as a standard. Bioassays for vir-inducing activity were performed according to Stachel et al. (1985b). Briefly, overnight cultures of strain A348 (pSM219) were centrifuged and resuspended in culture medium. As much as 100 llof material to be tested for vir-inducing activity was inoculated with 900 ll of bacteria culture at 0.1 absorbance unit ml -1 at 600 nm and incubated for 15 h at 28°C. The reporter b-galactosidase activity was measured, and the results were expressed in specific units calculated as previously described [START_REF] Miller | Experiments in molecular genetics[END_REF]Stachel et al. 1985b). All experiments were performed in triplicate and controls with known amounts of acetosyringone or without inducer were included.

HPLC analysis

Methanolic extracts from stem were analyzed using highperformance liquid chromatography (HPLC). Analyses were performed using C18 columns (Kromasyl, 4.6 cm 9 25 cm, 5 lm Phenomenex, Le Pecq, France and repeated on X-terra, 4.6 cm 9 15 cm, 5 lm, INTERCHIM, Montluc ¸on, France). The peaks were identified according to their retention time and spectral properties compared with commercial standards (Sigma-Aldrich). Eluted peaks were collected as independent fractions and tested for their vir-inducing potential.

FT-IR spectroscopy FT-IR spectra were recorded between 4,000 and 400 cm -1 at 4 cm -1 resolution on a Nicolet spectrophotometer using KBr discs containing 1% of the dry samples (lyophilized tissues) and corrected for the KBr background. The window between 800 and 2,000 cm -1 , which shows information on polysaccharides and lignin was selected to compare cell wall modifications between samples. All spectra were baseline corrected, then normalized at 1,650 cm -1 prior to spectra comparison. Average spectra were obtained from the cell wall analysis of the three replicates.

Statistical analysis

Statistical analysis was done using SPSS statistical software package (SPPS version 11.0.1 PC, Chicago, IL, USA). Genotypic and treatment effects were evaluated by two-way ANOVA (GLM procedure) using the following model: Y ijk = l ? G i ? T j ? (G i 9 T j ) ? e ijk ; where Y ijk are individual values, l is the general mean, G i is the effect of genotype i, T j is the effect of treatment j, G i 9 T j is the genotype by treatment interaction and e ijk is the error. All statistical tests were considered as significant at *P B 0.05, **P B 0.01 or ***P B 0.001.

Results

Induction of OMTs upon infection by Agrobacterium is decreased in antisense tobacco lines

Immunodetection of OMTs in stems of agro-inoculated controls (transformed with the empty plasmid) or OMT-suppressed plants using antibodies raised against COMTI, COMTII or CCoAOMT revealed four bands (Fig. 1a). The 39-kDa and 42-kDa isoforms corresponded to COMT proteins of class I and class II, respectively, while the 27-kDa and 32-kDa isoforms revealed two CCoAOMT isoforms [START_REF] Maury | Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different CCoAOMT and COMT classes have distinct substrate specificities and expression patterns[END_REF]. Quantification of these four isoforms in wounded-mock inoculated, wounded-agroinoculated or unwounded transgenic stems 6 h after the treatment is shown in Fig. 1b. A genotypic effect was

Repression of the phenylpropanoid pathway decreases the virulence of Agrobacterium tumefaciens

Typical tumors that developed on stems of agro-inoculated control and OMT-suppressed plants are shown in Fig. 2a.

The mass and area reached by the tumors 1 month after inoculation were used to evaluate bacterial virulence (Fig. 2b,c). These two variables were shown to be strongly correlated (r = 0.91 at P \ 0.05). Transgenic plants inhibited for COMTI, COMTII or COMTI and CCoAOMT, but not CCoAOMT alone, exhibited tumors with significantly smaller size and mass compared to those developing on control plants.

To find the causes of tumor size decrease on transgenic plants, two hypotheses were tested: (i) the ability of OMTsuppressed cells to divide. In vitro callus proliferation was measured and it was confirmed that transgenic tissues respond to growth regulators (similar to those produced in crown galls) in a similar way as control cells (Fig. 3a); and (ii) the ability of OMT-suppressed plants to activate Agrobacterium virulence. In planta tumor assays were performed with activated (or not) bacteria using acetosyringone before inoculation (Fig. 3b). In each plant, the vir induction was followed by an increase of tumor area (about 10-20%). The significant decrease of tumor size using non-activated bacteria was confirmed on transgenic plants inhibited for COMTI, COMTII or COMTI and CCoAOMT, but was suppressed using vir-induced bacteria compared to control plants.

To confirm that the effect on tumor development was mediated by a decrease in the biosynthesis of phenolic signals implicated in the activation of vir genes, we compared vir-inducing activities of phenolic stem extracts from the different tobacco genotypes (Fig. 4). An Agrobacterium tumefaciens strain carrying a pin-F::lacZ fusion gene was used as a reporter strain. In this strain, b-galactosidase expression is controlled by the vir locus and the relative amount of induced activity reflects the relative amount of vir-inducing activity to which the bacterial cell has been exposed (Stachel et al. 1985b). A stimulation of the vir activity was noticed in agro-inoculated control plants compared to unwounded ones (Fig. 4). Only a genotypic effect was observed for all transgenic plants. Transgenic plants inhibited for COM-TI, COMTII or COMTI and CCoAOMT, but not CCoAOMT alone, showed a decreased of their viractivities in healthy and/or agro-inoculated stems comto control plants. The lack of treatment effect reflected the absence of stimulation of the vir activity in OMT-suppressed plants. Linear correlations were established between tumor areas, vir-inducing activities and COMTI accumulation (Table 1). As vir-inducing activity inhibition paralleled the decrease in OMT stimulation in transgenics, this prompted us to analyze the phenolic composition of cell walls using FTIR spectroscopy and to estimate the total soluble phenolic fraction from stems (Suppl. materials 1 and 2, respectively). Several bands indicative of the main cell wall compounds such as polysaccharides (1,050 cm -1 ), proteins (1,560 cm -1 and 1,650 cm -1 ), esters (1,700 cm -1 ) and aromatic skeleton (1,420 cm -1 ) were observed by FTIR (Suppl. material 1). Overall, the fingerprint profile of FTIR spectra from the walls of 6-h post-treatment stems showed little variations compared to that of unwounded samples. Nevertheless, an increase of polysaccharides was observed in wounded control and in agro-inoculated dAS plants. In agro-inoculated CCoAOMT and dAS plants, an additional band was visible at 1,270 cm -1 and could possibly correspond to syringyl units. A genotypic effect was observed for total soluble phenolic contents but no treatment effect was detected (Suppl. material 2). dAS plants exhibited higher level of soluble content than control plants.

Soluble phenolic extracts from the different transgenic stems were separated by HPLC and 26 peaks were quantified in the extracts (Fig. 5). In control plants, the amounts of about half of them were affected either by wounding (peaks 4, 24 and 26), agro-inoculation (peaks 5, 7 and 13) or both treatments (peaks 6, 10, 14, 15, 18, 19, 21 and 23). Variations among transgenic plants were also observed for several peaks such as peak 1 that was increased in COMTIinhibited plants. Peak 15 was identified as acetosyringone, one of the major known vir inducer (Stachel et al. 1985a, b). This compound was not detected in COMTI and dAS Abbreviations used for genotypes are as in Fig. 1. Mean values ± SE (n = 4) are presented. Two independent experiments were performed. Significant differences between control and transgenic plants are indicated at *P B 0.05. In order to evaluate genotypic and treatment effects, two-way ANOVA analysis was performed: for each graph, g indicates the genotype effect, t the treatment effect and g 9 t the genotype by treatment interaction. Significant effects are indicated at *P B 0.05 or **P B 0.01 antisense plants (Fig. 5b,e) but was present in CCoAOMTrepressed tissues, thus demonstrating the function of COMTI in acetosyringone biosynthesis. In plants producing significant amount of acetosyringone, its quantities scaled positively with vir-inducing activities, COMTI and CCoAOMT levels (Table 1). Plants that do not produce detectable amounts of acetosyringone, such as COMTI and dAS plants (Fig. 5f), still exhibit vir-inducing activity due to unidentified compounds. Extracts from unwounded and wounded-agro-inoculated control or dAS transgenic genotypes were fractionated by HPLC to identify differences in active phenolic compounds. Each fraction was tested for its vir-inducing activity and the difference between values obtained for infected and unwounded plants was calculated (Fig. 6). Increased vir activities were found in control plants for fractions number 1, 2, 6 and 10, with highest changes in activity upon infection being recorded for fractions number 2 and 6. The compound responsible for inducing activity of fraction 2 could not be identified, but the active compound in fraction 6 was shown to be acetosyringone on the basis of elution time and absorbance spectrum compared to that of standard. Interestingly, some fractions issued from dAS profile displayed significant activity, while the corresponding control fractions were inactive (fractions number 7, 8 and 9; Fig. 6). These data suggest that OMT inhibition provoked the appearance of metabolic intermediates with inducing activity. The nature of these products remains to be elucidated.

Discussion

OMTs of the phenylpropanoid pathway are involved in wounding and agro-infection responses of tobacco plants COMTI and/or CCoAOMT-inhibited transgenic tobacco plants have been previously produced and characterized [START_REF] Atanassova | Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation[END_REF][START_REF] Martz | Modification de l'activite ´O-me ´thyltransfe ´rase dans des tabacs transge ´niques: conse ´quences sur la lignine et la re ´sistance au Virus de la Mosaı ¨que du Tabac[END_REF]Pinc ¸on et al. 2001a).

Their analysis showed different impacts on lignin synthesis that were changes in lignin composition with a decrease of syringyl units and/or a decrease of total lignin content. Similar effects of OMT inhibition were confirmed in other plant species [START_REF] Doorsselaere | A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity[END_REF][START_REF] Zhong | Dual methylation pathway in lignin biosynthesis[END_REF][START_REF] Do | Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid 3-O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis[END_REF]. In plants transformed with a double antisense construct (CCoAOMT/COMTI), stronger cumulative effects were obtained (Pinc ¸on et al. 2001a;[START_REF] Do | Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid 3-O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis[END_REF]). Down-regulation of OMT isoforms was confirmed in the present work in the stem of each type of unwounded transgenic plants. Furthermore, inhibition of one OMT isoform was accompanied, in some cases, by the co-repression of another OMT isoform by an unknown mechanism. A similar phenomenon has been reported for several enzymes of the phenylpropanoid pathway including OMTs (Pinc ¸on et al. 2001a, b). Here, we show that all OMT isoforms (COMTI, COMTII and CCoAOMT) are strongly and early induced in agro-infected stems of control plants. This result is in agreement with the model proposed by [START_REF] Veena | Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defence gene expression[END_REF] suggesting that tobacco responds in the first 6 h of infection, while Arabidopsis responds after 48 h [START_REF] Ditt | The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens[END_REF]. In transgenic tobacco plants, the targeted OMT(s) was downregulated even after infection. Up-regulation of the expression of genes encoding enzymes of the phenolic metabolism such as CCoAOMT by Agrobacterium infection has been shown in tobacco cells and Arabidopsis [START_REF] Ditt | The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens[END_REF][START_REF] Veena | Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defence gene expression[END_REF]. Similar early induction was previously reported for tobacco OMTS in wounded or TMV-infected leaves [START_REF] Jaeck | Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus[END_REF][START_REF] Pellegrini | Molecular cloning and expression of a new class of ortho-diphenol-Omethyltransferases induced in tobacco leaves by infection or elicitor treatment[END_REF][START_REF] Martz | cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme[END_REF][START_REF] Maury | Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different CCoAOMT and COMT classes have distinct substrate specificities and expression patterns[END_REF][START_REF] Toquin | Structure of the tobacco caffeic acid O-methyltransferase (COMT) II gene: identification of promoter sequences involved in gene inductibility by various stimuli[END_REF]. These results suggest a general involvement of OMTs in the responses of plants to wounding or pathogen attack.

Down-regulation of OMTs results in lower susceptibility of tobacco plants to agro-infection by decreasing virulence of Agrobacterium tumefaciens

In transformed cells, the expression of T-DNA genes that are involved in the synthesis of plant hormones results in Relative absorbance at 320 nm (arbitrary units) Relative absorbance at 320 nm (arbitrary units)

the growth of tumors on the host plant [START_REF] Gelvin | Agrobacterium virulence gene induction[END_REF][START_REF] Mccullen | Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer[END_REF]. We found that tumors developing on COMT-suppressed plants were significantly smaller than those on control plants. This phenotype was not related to an alteration of transgenic cells to grow and divide. Furthermore, the phenotype was suppressed if Agrobacterium was artificially activated with acetosyringone before inoculation. As few methoxylated phenolic compounds were known to be strong vir inducers (Stachel et al. 1985a;[START_REF] Melchers | Specificity of signal molecules in the activation of Agrobacterium virulence gene expression[END_REF][START_REF] Lynn | The structural characterization of endogenous factor from Vinca rosea crow, gall tumors that promote cell division of tobacco cells[END_REF][START_REF] Mccullen | Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer[END_REF], we tested the ability of phenolic extracts from agro-infected transgenic and control plants to induce vir genes. A strong inhibition of the vir induction was measured in COMT-suppressed plants and particularly in double transgenic plants, as has been previously shown for lignin biosynthesis (Pinc ¸on et al. 2001a). This result indicated that COMTI inhibition resulted in a strong decrease of vir-inducer biosynthesis.

In CCoAOMT-inhibited plants, no reduction of the virinducing activities was found, in good agreement with the lack of effect on the development of tumors on these transgenics. The strong impact in double transgenic plants could be due to the co-repression of COMTs and CCoAOMTs leading to the lowest residual accumulation of all OMTs as previously observed (Pinc ¸on et al. 2001a). Interestingly, in most of the transgenic plants, vir-inducing activities of wounded extracts were significantly different from the vir-inducing activities of agro-inoculated extracts, but not from those of unwounded controls. The linear correlation we established between COMTI accumulation, tumor size and vir-inducing activities suggests that COMTI suppression affects the production of vir inducers synthesized in response to Agrobacterium infection and consequently decreases the susceptibility of the plant to the bacteria.

OMTs are involved in the early production of acetosyringone

OMTs from the phenylpropanoid pathway are known to synthesize, upon infection by pathogens, methoxylated phenolics either soluble or cross-linked to the cell walls [START_REF] Nicholson | Phenolic compounds and their role in disease resistance[END_REF][START_REF] Iiyama | Covalent cross-links in the cell wall[END_REF][START_REF] Dixon | The phenylpropanoid pathway and plant defense-a genomics perspective[END_REF][START_REF] Mccullen | Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer[END_REF]. However, little is known about the partitioning between the soluble and cell wall-bound fractions in the early stages of infection or wounding. In this frame, FT-IR spectra analysis did not show significant cell wall cross-linking 6 h after wounding or agro-inoculation. It is likely that such phenomenon would take several hours or days as it has been shown in some cases [START_REF] Ascensao | Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f.sp. cubense[END_REF][START_REF] Hawkins | Defense lignin and hydroxycinnamyl alcohol dehydrogenase activities in wounded Eucalyptus gunnii hook[END_REF][START_REF] Hano | Differential accumulation of monolignolderived compounds in elicited flax (Linum usitatissimum) cell suspension cultures[END_REF]). Nevertheless, an increased incorporation of syringyl lignin units was observed in relation to CCoAOMT inhibition (CCoAOMT and dAS plants) after agro-inoculation in accordance with the results in Arabidopsis [START_REF] Do | Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid 3-O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis[END_REF]). Thus, we concentrated our efforts on the analysis of the soluble phenolic fraction. Quantitative variations were observed between transgenic plants but without significant impact on the treatment (Suppl. material 2). This result, at odds with the variations of vir-inducing activities upon wounding and infection, prompted us to perform HPLC separation of soluble phenolic extracts from treated plants. We found that the production of several phenolic compounds was stimulated in response to wounding and/or agro-infection, in good agreement with their vir-inducing activities.

The biosynthesis of acetosyringone was shown to be dependent on COMTI and to be particularly inhibited in plants repressed for both COMTI and CCoAOMT. This is certainly correlated to the presence of two methoxylated groups in acetosyringone molecule that may be introduced sequentially by the two OMTs, as demonstrated for the synthesis of syringyl units of lignin [START_REF] Atanassova | Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation[END_REF]Pinc ¸on et al. 2001a). Nevertheless, inhibition of COMTI is sufficient to obtain a significant decrease in acetosyringone content and this is reminiscent of the decrease in syringyl units observed in COMTI-suppressed plants [START_REF] Atanassova | Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation[END_REF][START_REF] Doorsselaere | A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity[END_REF].

Similarly, altered sinapoyl malate biosynthesis was found in an Arabidopsis COMTI-CCoAOMT double mutant [START_REF] Do | Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid 3-O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis[END_REF]. All these results demonstrate a clear biosynthetic relationship between acetosyringone and sinapate derivatives. We have previously characterized COMT and CCoAOMT substrate specificities toward monolignol precursors [START_REF] Maury | Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different CCoAOMT and COMT classes have distinct substrate specificities and expression patterns[END_REF][START_REF] Hoffmann | Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis[END_REF]) and it will be now interesting to measure their kinetic values toward 3,4,5-trihydroxyacetophenone and 5-methoxy, 3,4dihydroxyacetophenone to validate the putative involvement of the enzymes in acetosyringone biosynthesis.

Another possibility would involve a decarboxylation step of sinapate derivatives (sinapic acid, sinapoyl-CoA, sinapaldehyde or sinapyl alcohol). It is noteworthy here that inhibition of cinnamoyl-CoA reductase (which accepts sinapoyl-CoA as substrate) in CCR-suppressed transgenic tobacco leads to the incorporation of acetosyringone in plant cell walls [START_REF] Chabannes | Strong alteration in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl coA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants[END_REF]. Acetosyringone is usually not detectable in tobacco cell walls, but can be found, for example, in wheat straw after alkaline wet oxidation [START_REF] Klinke | Characterization of degradation products from alkaline wet oxidation of wheat straw[END_REF]. It was suggested that CCR inhibition has led to a change in the metabolic flux through the phenylpropanoid pathway.

The biological significance of phenolic release during wounding is poorly understood. It was proposed that acetosyringone could be related to lignin (cell wall?) repair in damaged cells or potentially toxic to other soil pathogens (Stachel et al. 1985a). This last assertion is supported by the recent report of inhibitory effects of acetosyringone on Pseudomonas syringae virulence [START_REF] Baker | Involvement of acetosyringone in plantpathogen recognition[END_REF]) and aflatoxin biosynthetic genes of Aspergillus flavus [START_REF] Hua | Inhibition of aflatoxin biosynthesis by phenolic compounds[END_REF][START_REF] Hua | Inhibitory effect of acetosyringone on two aflatoxin biosynthetic genes[END_REF]. This suggests that Agrobacterium tumefaciens has evolved an acquired resistance to acetosyringone. It was also recently shown that other phenolics like salicylic acid could attenuate the function of the VirA kinase domain and participate in defenses against Agrobacterium infection [START_REF] Yuan | The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium[END_REF]). On the other hand, several monocotyledonous plants have been shown to contain vir gene inducing factors as ethylferulate [START_REF] Messens | A nontransformable Triticum monococcum monocotyledonous culture produces the potent Agrobacterium vir-inducing compound ethyl ferulate[END_REF]), although they are recalcitrant to transformation by Agrobacterium tumefaciens. It has been proposed that this could be due to the local secretion around the root axis of MDIBOA (2-hydroxy-4,7-dimethoxybenzoxazin-3-one), a potent and specific inhibitor of the VirA-mediated induction of vir gene expression [START_REF] Zhang | At the maize/Agrobacterium interface: natural factors limiting host transformation[END_REF].

Agrobacterium tumefaciens affects few monocotyledonous and a large array of dicotyledonous plants, particularly fruit trees of economic interest. In our experiments, down-regulation of vir inducers in tobacco has led to an increased resistance to infection suggesting that this strategy could be applied to plants of economic interest. However, recent analyses of COMTI and CCoAOMT-inhibited double transgenic tobacco have shown that they developed larger necrotic lesions on tobacco mosaic virus infection [START_REF] Maury | Etude des O-me ´thyltransfe ´rases de la voie des phe ´nylpropanoı ¨des chez le tabac et modulation de leur expression dans des tabacs transge ´niques: conse ´quences sur la biosynthe `se de la lignine et d'autres compose ´s phe ´noliques et sur la re ´sistance aux agents pathoge[END_REF]Legrand et al. unpublished results). This increase of lesion size reflects a decrease of plant resistance to virus infection and suggests that some soluble and/or cell wallbound phenolic compounds are synthesized by OMTs and are important elements of plant defense against tobacco mosaic virus. These phenolic compounds will have to be identified and quantified in transgenic tobacco plants. Altogether, these results demonstrate the complexity and the variety of the roles of phenolic compounds in plant adaptation to its environment.

Fig. 1

 1 Fig. 1 Accumulation of OMT proteins in tobacco stems of various genotypes upon wounding and agro-infection. Treated stem tissues were harvested 6-h post-inoculation. Samples were immunoblotted with antibodies recognizing COMTI (gray bars), COMTII (white bars) or CCoAOMT (black bars). Protein amounts were quantified by scanning immunoblots. a Western blots showing the different OMT isoforms and their corresponding mass in kDa in agro-inoculated stems of transgenic plants. C control plants transformed with an empty plasmid. Antisense transgenic tobaccos were suppressed for COMTI (COMTI), COMTII (COMTII), CCoAOMT (CCoAOMT)o r for bothCOMTI and CCoAOMT (dAS). b Amounts of the three types of OMT. The calculated protein amounts represent the mean values ± SE of two independent experiments using three replicates. Significant differences between control and transgenic plants are indicated at *P B 0.05 or **P B 0.01 To evaluate genotypic and treatment effects, two-way ANOVA analysis was performed: for each graph. g indicates the genotype effect, t the treatment effect and g 9 t the genotype by treatment interaction. Significant effects are indicated at *P B 0.05 or **P B 0.01

Fig. 2

 2 Fig. 2 Impact of OMT repression on the development of Agrobacterium tumefaciens tumors. a Phenotype of typical tumors; genotypes and abbreviations are as in Fig. 1. b Tumor area. c Tumor mass values. Mean values ± SE (n = 40) are shown. Abbreviations used for genotypes are as in Fig. 1. Two independent experiments were performed. Significant differences between control and transgenic plants are indicated at *P B 0.05. Pearson coefficient (r) is given for the linear correlation between tumor area and weight values

Fig. 3 Fig. 4

 34 Fig. 3 Analysis of potential causes of the reduction of tumor size on OMT-suppressed plants (see Fig. 1 for abbreviations). Significant differences between control and transgenic plants are indicated at *P B 0.05 for at least two independent experiments. a Callus induction and proliferation in vitro of transgenic and control explants. Callus fresh mass (in g) was determined 6 weeks after induction. Pictures of calli and mean values of fresh mass ± SE (n = 16) are shown. b Tumor area after agro-inoculation with Agrobacterium cultures that have been vir activated (by acetosyringone) or not. Mean values ± SE (n = 40) are shown. Significant differences with mean tumor area obtained with non-activated bacteria on control plants are indicated at *P B 0.05

  correlations are indicated at *P B 0.05, **P B 0.01 or ***P B 0

Fig. 6

 6 Fig. 6 Fractionation of vir-inducing activity produced by tobacco stems infected with Agrobacterium tumefaciens. Extracts from unwounded or Agrobacterium tumefaciens-infected stems of control plants (C, white bars) and CCoAOMT/COMTI-suppressed (dAS, black bars) plants were fractionated by HPLC and tested for their ability to induce vir activity. The differences between values obtained for infected and unwounded plants of each genotype were calculated and are presented for each HPLC fraction. Acetosyringone was identified in fraction number 6 on the basis of its retention time and UV absorption spectrum

Table 1

 1 Pearson's coefficients (r) from linear correlations calculated between tumor areas, vir inducing activities, OMT proteins accumulations and acetosyringone amounts in 6-h post-treatment (unwounded, wounded and mock inoculated or wounded and Agrobacterium tumefaciens infected) transgenic plants (control, COMTI, COMTII, CCoAOMT and dAS plants; n = 80 except for acetosyringone where n = 25 corresponding to the number of plants with detectable amounts of this compound among the 80 samples)

		Tumor areas	Vir inducing activities	COMTI	COMTII	CCoAOMT	Acetosyringone
	Tumor areas	1	0.86 *	0.88 *
	Vir inducing activities		1	0.78 ***			0.70 *

Acknowledgments We are grateful to Professor F. Delmotte from LBLGC University of Orle ´ans and Le ´on Otten from IBMP du CNRS of Strasbourg for providing Agrobacterium tumefaciens strains. We acknowledge Dr G. Pinc ¸on, Dr F. Martz, Dr V. Toquin and Professor R. Atanassova for their previous works on OMT plants. We thank Professor E. Laine ´and Professor Franck Brignolas from LBLGC for helpful discussions. We are grateful to A. Guichard for taking good care of tobacco plants and to Gilles Moreau France for technical assistance. This work was supported by the Ministe `re de l'Education Nationale, de l'Enseignement Supe ´rieur et de la Recherche.