A El-Shibiny

P L Connerton

I F Connerton
email: ian.connerton@nottingham.ac.uk

Campylobacter Succession in broiler chickens

Keywords: Campylobacter, C. jejuni, C. coli, competition, poultry

The competitive ability of Campylobacter coli OR12 over C. jejuni OR1 has been examined in experimental broiler chickens following the observation that C. coli replaced an established C. jejuni intestinal colonisation within commercial chicken flocks reared outdoors (El-Shibiny, A.

Introduction

Members of genus Campylobacter are frequently responsible for human cases of enteric disease. It is estimated that there were around 350,000 cases in England and Wales in the year 2000 [START_REF] Adak | Trends in indigenous foodborne disease and deaths, England and Wales: 1992 to 2000[END_REF], with Campylobacter jejuni accounting for 90% of the cases and C. coli contributing around 25,000 cases [START_REF] Gillespie | A case-case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses[END_REF][START_REF] Tam | Campylobacter colian important foodborne pathogen[END_REF]. Human infections of either C. jejuni or C. coli result in intestinal inflammation leading to diarrhoea and abdominal pain [START_REF] Skirrow | Clinical aspects of Campylobacter infection[END_REF].

It one study of raw chicken meat sold in the UK, 80% was contaminated with Campylobacter [START_REF] Jorgensen | Prevalance and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods[END_REF] with 98% of isolates speciated as C . jejuni and 2 % C. coli. Various studies from around the world have shown that the numbers of C. coli isolated from poultry may range from 6 -50% [START_REF] Corry | Poultry as a source of Campylobacter and related organisms[END_REF] with the highest incidence associated with free-range birds and the lowest from birds reared in conventional barns [START_REF] Rivoal | Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free-range broiler farms and comparison with isolates of various origins[END_REF].

In a previous study [START_REF] El-Shibiny | Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens[END_REF] we reported the succession of C. jejuni by C. coli during the rearing cycle of organic and free-range broiler chickens. There were several possibilities that could explain the dramatic change in dominant species from C. jejuni to C. coli. The simplest explanation being that the C. coli had a competitive advantage due to more efficient use of the prevailing nutrients. However, differential sensitivity to bacteriophage predation or bacteriocins or other antagonistic metabolites may also have led to the decline in numbers of one species over the other [START_REF] Connerton | A longitudinal study of Campylobacter jejuni bacteriophage and their hosts from broiler chickens[END_REF][START_REF] Chaveerach | Antagonistic activities of several bacteria on in vitro growth of 10 strains of Campylobacter jejuni/coli[END_REF][START_REF] Humphrey | Inhibition of Campylobacter jejuni in vitro by broiler chicken caecal contents[END_REF][START_REF] Schoeni | Reduction of Campylobacter jejuni colonisation of chicks by caecum-colonising bacteria producing anti-C. jejuni metabolites[END_REF]. Alternatively host-associated factors such as developments in the immune system of the chicken might affect the dominant species, which could account for the common timing of the succession noted at 31 days-of-age.

Profound differences in the ability of individual strains of C. jejuni to colonise and persist in chickens have been demonstrated [START_REF] Cawthaw | Increased colonisation potential of Campylobacter jejuni strain 81116 after passage through chickens and its implication on the rate of transmission within flocks[END_REF]Ringoir and Korolik. 4 2003). The reasons for these variations are not clear but in vivo passage has been shown to enhance the colonisation ability of C. jejuni strains [START_REF] Cawthaw | Increased colonisation potential of Campylobacter jejuni strain 81116 after passage through chickens and its implication on the rate of transmission within flocks[END_REF][START_REF] Jones | Adaptation of Campylobacter jejuni NCTC11168 to high-level colonisation of the avian gastrointestinal tract[END_REF].

C. jejuni strains able to displace resident C. jejuni strains [START_REF] Chen | Competitive exclusion of heterologous Campylobacter spp. in chicks[END_REF]Korolik et al., 1998) or prevent colonisation in young birds [START_REF] Barrow | Inhibition of colonisation of the alimentary tract in young chickens with Campylobacter jejuni by pre-colonisation with strains of C. jejuni[END_REF] have been reported.

Of particular note fluoroquinolone-resistant strains of C. jejuni appear to be more fit than those sensitive to the antibiotic when co-inoculated in chickens [START_REF] Luo | Enhanced in vivo fitness of fluoroquinolone-resistant Campylobacter jejuni in the absence of antibiotic selection pressure[END_REF], and ciprofloxin resistant strains (often C. coli or a mixture of C. coli and C. jejuni) have been reported to persist and dominate in chicken flocks after the removal of antibiotic therapy that provoked their selection [START_REF] Humphrey | Prevelence and subtypes of ciprofloxin-resistant Campylobacter spp. in commercial poultry flocks before, during, and after treatment with fluoroquinolones[END_REF].

The aims of this work were: 1. to discover if there were any obvious differences in growth between isolates C. jejuni OR1 and C. coli OR12 previously observed to undergo succession [START_REF] El-Shibiny | Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens[END_REF]; 2. to investigate if the succession could be replicated in experimental birds; 3. to determine the time periods required to establish the competitive advantage of C. coli OR12 in order to investigate any link between the age of the birds and succession; 4. to investigate if the successful competitor C. coli OR12 could be used to displace other well characterised C. jejuni isolates colonising experimental birds and therefore be used to manipulate Campylobacter populations in birds.

Materials and Methods

Bacterial strains

Campylobacter jejuni OR1 and C. coli OR12 were isolated from a UK organic chicken farm [START_REF] El-Shibiny | Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens[END_REF]. C. jejuni HPC5 and C. jejuni GIIC8 were isolated from conventional barn-reared UK broiler chicken flocks [START_REF] Carrillo | Bacteriophage therapy to reduce Campylobacter jejuni colonisation of broiler chickens[END_REF] whilst C. jejuni 81116, a human derived isolate, was obtained from D. Newell, Veterinary Laboratories Agency Weybridge UK [START_REF] Cawthaw | Increased colonisation potential of Campylobacter jejuni strain 81116 after passage through chickens and its implication on the rate of transmission within flocks[END_REF]. The isolates were routinely cultured on horse 5 blood agar (BA, blood agar base No 2 CM0271, [Oxoid, Basingstoke, UK] with 5% defibrinated horse of blood [TCS, Buckingham, UK] added) under microaerobic conditions (5% O 2 , 5% H 2 , 10% CO 2 , 80% N 2 produced by the evacuation and replacement technique) at 42° C for 24 h. Isolates of Campylobacter were stored at -80° C on Microbank storage beads (Pro Lab Diagnostics, Cheshire, UK).

Enumeration of campylobacters in vitro and in vivo

Serial dilutions of either broth culture from growth curve experiments or caecal contents from chickens were made in maximum recovery diluent (MRD; CM0733, Oxoid) and enumerated from plate counts on modified Cefoperazone Charcoal Desoxycholate Agar (mCCDA) agar supplemented with cefoperazone (Pro-Lab Diagnostics) with additional agar (L13, Oxoid) to a total of 2% added to reduce swarming. Plates were incubated under microaerobic conditions at 42° C for 48 h before typical Campylobacter colonies were counted.

Discrimination between strains

Where co-cultures of C. jejuni and C. coli were performed it was necessary to be able to be able to accurately distinguish the strains from each other. Colony hybridisation was the primary method used for this purpose. Where Hybond-N (Amersham) membranes were placed onto the surface of agar plates with 50-200 colonies for 5 minutes and orientation marks applied. The blotting membranes were then placed, colony side up, on Whatman 3 MM paper soaked in 10% w/v sodium dodecyl sulphate and incubated for 5 minutes at room temperature to lyse the cells. The membranes were then placed, colony side up, on successive Whatman 3 MM papers soaked in denaturation solution (0.5 M NaOH, 1.5 M NaCl), then neutralisation solution (0.5 M Tris-HCl [pH 7.5], 3 mM NaCl) and then wash solution (300 6 mM NaCl, 30 mM Sodium citrate [pH 7]) for 5 minutes each. The membranes were then air dried for 30 min followed by UV cross-linking. DIG-labelled probes specific for the hipO gene of C. jejuni were synthesised using the PCR primers described by [START_REF] Linton | PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples[END_REF] in conjunction with digoxigenin-11-dUTP (Roche) to replace dTTP in a standard PCR reaction.

The distinction between C. jejuni and C. coli was confirmed by washing off the initial probe and re-hybridising with a 405bp PCR fragment based on the sequence located downstream of the C. coli aspartokinase gene identified by [START_REF] Linton | PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples[END_REF], which could be demonstrated to be specific for C. coli OR12 in these experiments. Hybridisation and detection were carried out using materials from Roche according to the instructions supplied. Colourmetric detection was carried out using nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate 4-toluidine (Roche) and the colonies were counted.

Isolates were also distinguished in parallel by their inherent antibiotic tolerances.

Colonies enumerated from the original mCCDA plates were replicated onto the mCCDA plates containing the additional antibiotics (all from Sigma Aldrich, Gillingham, UK) to suppress the competing isolates as follows: for C. jejuni OR1 and C. jejuni GIIC8, Tetracycline (50 µg/ml) was added to suppress C. coli OR12; for C. jejuni HPC5, Kanamycin (30 µg /ml) was added to suppress C. coli OR12; and for C. coli OR12 counts, Penicillin (20 µg /ml) was added to suppress C. jejuni OR1, C. jejuni GIIC8, and C. jejuni HPC5. The plates were then incubated as previously described. To confirm the distinction of C. jejuni 81116 from C. coli OR12 it was necessary to perform hippurate hydrolysis on individual colonies as no suitable antibiotic was available. The hippurate hydrolysis technique was validated using the PCR amplicons of [START_REF] Linton | PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples[END_REF].

In order to validate the accuracy of the three different methods, mixtures of cultures of know cell densities were prepared, treated as normal samples and then enumerated. All 3 techniques were found to accurately distinguish between the strains (only two techniques, 7 hippurate hydrolysis and hybridisation could be used to distinguish C. coli OR12 from C. jejuni 81116 because no antibiotic selection was possible for this combination).

Growth Curves

These experiments were performed by combining C. coli OR12 with C. jejuni in broth culture. A range of inoculation ratios were used to start the cultures, from which growth curves were plotted for each species. Campylobacters were grown in Nutrient Broth No 2 (CM 67, Oxoid) in conical flasks under microaerobic conditions, on an orbital shaker at 42° C. Samples were taken at intervals to obtain viable cell counts over 48 h. Growth was determined by viable count on mCCDA agar followed by replication on to antibiotic containing media to distinguish the input species from each other. The hippurate hydrolysis test was used additionally to further validate the distinction between C. jejuni and C. coli.

A c c e p t e d M a n u s c r i p t 8

Experimental birds

Commercial Campylobacter-free male Ross broiler chickens were obtained as hatchlings (P.D. Hook, Oxfordshire, UK). All experiments were performed in accordance with UK legislation and following independent ethical review. Birds were housed individually in a controlled environment in individual pens under strict conditions of biosecurity. To ensure that the experimental birds remained free of naturally occurring infection, faeces and cloacal swabs were taken each day and tested for Campylobacter by direct plating on mCCDA agar and for Salmonella by enrichment in Rappaport-Vassiliadis soya peptone broth (CM 866, Oxoid) then plating on Xylose-Lysine desoxycholoate agar (XLD) agar (CM 469,Oxoid). In order to ensure that no contaminating campylobacters had infected the birds in the containment facility, pulse field gel electrophoresis (PFGE) of Sma1 digests of genomic DNA was carried out [START_REF] Ribot | Rapid pulsedfield gel electrophoresis protocol for subtyping of Campylobacter jejuni[END_REF] from random isolates recovered from the chickens and compared to the known profiles of the test strains.

Competition experiments in broiler chickens using C. jejuni OR1 and C. coli OR12

The C. jejuni isolate, OR1 was suspended in phosphate buffered saline (PBS, BR14a, Oxoid) and diluted to contain log107 CFU/ml. Each chicken was dosed with 1 ml of bacterial suspension in PBS or PBS alone for the controls, by oral gavage at 21 days of age. The C. coli OR12 strain was administered in the same way to the pre-colonised chickens at different points during the rearing period from 25 days to 35 days. Chickens were killed at various intervals from 26 days to 46 days of age and the numbers of each Campylobacter species determined from the contents of the caeca as described previously [START_REF] Carrillo | Bacteriophage therapy to reduce Campylobacter jejuni colonisation of broiler chickens[END_REF].

The effect of administering C. jejuni OR1 at day 27 to birds that had been pre-colonised with C. coli at 21 days then sampling after 1, 5, 9 and 12 days to examine the short medium and 9 long term effects was also determined and campylobacters discriminated and enumerated by colony hybridisation and antibiotic resistance as described above.

Analysis of variance was performed using the software package Statistica (StatSoft Ltd, Bedford, UK).

Competition of C. coli OR12 with additional C. jejuni isolates

Experiments to determine competitive interaction between C. coli OR12 and C. jejuni strains HPC5, GIIC8 and 81116 were carried out with each isolate being administered as above but at 20 days of age to mimic natural infection since C. jejuni is usually first detected at around 3 weeks of age. C. coli OR12 was then administered to these birds at 30 days of age. These birds were culled at 40 days of age around the age at which they would normally be culled in commercial situations and the numbers of each Campylobacter species determined from the contents of the caeca as described previously above.

Results

In vitro Competition between C. coli OR12 and C. jejuni OR1

The in vitro competition experiments were carried out to determine if the succession in vivo could be attributed to any simple metabolic difference, for example one isolate might have a faster growth rate than the other. Growth curves were prepared using viable counts obtained from the different input ratios over 48 h. Figure 1 shows representative growth curves from some of the different input ratios tested. The exponential phase growth rates of C. coli OR12 (0.94±0.2 h -1 ) and C. jejuni OR1 (0.96±0.4 h -1 ) in co-culture were similar, and this was independent of their relative inoculum ratios.

When the input ratios were equal no significant differences in the growth curves were observed over the entire experimental period (Fig 1A). However, a significant change in the 10 behaviour of C. jejuni OR1 with C. coli OR12 was noted at higher input ratios, where C. jejuni OR1 counts started to fall at 12 h akin to death phase without any notable stationary phase, showing declines of log 10 1.6 CFU/ml, and log 10 4.3 CFU/ml at respective C. coli OR12:C. jejuni OR1 input ratios of 10 2 and 10 6 (Fig. 1D and1E). This did not occur when the situation was reversed with ratios of 10 2 :1 and 10 6 :1 C. jejuni OR1:C. coli OR12 (Fig 1 B and1C), implying that C. jejuni OR1 is sensitive to excess numbers of C. coli OR12 or its products in stationary phase.

In vivo competition of C. jejuni OR1 and C. coli OR12 in broiler chickens

Significant variation in the ability of Campylobacter isolates to colonise chickens has been reported [START_REF] Cawthaw | Increased colonisation potential of Campylobacter jejuni strain 81116 after passage through chickens and its implication on the rate of transmission within flocks[END_REF][START_REF] Ringoir | Colonisation phenotype and colonisation potential differences in Campylobacter jejuni strains in chickens before and after passage in vivo[END_REF]. The colonisation potential and ability to persist over time of each isolate were therefore determined individually in broiler chickens before commencing competition experiments. The first five columns of Fig. 2a Multivariate analysis of these data reveals the changes observed to be highly significant with respect to the change in species caecal count (p=5.8 x 10 -11 ) and the time post challenge (p=0.002), with clear interaction between these terms (p=5.5 x 10 -13 ). Partitioning of the data collected at 4 days post challenge demonstrated the importance of the age of the birds at this time with a significant fall in the C. jejuni caecal count for birds post 31 days of age (p=0.0001). To conclude, in order for C. coli OR12 to become dominant over C. jejuni OR1, a period of time of more than one day was necessary after introduction of the new species and 12 the birds needed to be greater than 31 days of age for this to occur. If these criteria were met C. coli OR12 became the dominant isolate otherwise co-colonisation occurred.

Following this dramatic decline in C. jejuni counts after the introduction of C. coli we elected to perform the reverse experiment to examine if the primary colonisation of chickens with C. coli at day 21 could prevent or hinder subsequent challenge with C. jejuni at day 27.

Birds were sampled after 1, 5, 9 and 12 days to examine the effects of the challenge on 27 day old birds. Figure 2B shows that when birds were pre-colonised with C. coli OR12 this did not prevent initial co-colonisation with C. jejuni OR1, although this was always at a reduced level compared to controls (first five columns of Fig2A). It was also notable that the caecal counts of C. coli OR12 were reduced as compared to unchallenged controls (Fig. 2B). However, at 12 days post administration, the C. jejuni OR1 caecal count was observed to show a significant decline to log 10 2.9 (± 0.9) CFU/g (p=0.02) accompanied by a rise in the C. coli OR12 mean caecal count from log 10 5.4 after 9 days to log 10 7.0. Thus following a period of co-colonisation C. coli OR12 became the dominant species, as had been observed in the previous experiments.

The displacement of alternative C. jejuni isolates in competition with C. coli OR12

It was our aim in this series of experiments to examine if C. coli OR12 could compete with independent isolates of C. jejuni capable of efficiently colonising broiler chickens. The timing of infection was selected to mimic intensively rearing of birds rather than free-range rearing that we had studied in the previous experiments. Chickens were pre-colonised at 20-days-ofage with log 10 7 C. jejuni from one of the following C. jejuni isolates: GIIC8, 81116 or HPC5.

They were then challenged with log 10 7 C. coli OR12 at day 30. The birds were culled at day 40 and the Campylobacter species enumerated from their intestinal contents. These results recorded in Fig. 3 show that control chickens maintained C. jejuni colonisation until day 40 in

Discussion

Chickens exposed to the environment are likely to encounter more than one strain or species of Campylobacter capable of establishing a persistent commensal relationship during the lifetime of the bird. Previously we and others have shown that the dominant strain may change several times throughout the rearing cycle of organic and free-range birds exposed to environmental sources [START_REF] El-Shibiny | Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens[END_REF][START_REF] Rivoal | Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free-range broiler farms and comparison with isolates of various origins[END_REF]. Barn reared broiler chicken flocks are likely to be exposed to a somewhat more limited set of Campylobacter types but these have also been reported to exhibit the dominance of one Campylobacter type only to be replaced with another [START_REF] Bull | Sources of Campylobacter spp. colonising housed broiler flocks during rearing[END_REF].We currently have little idea of the factors that enable one species or strain to become the dominant type in the chicken gut but it appears to be more complicated than being just a matter of being the first strain to establish colonisation. It is also not unusual to isolate more than one species or subtype from the same bird [START_REF] Schouls | Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination[END_REF][START_REF] Höök | Genotype dynamics of Campylobacter jejuni in a broiler flock[END_REF]. In these experiments we have shown that the C. coli OR12 was able to co-colonise chickens with C. jejuni isolate OR1 until the birds were 35 days of age, at which stage C. coli OR12 began to dominate the intestinal population. It does not appear to be possible to identify a potential dominant strain on the basis of it's growth rate in vitro, since in broth co-cultures C. coli and C. jejuni grew in a compatible manner with similar growth rates. However, we did note a significant difference in behaviour of C. jejuni OR1 compared to C. coli OR12, which was observed to go into decline after peaking at the A c c e p t e d M a n u s c r i p t 14 end of the exponential growth phase when cultured as a minority population with C. coli OR12.

A good deal of effort has gone into the identification of Campylobacter genes that are necessary to enable the colonisation of poultry [START_REF] Hendrixson | Identification of Campylobacter jejuni genes involved in commensal colonisation of the chick gastrointestinal tract[END_REF]. These studies have provided some intriguing insights into the structures and metabolic processes that support growth in the avian intestine but they have not shed light on the key competition factors that enable Campylobacter species and subtypes to dominate populations, or indeed the strategies that are adopted so that minority sub-populations be retained in the presence of a dominant competitor in order that they may proliferate again later. The enigmatic nature of the processes in operation may be exemplified by the recent observations of the high frequency loss of C. jejuni signature-tagged transposon mutants at random from chickens of signature-tagged pool members that later proved to be colonisation proficient [START_REF] Grant | Signature-tagged transposon mutagenesis studies demonstrate the dynamic nature of caecal colonisation of 2-week-old chickens by Campylobacter jejuni[END_REF].

The overall conclusions from the series of in vivo experiments in broiler chickens were that, both C. jejuni OR1 and C. coli OR12 could colonise and compete with each other equally until the birds were 35 days old, regardless of the order or day of exposure. Beyond this time point the numbers of C. jejuni OR1 declined leaving C. coli OR12 as the dominant isolate. Without competition C. jejuni OR1 did not decline at this time point so the observed decline must at least in part be due to competition with C. coli OR12 but is also likely influenced by developmental changes occurring in the birds at that time.

Having established that C. coli OR12 was a particularly good competitor against C. jejuni OR1 it was important to determine if it could compete against other C. jejuni isolates in a time scale more typical of intensively reared birds. Our aim was to see if it might be possible to manipulate the dominant strain present in commercially reared birds at the time they are sent for slaughter by using a particularly competitive isolate. C. coli OR12 was found to be able to replace C. jejuni broiler chicken isolates HPC5 and GIIC8 as well as a C. jejuni isolate of human origin (81116), all of which were efficient colonisers of broiler chickens beyond 35 days-of-age without challenge.

Changes in host immunity may contribute to the competitive succession of C. coli OR12 either through responses directed at the C. jejuni isolate or through host-mediated changes in the gut flora that result in the antagonism of C. jejuni. Studies demonstrate that there are profound changes in the intestinal flora of chickens during development [START_REF] Lu | Diversity and succession of the intestinal bacterial community of the maturing broiler chicken[END_REF], and how these bacterial communities vary are likely the result of interactions between the diet and immunological competence of the birds. These studies were carried out on male Ross broiler chickens but it would be of interest to examine if the succession of these C. jejuni by C. coli isolates could also occur with in-bred chicken lines, and if so whether the succession occurred at a similar stage of development. From these studies it might be possible to assess if a genotypic basis can be ascribed to the intolerance of the birds to the selected C. jejuni isolates when co-colonised with C. coli OR12.

Our experimental findings on chickens reared under conditions of strict biosecurity and fed ad libitum indicate that it is possible that the organic chickens in our original study [START_REF] El-Shibiny | Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens[END_REF] may have demonstrated the succession of C. coli OR12 independent of any external factor, and that this event could have occurred whether both species were acquired from the same source at the same time or through later exposure to C. coli OR12 from another source. We were prompted to perform these experiments since our original study [START_REF] El-Shibiny | Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens[END_REF] demonstrated that bacteriophage appeared at the same time as the change in dominant species leading to the possibility that they could have been involved in, or indeed even provoked the succession process. This appears not to be the case; instead we have highlighted a more general process whereby chickens co-colonised by C. coli OR12 and one of several C. jejuni isolates tested showed a marked change in their respective expense of the C. jejuni isolate. In this context it is notable that adult breeding hens have been reported to be readily colonised by C. coli [START_REF] Peterson | Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks[END_REF].

The timing of the change in species dominance is of particular note, as conventional barn-reared broilers are often slaughtered from 35 days old. Organic and free-range birds are reared over a longer period and are at the same time exposed to a wider variety of environmental campylobacters, and other microbes producing metabolites that may be differentially prejudicial to the campylobacters colonising the intestinal tracts of the chickens.

The ability to understand which factors allow one species or strain to dominate over others may lead to strategies to reduce colonisation of chickens by campylobacters. We have studied a C. coli isolate based on the observation of it's dominance in our previous work and this allowed the competition between pairs of strains to be examined as they were easily discriminated on the basis of DNA hybridisation, antibiotic sensitivity and ability to hydrolyse hippurate. It is possible that there are also successful strains of C. jejuni that are destined to dominate. Our experiments have demonstrated that replacement of one isolate by another is not always a random event but can be readily repeated in experimental birds using specific strains. It may be feasible to replace antibiotic resistant C. jejuni colonising strains with sensitive ones (either C. jejuni or C. coli) thus reducing their presence on poultry meat and the likelihood of them infecting humans. In the future, if specific human pathogenic traits can be identified, strains that express these traits might be replaced with more benign strains.

Figure Legends

  Fig. 2A show that irrespective of the age of the bird to which C. coli OR12 was introduced, the strain was able to co-colonise with the existing C. jejuni OR1, to establish C. coli OR12 populations comparable with those recorded for controls birds colonised by C. coli OR12 alone (the first five columns of Fig 2B). However, after 11 days there was a striking reduction in the C. jejuni counts of >4 log 10 CFU per gram of caecal contents, to leave C. coli OR12 as the dominant species. At 4 days post challenge the ability of C. coli OR12 was dependant on the age of the birds but not on the time between administration and sampling. In the youngest birds (those administered with C. coli OR12 at 25 or 27 days of age) the counts of C. jejuni OR1 remained at normal levels compared with the controls. However, there was a greater than 2 log10 CFU /g reduction in C. jejuni OR1 in those birds administered with C. coli at 31 and 35 days of age and sampled 4 days later at 35 and 39 days of age respectively.

A

  c c e p t e d M a n u s c r i p t 13 the absence of C. coli but following inoculation with C. coli OR12 at day 30 the birds showed a dramatic fall in their caecal populations of C. jejuni compared to the controls. In contrast the C. coli OR12 caecal counts were not significantly different to the chickens colonised with C. coli OR12 alone.

Fig. 1

 1 Fig. 1 Growth in vitro of C. jejuni OR1 (dashed lines) in competition with C. coli OR12 (solid lines) with the following starting ratios: A) 1 C. coli:1 C. jejuni; B) 1 C. coli:10 2 C. jejuni; C) 1 C. coli:10 6 C. jejuni; D)10 2 C. coli: 1 C. jejuni; E) 10 6 C. coli: 1 C. jejuni.

Fig. 2

 2 Fig. 2 Competitive colonisation of broiler chickens with campylobacters: A) Effect of introducing C. coli OR12 (black columns) at: 25, 27, 31 and 35 days old and sampled after 1, 4 and 11 days, to chickens that had been pre-colonised with C. jejuni OR1 (unshaded columns); B) Effect of introducing C. jejuni OR1 (unshaded columns) at 27 days old and sampled at 1, 5, 9 and 12 days to chickens that had been pre-colonised with C. coli OR12 (black columns) at 21 days of age. Each sample point point postchallenge represents the means and standard deviations of the caecal counts for each Campylobacter species from n=12 independent birds. For unchallenged birds with single Campylobacter species colonisation n=9.

Fig. 3 .

 3 Fig. 3. The displacement of alternative C. jejuni isolates GIIC8, 81116 or HPC5 used to pre-colonise chickens at 20 days, then challenged with C. coli OR12 at 30 days and sampled at 40 days of age. The data are recorded as the means and standard deviations of the caecal counts for each Campylobacter species from n=9 independent birds.

  A c c e p t e d M a n u s c r i p t

Age at Sample point: 24 35 39 42 46 26 28 32 36 29 31 35 39 36 38 42 46 (days) Campylobacter count (log 10 CFU/g) Campylobacter count (log 10 CFU/g)

	A)
		9
		8
		7
		6
		5
		4
		3
		2	0	1		4
	Days post challenge:		Unchallenged	1		4	11
	Age at challenge:			25 27 31 35		25 27 31 35	25 27 31 35
	(days)
	Days post challenge: B)	Unchallenged 2 3 4 A c c e p t e d 1 2 3 4 5 6 7 8 9 0 1 5 6 7 8 9 M a n u s c r i p t 5 9 10 11 12 13 14 15	16	18
	Age at Sample point:		24 35 39 42 46	28	32	36	39
	(days)

Single colonisation Paired Colonisation C. coli OR12 C. jejuni GIIC8 C. jejuni HPC5 C. jejuni 81116 C. coli OR12 C. coli OR12 C. coli OR12 C. jejuni GIIC8 C. jejuni 81116 C. jejuni HPC5 Campylobacter count (log 10 CFU/g)

	coli	GIIC8	81116	HPC5 A ve	HPC5 coli	jej	GIIC8 coli	jej	81116 coli	jej

Acknowledgements

AE acknowledges the support of the Egyptian Government.


Nicolas Amelot

Audrey Carrouche

Saïda Danoun

Stéphane Bourque

Jacques Haiech

Alain Pugin

Raoul Ranjeva

Jacqueline Grima Pettenati

Christian Mazars

Christian Brière
email: briere@scsv.ups-tlse.fr

Audrey Carrouché

Jacqueline Grima-Pettenati

Cryptogein, a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium-dependent manner

Keywords: calcium signalling, transcription regulation, phenylpropanoid, modelling, Nicotiana tabacum (tobacco BY-2), cryptogein PAL, phenylalanine ammonia-lyase, C4H, cinnamate 4-hydroxylase, 4CL,

Cryptogein, a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium-dependent manner.

INTRODUCTION

Plant cells use calcium-based signalling pathways to transduce a variety of external and/or internal stimuli into adaptive responses (for review, [START_REF] Hetherington | The generation of Ca 2+ signals in plants[END_REF][START_REF] Kudla | Calcium Signals: The Lead Currency of Plant Information Processing[END_REF][START_REF] Lecourieux | Calcium in plant defence-signalling pathways[END_REF][START_REF] Mcainsh | Shaping the calcium signature[END_REF][START_REF] Sudha | Involvement and interaction of various signaling compounds on the plant metabolic events during defense response, resistance to stress factors, formation of secondary metabolites and their molecular aspects[END_REF][START_REF] White | Calcium in plants[END_REF]. A paradigm establishing the central role of calcium in a complex regulatory network with multiple interacting components has been inferred, at the single cell level, from the multifaceted approach used to study guard-cell physiology (Allen et al. 2001a, Allen & Schroeder 2001b[START_REF] Israelsson | Guard cell ABA and CO2 signaling network updates and Ca 2+ sensor priming hypothesis[END_REF], Schroeder et al. 2001). As a general feature, in response to variations of abiotic (light, temperature, drought, etc.) and/or biotic (symbionts, pathogens, etc.) environment including stressful conditions that plants have to face, free calcium concentration varies in cells [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF][START_REF] Knight | Control of free calcium in plant cell nuclei[END_REF][START_REF] Ward | Roles of ion channels in initiation of signal transduction in higher plants[END_REF][START_REF] Xiong | Calcium signaling in plant cell organelles delimited by a double membrane[END_REF]). These changes control basic cellular functions including ion and molecule transport and most probably gene expression [START_REF] Kaplan | Rapid transcriptome changes induced by cytosolic Ca 2+ transients reveal ABRErelated sequences as Ca 2+ -responsive cis rlements in Arabidopsis[END_REF][START_REF] Kim | Functional analysis of a calcium-binding transcription factor involved in plant salt stress signaling[END_REF][START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF][START_REF] Moscatiello | Transcriptional analysis of calcium-dependent and calcium-independent signalling pathways induced by oligogalacturonides[END_REF]. Indeed, the identification of transcriptional regulators modulated by calcium and calmodulin [START_REF] Kim | Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants[END_REF] supports the importance of calcium in the transcriptional process during plant responses to endogenous and exogenous stimuli. Also interesting, is the evidence that the calcium signature itself and some of its components (e.g. amplitude, duration) are correlated to gene expression. For instance, during the establishment of symbiosis in root hairs [START_REF] Miwa | Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell[END_REF] showed a correlation between the number of Ca 2+ spikes and the transcript level of the Early Nodulation 11 (ENOD11) gene.

They suggested that a minimum of 36 spikes is required for ENOD11 induction. Similarly, the shape of the calcium signal in response to salt stress was shown to be very important for the control of salt-stress genes. Using the Ca 2+ -ATPase loss of function mutant in the moss Physcomitrella patens, a sustained elevation of free calcium was observed instead of a 09/02/10 transient elevation. This sustained level of calcium down-regulates salt-stress-induced genes rendering the mutant plants more susceptible to salt stress [START_REF] Qudeimat | A PIIB-type Ca 2+ -ATPase is essential for stress adaptation in Physcomitrella patens[END_REF]. These examples illustrate how the calcium-dependent processes underlying the calcium signature can control gene expression. It may thus be hypothesized that calcium, as a signal, interferes with the various steps involved in the conversion of a stimulus into a biological output.

Surprisingly little is known about the relationships linking calcium to transcriptional regulation, protein synthesis and enzyme activities, and in fine the remodelling of the biochemical processes downstream [START_REF] Kudla | Calcium Signals: The Lead Currency of Plant Information Processing[END_REF].

For a better understanding of the connections between these different events and to identify critical regulatory steps [START_REF] Weckwerth | Metabolomics in systems biology[END_REF] or signalling hubs, we took advantage of the biological model consisting of tobacco cell suspension cultures challenged with cryptogein.

Cryptogein, a polypeptide elicitor secreted by the oomycete Phytophthora cryptogea, induces free calcium changes in the cytosol ( [Ca 2+ ] cyt ) in tobacco cells and enhances the transcription of the gene encoding phenylalanine ammonia lyase (PAL), the enzyme catalysing the first committed step of the phenylpropanoid pathway [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF]. Although this biological model has been extensively studied at different levels of the signalling pathway [START_REF] Garcia-Brügger | Early signaling events induced by elicitors of plant defenses[END_REF], it is still not known if other genes of the multi-branched pathway are differentially regulated in response to cryptogein, and there are no data showing changes in enzyme activities or increases in the amounts/concentrations of phenolic compounds.

In the present paper, we made use of this simplified biological system to evaluate qualitative and quantitative changes in successive steps of elicitor-induced signalling pathways, including calcium-transient generation, transcriptional regulation and metabolism reorientation.

Our work aimed at bridging the different steps involved in the signalling pathway that converts the initial perception of cryptogein (input) into the final response (output) that 09/02/10 consists here of the changes induced in the metabolism of phenolic compounds (Fig. 1). We reasoned that quantitative parameters and time-dependence are essential to get a deeper insight into the regulatory processes and may shed light on overlooked properties. In this way, we have addressed the following questions:

-How do free calcium concentrations vary with time when cells are challenged with increasing concentrations of cryptogein?

-Are all genes coding for the enzymes of the phenylpropanoid pathway induced by cryptogein in a calcium-dependent manner?

-How does cryptogein remodel phenylpropanoid metabolism?

On these grounds we propose an integrated model that could explain key features of the input/output network linking cryptogein signal perception to gene expression through calcium signalling.

MATERIAL AND METHODS

Products

Cryptogein was purified according to [START_REF] Bonnet | Acquired resistance triggered by elicitins in tobacco and other plants[END_REF] and dissolved in water as a 100 µM stock solution. [ 3 H]-S-adenosyl-L-methionine (specific activity 0.37 TBq/mmol) was from MP Biochemicals (Illkirch, France) and [ 14 C(U)]-L-phenylalanine (specific activity 6.8 GBq/mmol) was from Perkin-Elmer (France). A 5 mM stock solution of native coelentarazine (Interchim, Montluçon, France) in methanol was stored at -20°C in 10 µL aliquots. All other compounds were from Sigma-Aldrich.

Radiolabelled trans-cinnamic acid was obtained by the enzymatic conversion of 0.185 MBq of [ 14 C(U)]-L-phenylalanine by 0.007 units of phenylalanine ammonia lyase (Sigma-Aldrich) in Tris-HCl buffer pH 8.5 at 30°C for 2 hours. After stopping the reaction with 6µL 6M HCl, 09/02/10 cinnamic acid was extracted in 2x1 mL diethyl ether, evaporated to dryness and taken up in 50µL MeOH.

Cell culture and luminescence measurements

Transgenic tobacco cells (Nicotiana tabacum L. cv Bright Yellow BY-2 lines) expressing cytosolic apo-aequorin [START_REF] Takahashi | Hypoosmotic shock induces increases in cytosolic Ca 2+ in tobacco suspension-culture cells[END_REF] were grown and processed according to Pauly et al. (2001). Cells harvested at late exponential growth phase were washed twice and resuspended at a density of 20% (Packed Cell Volume, PCV) in the suspension buffer (175 mM mannitol, 0.5 mM K 2 SO 4 , 0.5 mM CaCl 2 , 2 mM MES KOH, pH 5.8), supplemented with 2 µM coelenterazine to reconstitute active aequorin for luminescence measurements. Subsequently, cells were incubated at room temperature overnight in the dark on a rotary shaker (130 rpm). Measurements of luminescence and calcium calibration were carried out as previously described [START_REF] Knight | The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells[END_REF].

Elicitor treatments

Cells in suspension medium were transferred into a glass vial and challenged with the indicated concentrations of effectors. After given times of incubation, cell aliquots were collected and filtered under vacuum on a 37 µm mesh nylon membrane and immediately frozen in liquid nitrogen.

Quantitative real-time RT-PCR

Tobacco BY-2 cell samples were ground in liquid nitrogen and total RNAs isolated using the Nucleospin II RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. First-strand cDNA was synthesized from 5µg total RNA in a 20µl reaction volume containing 50ng random primers, 10 mM dNTPs, 1µl RNasOUT (40 units) and 200 units of SuperScript III reverse transcriptase, according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). After 50 minutes at 50°C followed by 5 minutes at 85°C, cDNAs were purified using the PCR Clean-Up system (Promega, Madison, USA). Two µL cDNAs were used as templates in a quantitative real-time PCR assay (10 µL) performed on the sequence detection system ABI PRISM 7900HT (Applied Biosystems, Foster City, USA) using the SYBR Green PCR Master Mix (Applied Biosystems). After an initial denaturation of 10 minutes at 95°C, 40 cycles of 15 sec at 95°C followed by 1 minute at 60°C were performed. Amplification specificity was checked by melting-curve analysis, and PCR efficiency was determined using standard curves constructed with serial dilutions of PCR products as templates. 18S rRNA was used as an internal control. Quantification of expression ratios was performed according to the mathematical model developed by [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

Primers and amplicon sizes are given in Supporting Information (table S1). In the case of multigene families, primers have been designed to hybridize either to all known genes of the class postulated to have a role in lignin biosynthesis and/or specifically to one gene member (table S1).

Measurement of enzymatic activities of PAL and CCoAOMT

Cell samples were ground in liquid nitrogen and weighed (approx. 2g/sample) before being extracted for 30 minutes in 4 mL of protein extraction buffer (2 mM MgCl 2 , 10 mM DDT, antiprotease (P9599 Sigma), 10% glycerol, 0.1M Tris-HCl pH 7.5) at 4°C under gentle shaking. After a 30 minute centrifugation (10 000 x g, 4°C) the supernatant was collected and desalted on a PD10 column equilibrated with the same buffer at pH 7.5 for CCoAOMT activity or pH 8.8 for PAL activity measurements. PAL activity was assayed by spectrophotometry. The reaction mixture (1 mL) contained 50µg protein extract and 1 mM Lphenylalanine (Sigma) in 0.1M Tris-HCl, pH 8.8 buffer. The formation of cinnamic acid was monitored for 10 minutes by measuring absorption at 290 nM and PAL activity was determined from the slope of the absorption curve using the extinction coefficient of cinnamic acid at 290 nm = 10000 M -1 cm -1 09/02/10

CCoAOMT activity was determined by radiometric assay. The reaction mixture contained 10µL of protein extract, 3.7kBq [ 3 H]-S-adenosyl-L-methionine, 40 µM S-adenosyl-Lmethionine, 40 µM caffeoyl-CoA in 0.1M Tris-HCl, pH 7.5 (total volume 50µL). After 30 minutes of incubation at 30°C the reaction was stopped with 10µl 3M NaOH and incubated for a further 10 minutes at 37°C to allow feruloyl-CoA hydrolysis before adding 40µL 1M HCl to stop the reaction. Ferulic acid was extracted 10 minutes in 200µL of hexane/ethyl acetate (1:1 v/v) and 150µL of the organic phase was collected and evaporated overnight before addition of 8 mL of scintillation cocktail for radioactivity counting.

Radioactivity measurements

[ 14 C]-cinnamic acid was added to the cells (0.007MBq. g -1 fresh cells) immediately before treatment with cryptogein. After 4 hours of incubation at 20°C in the dark and under shaking, cell aliquots were filtered under vacuum and frozen immediately. A 1 mL aliquot of filtrate was collected and transferred into a scintillation vial containing 8 mL of Ready Safe cocktail (Beckman Instr., Fullerton CA USA) to measure residual radioactivity in the medium. Frozen cell samples were weighed before grinding in 80% EtOH with glass beads (Fastprep system, MP Biomedicals, Illkirch, France). Ground cells were then extracted for 30 minutes at 4°C in 5 ml 80% EtOH under gentle shaking, and centrifuged 10 minutes at 3000 x g. A 1 mL aliquot of supernatant was collected for measuring the radioactivity of ethanol-soluble compounds (total radioactivity). Another 1 mL aliquot of the supernatant was added to a PVPP column, which selectively complexes phenolic compounds (radioactive soluble phenolics). Radioactive non-phenolic products were recovered in the pass through (radioactive soluble non-phenolics) and the distribution of the radiolabel was calculated as: radioactive soluble phenolics = total radioactivityradioactive non-phenolics. The remaining supernatant was discarded and the cell pellet resuspended in water, and a 200µL aliquot collected for counting the radioactivity incorporated into the non-soluble fraction.

Extraction and analysis of soluble and wall-bound phenolics

Frozen cell samples were ground in liquid nitrogen, and the powder weighed before being extracted in 5 mL 80% EtOH for 30 minutes at 4°C under gentle shaking in the dark. After 10 minute centrifugation at 3000 x g the supernatant was collected for analysis of soluble phenolics. Phenolic material contained in the ethanol-insoluble fraction was extracted according to [START_REF] Bolwell | Metabolic changes in elicitor-treated bean cells. Enzymic responses associated with rapid changes in cell wall components[END_REF]. The pellets were resuspended in 5 mL of 4.25M NaOH and hydrolyzed for 6 days at 4°C followed by 1 day at 37°C in the dark under gentle shaking.

After 15 minute centrifugation at 4500 x g the supernatant was collected, acidified to pH 1 with 12M HCl, then extracted twice in ethyl acetate. The organic phase was collected, and evaporated to dryness under N 2 . The residue was re-dissolved in 100 µL of water/methanol (1/1 v/v) for analysis by UPLC-QTOF using a Waters ACQUITY UPLC System (Waters, Milford MA, USA). Separation was achieved using an ACQUITY UPLC BEH C18 column (100 mm, 2.1 mm i.d., 1.7 µm particle size; Waters) maintained at 40°C, with a mobile phase flow rate of 0.30 mL.min -1 in isocratic mode. The mobile phase contained 32 % methanol and 0.1 % formic acid in water. The injection volume was 2 µL with partial loop. A time-of-flight mass spectrometer with an electrospray ionization (ESI-MS) interface was used for mass spectrometry analysis (Micromass, Manchester, UK). Data acquisition was performed using negative ion mode over a m/z range 50-1000. The general conditions were: source temperature 130 °C, capillary voltage -2.8 kV and cone voltage 40 V, extraction cone -4 V, ion guide -1 V, the collision energy 5 eV, desolvatation temperature 350 °C, desolvatation flow 750 L.h -1 , the nebulizer 30 L.h -1 and data were acquired using a scan time of 0.5 s.

Leucine-enkephalin (M-H) -(m/z 554.2615) was used for the lock mass of the lock spray.

Statistical analysis

Statistical tests and multivariate analysis (Principal Component Analysis) were carried out using the R statistical software (R Development Core Team 2009).

RESULTS

Cryptogein induces a biphasic calcium signature in tobacco BY-2 cells

Cryptogein induces biphasic calcium variations in the cytosol of BY2 cells (Fig. 2a). This response is characterized by an increase of free cytosolic calcium [Ca 2+ ] cyt starting as early as 2 minutes after elicitation, reaching a maximum after about 5 min, followed by a sustained elevation (plateau) of cytosolic free calcium lasting for more than an hour. This cytosolic calcium variation was inhibited in cells co-incubated with 1 mM La 3+ , illustrating the absolute requirement of an external calcium influx. The maximum calcium elevation (peak value) and the plateau value increased in a dose-dependent manner but with different EC 50 values (Fig. 2b). Thus, the maximum plateau value is reached for very low cryptogein concentrations (less than 100 nM), while the peak value continues to increase for cryptogein concentrations above 1 µM. Fitting a simple one-site binding model to experimental data enabled us to estimate the EC 50 values at 128 nM cryptogein for the calcium peak (Bmax = 0.9 µM) and to 17 nM cryptogein for the calcium plateau (Bmax = 0.1 µM), a value close to the measured Kd of cryptogein binding to cell membranes [START_REF] Bourque | Characterization of the cryptogein binding sites on plant plasma membranes[END_REF]. This suggests either the existence of two cryptogein receptors, with low and high affinities, and involved in the generation of the calcium-peak or the sustained plateau respectively, or the existence of only one type of cryptogein receptor, but interacting with two types of protein complexes with different affinities.

Cryptogein strongly increased the number of transcripts of genes of the common phenylpropanoid biosynthetic pathway

We measured the transcript levels of genes encoding enzymes involved in the synthesis of phenylpropanoids and lignin (PAL, C4H, 4CL, HCT, C3H, COMT, CCoAOMT, F5H, CCR and CAD), as well as of three genes known to be involved in defence responses: HSR203J, a cell death response marker [START_REF] Pontier | hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactions[END_REF], and two PR genes, PR1 and PR3 [START_REF] Ward | Coordinate gene activity in response to agents that induce systemic acquired resistance[END_REF]. Transcripts levels in tobacco BY-2 cells were measured using RT-qPCR after treatments of cells with various concentrations of cryptogein (from 10 nM to 1 µM) and different durations (from 30 minutes to 8 hours). The ratios of transcripts levels in treated to that in control cells are presented in Fig. 3.

After a cryptogein treatment of 3 hr, the transcript levels of the majority of the genes tested were strongly enhanced, but to different extent, ranging from 4-fold for C4H to about 200fold for CCoAOMT and F5H. In the core pathway, PAL, C4H and 4CL transcript accumulation was increased on average by 26-, 4-and 15-fold respectively. In contrast transcript accumulation of CAD, the last gene of the monolignol biosynthesis pathway, was decreased by about 2-fold. Interestingly, cryptogein had no effect on transcription of the two defence marker genes (PR1 and PR3) while the expression of the cell death response marker HSR203J was strongly up-regulated (55-fold on average).

Transcript accumulation of tested genes was strongly inhibited when cells were challenged with cryptogein in the presence of 1 mM La 3+ , a calcium channel blocker (Fig. 3a), or in the presence of the calcium chelator EGTA (Supplemental information S2), showing that calcium influx was required for the transcriptional response. 09/02/10

De novo synthesis of proteins was also required to allow the transcriptional response to cryptogein, as shown by its complete suppression by a 30 minute pre-treatment of the cells with cycloheximide prior to the addition of cryptogein (Supporting Information Fig. S3).

Because many genes of the phenylpropanoid pathway belong to small gene families, we examined transcript accumulation in response to cryptogein for each known isoform of three strongly induced genes. For PAL and 4CL, only the expression of one gene member was upregulated, PAL A and 4CL2, respectively. Transcript levels of CCoAOMT genes were highly and equally up-regulated upon cryptogein treatment except CCoAOMT5 which was only weakly activated in comparison to the other family members (Supporting Information Fig. S4). These data show that the intensity of the transcriptional response within a gene family may be specific either for a particular gene or for a set of genes.

The magnitude of the transcriptional response to cryptogein increased with time and depended upon the intensity of the stimulus. As exemplified with PAL, CCoAOMT and HCT, the transcript levels of the most activated genes increased after the cryptogein treatment as early as 30 minute and continued increasing for several hours (Fig. 3b). Different induction kinetics patterns were observed depending on the gene considered. Transcripts levels of some genes like CCoAOMT reached a maximum after 4 hours of treatment then decreased, while transcripts of other genes like COMT and HSR203J continued to accumulate for 8 hours.

Furthermore, the intensity of the transcriptional response was strongly dependent upon the intensity of the stimulus (Fig. 3c). Similar dose-response curves were observed for the different genes of the phenylpropanoid pathway, showing a strong correlation between these genes in terms of dose dependence. Dose-response curves were non-linear and biphasic: the effect of cryptogein began to be significant at 20 nM, reached a maximum between 50 nM and 100 nM (depending on the gene considered), and then decreased fairly sharply while increasing the cryptogein concentration (Fig. 3c). As observed for the kinetics patterns, differences in the dose-response curves were observed between genes. Thus, in order to analyse the correlations between gene transcript ratios in dose-response experiments, we performed a Principal Component Analysis (PCA) of the correlations between kinetic data obtained for all studied genes. Briefly, this multivariate analysis allows representing graphically the correlations between variables and/or the proximities between observations on planes with a minimal loss of information. "Calcium-peak" and "Calciumplateau" values measured at different concentrations of cryptogein were included in this analysis in order to analyze the correlations between transcript ratios and these two characteristics of the calcium signature. This analysis led to the definition of two groups of up-regulated genes in addition to the group formed by CAD, PR1 and PR3 that are not up-or even down-regulated (Fig. 4). The first group (4CL, C4H, C3H, COMT, F5H, CCR) is correlated with the "Ca-peak" variable and is colocalized with observations made at 100nM, 200nM and 1µM cryptogein., It corresponds to genes whose transcript accumulation reached a maximum for cryptogein concentrations above 100 nM. The second group (PAL, HCT, CCoAOMT, and HSR203J), correlated to the "Ca-plateau" variable, corresponds to genes whose activation was maximal at 60 nM cryptogein and then decreased for higher concentrations.

Cryptogein-induced enhancement of transcription correlates with increased activities of enzymes of the phenylpropanoid pathway

In order to evaluate whether transcriptional activation was correlated with the activation of the corresponding enzymes, we assayed the enzymatic activities of PAL and CCoAOMT whose corresponding genes were among the most activated. These activities were strongly increased (2.5x for PAL, 6x for CCoAOMT) in BY2 cells challenged with 0.1µM cryptogein for 4 hours as compared to control cells (Fig. 5). These increases were totally inhibited when the cells were treated by 1mM La 3+ (Fig. 5 inset). A kinetic study showed that the cryptogeininduced increase of these enzyme activities began as early as 30 minute after elicitation for CCoAOMT (2 hours for PAL) and increased continuously during the whole experiment (8 hours). This continuous increase is consistent with the increases in transcript levels of the corresponding genes (Fig. 5), indicating that cryptogein-induced enhancement of transcript accumulation resulted in an increase of enzymatic activities.

Cryptogein induces accumulation of cell-wall associated phenolic compounds

From the results presented above, we reasoned that the distribution of phenolics (metabolic remodelling) might be significantly altered in cells challenged with cryptogein. To address this question, cells were fed with [ 14 C(U)]-cinnamic acid and incubated with cryptogein. After 4 hours of incubation, the cell suspension was filtered and radioactivity was measured in different fractions: external medium, phenolic soluble fraction, non-phenolic soluble fraction, and insoluble fraction (containing insoluble phenolics). Compared to control cells, cryptogein-treated cells showed a slight decrease of residual radioactivity in the external medium and in the soluble fraction containing non-phenolic compounds (including radiolabelled free cinnamic acid), but an increase in the insoluble fraction (Fig. 6). The variations of radioactivity in the different fractions were inhibited by 1mM La 3+ suggesting that, in line with what we showed for gene expression and enzymatic activities, the putative remodelling of phenolic metabolism is a calcium-dependent process. The decrease of radioactivity observed in the soluble non-phenolic fraction and the concomitant increase measured in the insoluble fraction in response to cryptogein strongly suggest an accumulation of phenolics in the cell walls.

In order to identify these insoluble phenolic compounds, cell wall pellets were hydrolyzed with NaOH in order to release associated esterified phenolics that were further analyzed by 09/02/10 MS/MS. Intermediate compounds of the "core" phenylpropanoid pathway were found to be significantly increased in cryptogein-treated samples as compared to controls i.e.: p-coumaric acid, ferulic acid, and 5-hydroxy-ferulic acid which was found only as traces in control samples but at a high level in cryptogein-treated cells. In addition, a small increase in 4hydroxybenzaldehyde was also observed in treated cells (table 1).

Calcium controls the cryptogein-induced response

The data reported above are consistent with the existence of non-linear relationships between different steps of cryptogein signalling in tobacco cells and highlight the crucial role of the steps associated with the generation of the calcium signature. They further suggest the possible co-existence of regulatory modules differing in their sensitivity to calcium and/or effector concentration, and able to modulate the response at the transcriptional level. These results raise questions on the respective roles of the two phases of the cryptogein-induced calcium signature and on the potential involvement of calcium sensor proteins.

To address these questions, we disturbed the cryptogein-induced response either by modulating the calcium response at a key step of the time course, or by using inhibitors of calcium-sensor proteins and we further investigated the effects of these perturbations on the transcript levels of representative genes.

Sustained calcium elevation plays a key role in the cryptogein-induced response

In order to determine the relative importance of the transient peak and of the sustained phase of calcium elevation (plateau) in cryptogein-induced transcriptional regulation, we applied 1mM La 3+ either just before the cryptogein treatment, thus suppressing the whole calcium response (light grey curve, Fig. 7a), or 10 minutes after cryptogein treatment (arrow) in order to maintain the first peak while suppressing the sustained plateau (dark grey curve, Fig. 7a).

After a 3-hour treatment, we measured the accumulated level of transcripts of phenylpropanoid genes in each condition (Fig. 7b). 09/02/10

Adding lanthanum just before or 10 minutes after the cryptogein treatment resulted in both cases in a strong reduction of transcript levels of PAL, HCT or CCoAOMT (Fig. 7b) as compared to cryptogein treatment alone (see also Fig 3a). Furthermore no significant difference (p=0.05) was observed between the inhibitory effects of the two treatments with lanthanum. In contrast, cryptogein-induced accumulation of transcripts of C4H, 4CL, C3H, COMT and CCR was only slightly modified by addition of lanthanum after 10 min, suggesting that transcriptional regulation of these genes is mainly dependent of the early calcium influx. It is noteworthy that these two subsets of genes are identical to those identified by the PCA analysis, suggesting a possible link between the intensity of the stimulus (PCA analysis; figure 4), the two different phases of the calcium response (figure 7) and gene expression.

Regulators with low and high affinity for calcium control the transcriptional response

To assess the possibility of regulatory effects downstream of the calcium signal, we evaluated the effects of compounds able to interact with calmodulins or CaM-like proteins on cryptogein-induced responses. We used W-13 known as a calmodulin inhibitor (Kd = 50 µM) and W-12 (a molecule of the same family as W-13 with a H/Cl substitution and a lower affinity for CaM, Kd = 250 µM) as negative control.

By themselves, these drugs provoked changes in cellular calcium [START_REF] Dagher | Calcium fingerprints induced by Calmodulin interactors in eukaryotic cells[END_REF] but in a differential manner (Supporting Information Fig. S4). Within two minutes, the calmodulin inhibitor W-13 induced a transient dose-dependent increase in cytosolic calcium, followed by a rapid decrease to the resting level. In contrast, W-12 here considered as the negative counterpart of W-13, induced significantly lower calcium variations in the cytosol. In subsequent experiments these two compounds were used at a final concentration of 50 µM for which the calcium level returned to the basal level within 10 minutes. In order to investigate the effect of these two drugs either on the calcium response or on the transcriptional response induced by cryptogein, W-13 or W-12 were added to the cell suspension 10 minutes before cryptogein treatment. Then, cytosolic calcium variations were recorded for 15 min, and the cells were collected after 4 hours for transcriptional analysis. The calcium response induced by high concentrations of cryptogein (0.25 µM and above) was not modified by either inhibitor, but when cryptogein was added at low concentrations (< 50 nM) the sustained calcium elevation (plateau value), and to a lesser extent the peak value, were significantly increased by W13 (Fig. 8a), but not by W12 (not shown). When added alone, these compounds had no effect on the transcript level of phenylpropanoid genes (not shown). When added before cryptogein-treatment, they had no effect on the transcript increase induced by concentrations of cryptogein above 100 nM, but a strong enhancement of the cryptogein effect was mediated by the CaM inhibitors at low concentrations of cryptogein i.e. 25 nM (Fig. 8b). As expected, this effect was higher with the calmodulin inhibitor W-13 than with its negative counterpart W-12.

These results confirm the importance of the plateau phase of the calcium signature in the increase of transcript levels induced by cryptogein. Furthermore, the differential effect of anti-CaM compounds, between low and high cryptogein treatments, is consistent with the hypothesis that two calcium decoding modules, one with low affinity and the other with high affinity for calcium, are involved in the regulation of this response.

Modelling the coupling between cryptogein-induced calcium and transcriptional responses

The results presented above show the existence of correlations between the different phases of the cryptogein-induced calcium signature and the transcriptional response: the calciumplateau value correlates positively with transcript accumulation, while an increase of the calcium-peak value above a threshold correlates negatively with transcript accumulation. Furthermore they support the hypothesis of two decoding modules (involving different calcium sensors) able to modulate the response to cryptogein and which could explain the observed correlations.

To test this hypothesis and verify its coherence with the experimental data we used computer modelling and built a model of the non-linear system coupling the cryptogein signal to calcium and transcriptional regulation. The model can be described as follows (see scheme Fig. 9).

Perception of cryptogein triggers opening of calcium channels, which results in an influx of external calcium. This calcium influx activates calcium-dependent mechanisms that depends upon the strength of the stimulus (= cryptogein concentration). At low concentrations of cryptogein (10 nM -50 nM), the concerted activation of calcium channels and transporters results in a small but sustained elevation of free cytosolic calcium concentration (=plateau value). This increase (or influx) is nevertheless sufficient to activate calcium-sensors belonging to a high affinity decoding module (module 1), which in turn will enhance transcription of response genes. For higher cryptogein concentrations (> 50 nM), the larger calcium influx triggers the release of calcium from internal stores and/or the opening of additional plasma-membrane calcium channels, resulting in a large transient increase of cytosolic calcium (=peak value) followed by a slow return to the plateau value through the action of homeostatic mechanisms. This has two consequences: 1) the activation of high affinity module 1 due to sustained calcium elevation, and thus differential expression of responsive genes, and 2) the activation of other calcium responsive effectors of a low-affinity module (module 2), which have an inhibitory effect on the transcription of the same genes. This results in equilibrium between activation-inhibition mechanisms through the activation of these two modules, linking the transcriptional response to the cryptogein concentration in a non-linear manner.

We used a simple phenomenological approach to formalize this model using a twocomponent system derived from chemical binding kinetics (Supporting Information S7). In this model each component (modules 1 and 2) is characterized by an "apparent" affinity constant for cryptogein (K1 and K2 resp.), a parameter defining its maximum activity (S1 and S2 resp.), and a Hill coefficient (n1 and n2 resp.) to account for cooperativity. This model integrates, in a simple way, all the steps between cryptogein binding and transcriptional activation, and thus its parameters depend implicitly on the characteristics of the calcium decoding modules. This model fits well to experimental dose-response curves obtained for transcription of PAL, HCT, and CCoAOMT (Fig. 10a). Interestingly, the K values for cryptogein estimated for each component were both very close to the Km value of the doseresponse curve observed for the calcium plateau (30 nM), which is in the range of IC 50 values measured for cryptogein binding to its receptor. In order to test the model further, we used it to fit the effect of calmodulin inhibitors on cryptogein-induced transcription. A good fit to experimental curves was obtained (Fig. 10b) by increasing the affinity parameter K1 that corresponds to the "apparent" affinity of the "activation" module (the other parameters being held constant). Interestingly, the K1 value estimated for the W13 experimental curve was greater than its value estimated for W12.

The hypothetical mechanisms at the basis of this simple model seem to be sufficient not only to fit dose-response curves but also the effect of anti-calmodulin compounds on the transcriptional response to cryptogein. It is worth noting that this model was built on assumptions derived from observed correlations between calcium and expression data only. However, by modifying only two parameter values, the model was able to simulate (at least qualitatively) the effect of anti-calmodulin compounds. Considering that data from anti-CaM experiments were not used to derive the model, this last result is in some extent a first step in the validation of this model.

DISCUSSION

In this work our aim was to build an integrated view of the relationships linking the calcium response to the short-term responses induced at the transcriptional and metabolic levels by the necrotizing elicitor cryptogein in BY-2 cells. For this purpose, we first carried out a quantitative analysis of the biological responses at different levels, and then exploited these data to propose a simple coupling model between calcium variations and transcriptional regulation of genes of the phenylpropanoid pathway.

Cryptogein increases transcription of most genes of the phenylpropanoid pathway

In BY-2 cells cryptogein induced an increase of transcript levels for most of the genes of the phenylpropanoid pathway, with expression ratios ranging from 4 (e.g. C4H) to more than 100 (e.g. CCoAOMT), but with the noticeable exception of CAD, which catalyses the last step leading to lignin monomers, that was not induced or was even repressed for high concentrations of elicitor. These transcriptional responses occurred early after elicitation (less than 30 minutes for the most activated gene CCoAOMT) and transcript accumulation continued increasing for several hours, reaching a maximum after 4 hours for PAL, HCT and CCoAOMT. For other activated phenylpropanoid genes like COMT, their transcripts continued to accumulate for at least 8 hours after treatment. Depending on the gene, these responses were totally or partially, suppressed when calcium influx was blocked by lanthanum. This is consistent with the calcium-dependence of the biological responses to cryptogein shown in previous works [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF]).

An increased accumulation of transcripts of genes coding for key enzymes of the phenylpropanoid pathway (PAL, 4CL, CCoAOMT, C4H) in response to elicitors has been previously reported in various plant species [START_REF] Dixon | Stress-induced phenylpropanoid metabolism[END_REF][START_REF] Hano | Differential accumulation of monolignol-derived compounds in elicited flax ( Linum usitatissimum) cell suspension cultures[END_REF], Kauss et 09/02/10 al. 1993[START_REF] Shinya | Comprehensive analysis of glucan elicitor-regulated gene expression in tobacco BY-2 cells reveals a novel MYB transcription factor involved in the regulation of phenylpropanoid metabolism[END_REF][START_REF] Suzuki | Comprehensive analysis of early response genes to two different microbial elicitors in tobacco cells[END_REF]. In particular, cryptogein activates transcription of the phenylalanine-ammonia lyase (PAL) gene in tobacco cells [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF][START_REF] Suty | Early changes in gene expression in tobacco cells elicited with cryptogein[END_REF]. In this work, we extended the study to a larger set of genes belonging to the phenylpropanoid pathway and quantified corresponding transcript levels.

Transcription of HSR203J, a marker of hypersensitive response in tobacco [START_REF] Pontier | hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactions[END_REF], was strongly increased by the cryptogein treatment as previously shown [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF]. In contrast PR1 and PR3, two genes involved in defence responses through the salicylic acid pathway (Keller et al. 1996a[START_REF] Ward | Coordinate gene activity in response to agents that induce systemic acquired resistance[END_REF], were not differentially expressed in BY-2 cells. This is in agreement with the known biological effect of cryptogein in tobacco, which induces cell death through a hypersensitive response, while the salicylic pathway has been shown not to be involved in the HR response (Keller et al., 1996).

Phenolic metabolism changes in response to cryptogein

In tobacco BY-2 cells, cryptogein induced an early accumulation of cell-wall bound phenolic compounds (p-coumaric acid, ferulic acid, 5-hydroxyferulic acid, 4-hydroxybenzaldehyde), whereas no consistent variation of free soluble phenolics was detected after a 4-hour treatment. The accumulation of cell-wall-bound phenylpropanoid derivatives has often been described as a form of biochemical defence in response to elicitors. For instance, [START_REF] Bolwell | Metabolic changes in elicitor-treated bean cells. Enzymic responses associated with rapid changes in cell wall components[END_REF] reported an accumulation of phenolic material bound to cellulosic and hemicellulosic fractions and of free hydroxycinnamic acids in bean cells. Some of the phenolicbound esters identified in this study were also found in cells treated with elicitor fractions from other oomycetes. It is the case of extracts from Phytophthora megasperma that induced accumulation of cell-wall-bound 4-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, 4hydroxybenzaldehyde and vanillin [START_REF] Kauss | Conditioning of parsley (Petroselinum crispum L.) suspension cells increases elicitorinduced incorporation of cell wall phenolics[END_REF]. Cell-wall-bound 4hydroxybenzaldehyde and tyramine amides were also detected in suspension cultures of potato treated with an elicitor extract of Phytosphora infestans (Keller et al. 1996b).

Since in response to cryptogein, CAD transcript levels are slightly down-regulated whereas most upstream genes are up-regulated, it is conceivable that the CAD substrate (coniferaldehyde) could be (at least in part) not converted into coniferyl alcohol, but rather redirected to ferulic acid by oxidation. Such a hydroxycinnamaldehyde dehydrogenase (REF1, AtALDH2C4) activity has been demonstrated in Arabidopsis thaliana [START_REF] Nair | The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis[END_REF]. The identification of this enzyme inverted the conventional model of flux, ferulate and sinapate being products of coniferaldehyde and sinapaldehyde oxidation, rather than being precursors in their synthesis. In support of the hypothesis that cell-wall-bound ferulate could originate from coniferaldehyde, we recently identified within the publicly available tobacco ESTs, a sequence highly homologous to AtALDH2C4. Furthermore, we have shown that this gene was significantly over-expressed by 5 times in BY-2 cells challenged with cryptogein (Amelot et al, in preparation).

An increased amount of 4-hydroxybenzaldehyde was also observed in cryptogein-treated cells. Although the origin of this compound is unknown, one could hypothesize the existence of a 4-hydroxybenzaldehyde synthase activity in tobacco. Such a synthase activity, which converts 4-coumaric acid to hydroxybenzaldehyde, was found in tissue cultures of Vanilla planifolia [START_REF] Podstolski | Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia[END_REF] and in hairy root cultures of Daucus carota [START_REF] Sircar | Evidence for p-hydroxybenzoate formation involving enzymatic phenylpropanoid side-chain cleavage in hairy roots of Daucus carota[END_REF].

Taken together, our results clearly show a reorientation of the phenylpropanoid metabolism in response to cryptogein, resulting in the incorporation of new cell wall phenolic compounds which likely reinforce the cell wall barrier. Moreover they suggest a new possible route accounting for the accumulation of ferulic acid in tobacco BY-2 cells.

Calcium controls the cryptogein-induced responses

Our results showed that the phenylpropanoid genes belong to two distinct subsets that respond differentially to cryptogein treatment. The regulation of these two subsets differs by their sensitivity to stimulus intensity (i.e. cryptogein concentration) as well as by their correlation with one particular phase of the calcium signature (peak and/or plateau). Indeed, the long lasting influx of calcium responsible for the sustained increased level of cytosolic calcium (plateau) is necessary for the appropriate transcriptional response of genes induced by low concentrations of cryptogein (i.e. PAL, HCT and CCoAOMT). Thus, a positive correlation is observed at low cryptogein concentrations between the sustained calcium elevation and transcript accumulation of these genes, while at higher concentrations the calcium peak value was negatively correlated with transcription. On the other hand, C4H, 4CL, C3H, COMT and CCR genes activation seem to rely on higher cryptogein concentrations. Furthermore, it appears that the early phase of the calcium response is mainly responsible for their transcriptional response. Such differential responsiveness to calcium response would allow the plant to selectively activate genes of the phenylpropanoid pathway.

To explain these results one might consider that different calcium stores involved in the generation of the cryptogein-induced calcium signature [START_REF] Garcia-Brügger | Early signaling events induced by elicitors of plant defenses[END_REF][START_REF] Kadota | Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca 2+ transients, anion efflux and production of reactive oxygen species[END_REF][START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF] contribute differently to the subsequent biological responses. However, computer simulations of our two-component model rather suggest the existence of two different calcium decoding modules, with low and high affinity for calcium, associated with the main calcium peak and the sustained phase (plateau) respectively, and displaying either an inhibiting or an activating effect. This model is also supported by the specific effect of calmodulin inhibitor W13 on the transcriptional response to low cryptogein concentrations only, which suggests the involvement of calmodulins or calmodulin-like proteins in the transduction process. This effect could be simulated with our model by increasing the value of the "apparent" affinity coefficient of the "activation" component (Fig. 10). One may thus hypothesize that W13 inhibits calcium-binding proteins (such as CaMs, CMLs or potentially other EF-hand proteins) involved either in the control of calcium homeostasis (channels or pumps) and/or in signal transduction. It is known that some Ca-channels are regulated by calmodulin [START_REF] Tadross | Mechanism of local and global Ca 2+ sensing by calmodulin in complex with a Ca 2+ channel[END_REF], Ca 2+ -CaM is also involved in the control of calcium pumps [START_REF] Boursiac | The origin and function of calmodulin regulated Ca 2+ pumps in plants[END_REF]. Thus, according to our model, increasing the channel activity or inhibiting pump activity would result in an increase of the calcium resting level (plateau value), which could in turn activate calcium-dependent transcription complexes. When the cryptogein concentration is higher (> 0.1 µM), activation is already at its maximum, and thus cannot be enhanced further, but for genes like PAL or CCoAOMT inhibition can still be increased, leading to a transcriptional response lower than the one obtained at low cryptogein. Globally, anti-CaMs appear to shift the cryptogein concentration at which the transcriptional response is maximal towards lower values.

The non-linear relationship between the calcium signal and downstream responses highlights the complex regulatory properties of calcium and reinforces the concept of calcium signature, considered here as an "observable" giving an overview at the whole population level of the underlying calcium-dependent processes thought to encode/decode information required for the specificity of the final responses As the calcium signature observed at the level of a population represents the integral of calcium signals occurring in each individual cell, the possibility to measure calcium signals at the single cell level will be invaluable to further interpret the observed data at the mechanistic level. Finally, the identification of calcium-or CaM-dependent transcription factors involved in the regulation of genes of the phenylpropanoid pathway in tobacco should bring new insights in understanding cryptogein signalling.

Table 1

Cryptogein effect on the accumulation of insoluble phenolic compounds in tobacco BY-2 cells. For each compound, ratio between treated versus control cells was calculated based on their relative amounts measured by MS/MS. Data are means and standard-errors from 4 independent experiments. Paired-t-test was used for statistical comparisons between amounts (Log transformed).

Phenolic compound

Ratio

Supporting Information:

Table S1: primers and amplicons sizes.

  MassLynx 4.1 software (Waters, Manchester, UK) was used for data analysis. 09/02/10 Phenolic compound identification was based on comparison of retention times of commercial standards, accurate mass measurements, and MS/MS fragmentation analysis (collision energy: 15 eV).

Figure 2 .

 2 Figure 2. Cryptogein-induced calcium signatures in tobacco BY-2 cells. (a) Time-course of

Figure 3 .

 3 Figure 3. Effects of cryptogein on transcript accumulation of phenylpropanoid genes in

Figure 4 .

 4 Figure 4. Principal Component Analysis (PCA). Analysis of transcription ratio data plus

Figure 5 .

 5 Figure 5. Time course of relative enzymatic activities of PAL and CCoAOMT. Activities

Figure 6 .

 6 Figure 6. Distribution of radioactivity in tobacco BY-2 cell suspensions incubated for 4 hours with [ 14 C(U)]-cinnamic acid. At t=0 hour, cells were treated with water (control, white bar), 0.1µM cryptogein (black bar), or 0.1µM cryptogein + 1mM La 3+ (grey bar). Relative

Figure 7 .

 7 Figure 7. Effect of inhibiting calcium influx on cryptogein-induced calcium variations (a) and transcript accumulation (b) in tobacco BY-2 cells. 1mM La 3+ was added at t=0 (black line and

Figure 8 .

 8 Figure 8. Effects of calmodulin inhibitors W12 and W13 on the cryptogein-response. (a)

Figure 9 .Figure 10 .

 910 Figure 9. Proposed model for regulation of cryptogein-induced transcriptional response by

Figure S2 :

 S2 Figure S2: Comparison between lanthanum chloride and EGTA pre-treatments on cryptogein-

Figure S3 :

 S3 Figure S3: Effects of a cycloheximide pre-treatment on cryptogein-induced transcript

Figure S4 :

 S4 Figure S4: Effect of cryptogein on transcript accumulation of genes coding for isoforms of

Figure S5 :

 S5 Figure S5: Cytosolic calcium responses to calmodulin inhibitors in tobacco BY-2 cells.

Figure S6 :

 S6 Figure S6: Assessment of cryptogein-induced cell death in tobacco BY-2 cells.

Figure

  Figure S7: Model formulation.

S2.

  Comparison between lanthanum chloride and EGTA pre-treatments on cryptogein-induced phenylpropanoid genes expression in tobacco BY-2 cells.

Figure S2 .

 S2 Figure S2. Comparison between lanthanum chloride and EGTA pre-treatments on cryptogeininduced phenylpropanoid genes expression in tobacco BY-2 cells. (a) Time course of cytosolic calcium variation in response to 0.1µM cryptogein in the absence (dark grey trace) or presence of either 1mM La 3+ (black trace) or 2mM EGTA (light grey trace). Data show one representative experiment out of three. (b) Relative transcript levels assessed by RT-qPCR in cells treated for 3 hours with 0.1µM cryptogein in the absence (dark grey bars) or presence of either 1mM La 3+ (black bars) or 2mM EGTA (light grey bars). Transcript levels are expressed as means ±SE (n=3) of transcript ratios relative to control cells (H 2 O treatment).

  Figure 1. Biosynthetic routes for phenolic compounds adapted from the model proposed by Nair et al. (2004) for A. thaliana. Encircled compounds accumulate in tobacco BY-2 cell walls in response to cryptogein. Light grey arrows indicate routes that are unlikely to occur in

	Figures legends
	planta. Dotted arrow indicates a hypothetic route for the biosynthesis of 5-hydroxyferulate
	(see results section). PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase;
	4CL, 4-coumarate-CoA ligase; C3H, p-coumarate 3-hydroxylase; HCT, hydroxycinnamoyl
	P value CoA:quinate/shikimate hydroxycinnamoyltransferase; CCoAOMT, caffeoyl-CoA 3-O-(mean SE)
	p-coumaric acid methyltransferase; COMT caffeic acid/5-hydroxyferulic acid O-methyltransferase; F5H, 0.04 3.3 1.0
	ferulic acid	1.9 0.3	0.05
	ferulate 5-hydroxylase; CCR, cinnamoyl CoA reductase; CAD, cinnamyl alcohol
	5-hydroxyferulic acid	184 112	0.005
	dehydrogenase. REF1, Reduced Epidermal Fluorescence1.
	4-hydrobenzaldehyde	1.8 0.3	0.06

Table S1 : Primers and amplicon sizes:

 S1

	Gene	Amplicon size	upper 5'	lower 5'
	18S rRNA	171bp	cgcgctacactgatgtattc	gtacaaagggcagggacgta
	PAL	94bp	gacaaagtgttcacagcaatg aaacagatwggaagaggagca
	PAL A	228bp	cctcgtcgaccacgccttgc	taattcagctccaagttctc
	PAL B	242bp	acacattgccacattcagca	caccattgggaccaacagcc
	C4H	124bp	tcaacacaatggtggaatgc	actttgggacgtttggttca
	4CL	89bp	cttctcaaccatcccaacatt	ctaacaacaaaagccactgga
	4CL1	178bp	ttcctggagttaatacagaag	gggaatttggttctcacagcg
	4CL2	179bp	tttcttggagttgatacaacg	ggaaatttggctcgaacagta
	COMT	132bp	cctgcaaatgggaaggtgat	cagtcctttctttgcctcct
	C3H	142bp	tggctgaggtgatcaagaac	tatgggaggaaggggaagtc
	HCT	127bp	ggctgccaatccatgatgct	gcaacagattgactgccatca
	CCoAOMT	96bp	acaccctatggaatggatca	ccttgttgagttccaatacga
	CCoAOMT 1	89bp	aatttgccagctacctgttggt	ttggttgccacaaatagacgat
	CCoAOMT 2	92bp	gaactattgtccatttgtggca acgaatgatacagaacagggga
	CCoAOMT 3	130bp	tttacagggctagttcatggca	ggtaacatcgacaatgcaacct
	CCoAOMT 4	102bp	cattggctgctgattctaga	aaatcctccattgcctgtaa
	CCoAOMT 5	113bp	acgtatgatttcatatttgtggat caatccttggatcaacggctagg
	CCoAOMT6	121bp	cacatatgatttcattttcgtgg	caattctgggatcagctgccaaa
	F5H	100bp	agcccggacaaggaagat	cccatccaagccaaggta
	CCR	99bp	gacttctgcaaaaacaccaa	caccaaatcgactcctttt

ACKNOWLEDGEMENTS

Article Amelot.docx C. Brière Page 26 09/02/10 The authors thank Sabine Grat for her technical assistance in cell culture maintenance and Nathalie Ladouce for her help in RT-qPCR analysis. Raoul Ranjeva thanks the Miller Institute for Basic Research (UC Berkeley) for supporting him with a Miller Visiting Professorship. Nicolas Amelot thanks the Association pour la Recherche sur les Nicotianae for financial support. This work was supported by a grant from the Agence Nationale pour la Recherche (ANR-06-BLAN-370) and by the Centre National de la Recherche Scientifique.

S3. Effects of a cycloheximide pre-treatment on cryptogein-induced phenylpropanoid genes in BY-2 tobacco cells

S7. Model formulation

The model used to describe the relationship between cryptogein concentration "C" and transcript ratio "R" (measured 4 hours after treatment) was inspired from enzymatic kinetic laws for one ligand and two binding sites (Adair-Klotz model) and Hill phenomenological equation. Here the ligand is cryptogein, the observable variable is the transcript ratio, and the binding sites correspond by analogy to the two putative decoding modules. Thus the equilibrium reads as follows:

(1)

Where K1, S1, and n1 are the association constant, the maximum response and the Hill coefficient for component 1(respectively K2, S2, and n2 for component 2). For a given condition Ki values and Hill coefficients were supposed to be the same for all genes, while coefficients S1 and S2 were gene dependent.

Estimated values of the coefficients for three genes (data from Fig. 10a): K1 = 23.3 µM -1 ; K2 = 24.9 µM -1 ; n1 = 3.9; n2 = 2.