Concord grape (Vitis labrusca) cell suspensions were cultured in Murashige and Skoog (MS) medium (pH 5.8) containing vitamins (×1.5), sucrose (30 g l^-1), 2,4-dichlorophenoxyacetic acid (2,4-D, 0.2 mg l^-1), 6-benzylaminopurine (BAP, 0.5 mg l^-1) and were propagated in the dark at 25°C under shaking at 120 rpm. They were sub-cultured every 7 days to be maintained in exponential phase. For experiments, 30 ml of cells sub-cultured for 7 days were collected and resuspended in fresh MS medium for 24 h before treatment (Aziz et al., 2004).

Bacteria were collected by centrifugation (3,000 × g for 15 min) and washed twice with a phosphate-buffered saline (PBS) (10 mM, pH 6.5). The pellet was resuspended in the PBS and used as inoculum. The bacterium S. anulatus S37 concentration was estimated by the spectrophotometer (600 nm) and adjusted to 10⁶ colony forming units per ml (cfu ml^-1) with the PBS (Loqman et al., 2009).

Botrytis cinerea strain 630 used in this study was provided by Dr. Brygoo (INRA, Versailles, Grignon, France) and maintained on the potato dextrose agar (PDA, Difco, United States). The B. cinerea inoculum was initiated by growing the fungus on fresh PDA medium to obtain abundant hyphal swellings. After 3 weeks, conidia collected from the PDA medium with sterile distilled water were filtered through a sterile 25 μm filter. The density of B. cinerea conidiospores was then determined and adjusted to 10⁵ spores ml^-1 for all bioassays.

Cells were collected during the exponential growth phase and washed by filtration in a suspension buffer containing 175 mM mannitol, 0.5 mM K2SO4, 0.5 mM CaCl2, and 2 mM MES, pH 5.5. Cells were resuspended at 0.1 g FW ml^-1 with a suspension buffer and equilibrated for 2 h on a rotary shaker (120 rpm, 25°C). Grapevine cells were then used to analyze the extracellular pH, the H₂O₂ production, the MAP kinase assay, the cell death, the defense-related gene expression, and the phytoalexin production after a treatment with the S. anulatus S37 and/or the B. cinerea. Control cells were incubated under the same conditions without treatment. The EGTA (Sigma) was supplied at 3 mM, for 10 min, before an inoculation with the S37 and/or the B. cinerea.

The extracellular pH variation was analyzed according to Felix et al. (1993) in 10 ml of agitated cell culture using a glass pH electrode (Basic, Denver Instrument, Gottingen, Germany). The production of H₂O₂ was analyzed by chemiluminescence from the ferricyanide-catalyzed oxidation of luminol using a luminometer (Lumat LB 9507, Berthold) as described previously (Aziz et al., 2004). The chemiluminescence was integrated and expressed as nmol H₂O₂ per g FW, using a standard calibration curve with H₂O₂.

The refractory state experiments on grapevine cell suspensions were analyzed by the extracellular pH change after a successive addition of the S. anulatus S37 and the B. cinerea. Cells were first treated at time 0 with the S. anulatus S37 at 10⁶ cfu ml^-1 or with the B. cinerea at 10⁵ spores ml^-1, washed at 50, 100, and 150 min with a fresh medium, and then treated, at 150 min, a second time with the S. anulatus S37 or the B. cinerea at the same concentrations.

In vivo experiments followed by in-gel kinase assays were performed as previously described (Vandelle et al., 2006) using myelin basic protein (MBP) as a MAP kinase substrate at the final concentration of 0.2 mg ml^-1.

The cell death was visualized by the FDA (fluorescein diacetate; Sigma–Aldrich) staining. Fresh cells were incubated in FDA (10 μg ml^-1 of PBS) in the dark to maximize the formation of the fluorescein. The fluorescent FDA signals were detected with a fluorescence microscope (Olympus BX 51, Olympus, Japan). Only cells that exhibited bright green fluorescence from their cytosol were considered to be viable.

The cell death was also evaluated by analyzing caspase-like activity in grapevine cell suspensions, using the Muse MultiCaspase-7-AAD Assay as described by the manufacturer (Millipore, Molsheim, France). The assay determines simultaneously the percentage of cells showing a caspase activity and cell death fraction. Briefly, after each experimental condition, cells are resuspended at a density of 2–5 × 10⁵ cells ml^-1 in the 1X Assay Buffer. The 50 μl of cell suspension were then added to each measurement tube. Then, 5 μl of Muse^TM MultiCaspase Reagent working solution were added to each tube. After vortexing at a medium speed for 3 to 5s, tubes were incubated for 30 min in the 37°C incubator with 5% CO₂. Subsequently, 150 μl of Muse^TM 7-AAD working solution were added to each tube. After vortexing as indicated before, tubes were incubated at room temperature for 5 min, protected from light. After cell analysis, percentages of gated cells were calculated. Data indicate viable cells without caspase activity, cells exhibiting caspase activity without death marker, cells in the late stages of caspase activity with death marker, and cells that have died via necrosis but not through the caspase pathway.

Cells were ground in the liquid nitrogen and total RNA was extracted from 50 mg of ground powder following the Concert PlantRNA reagent protocol according to the manufacturer’s instructions (Life Technologies). The RNA pellets were resuspended in 30 μl of RNAse-free water and incubated 2 h at -20°C for solubilization. The genomic DNA was removed by a DNAse treatment according to the manufacturer’s instruction (RQ1 RNase-Free DNase – Promega). The first-strand cDNA was synthesized from 150 ng of total RNA using the Verso cDNA Synthesis kit (Thermo Scientific). The quantitative RT-PCR was performed as described in Gruau et al. (2015), with Absolute Blue QPCR SYBR Green ROX Mix (Thermo Scientific) using a CFX96 system thermocycler (Bio-Rad). PCR reactions were carried out in duplicates in 96-well plates in a 15 μl final volume containing Absolute Blue SYBR Green ROX mix (including Taq polymerase, dNTPs, and SYBR Green dye), 280 nM forward and reverse primers and 30-fold diluted cDNA. The specificity of PCR reaction was also checked and EF1α and 60RSP genes were used as internal controls for the normalization (Gruau et al., 2015). Results are expressed as the folds increase of transcript level relative to untreated cells as the control sample (1x expression level). The gene-specific primers (Supplementary Table S1) were designed based on sequences present in databases (Gruau et al., 2015).

Stilbenic phytoalexins were extracted from 200 mg of freeze-dried cell powder with 2 ml of methanol:water 85% v/v. Tubes were placed in the shaker for 1 h at the room temperature and then centrifuged for 10 min at 8000 ×g. The supernatant was dried under a nitrogen stream and residues were solubilized with 1 ml of methanol, filtered through 0.22 μm PTFE filters. Stilbenes were analyzed using an Acquity^TM UPLC system (Waters Corporation, Milford, CT, United States) as described in Hatmi et al. (2014), identified and quantified with reference to retention time and calibration with external standards.

Each experiment was repeated at least three times, unless indicated elsewhere. For gene expression, medium alkalinization, H₂O₂ production and phytoalexin production, results are expressed as the mean ± SE of a triplicate from one representative experiment out of three independent experiments (in each experiment three different extractions were pooled). For cell death, MAP kinase and Caspase-like activity, the data have been presented as a representative of three independent experiments. The refractory state experiment, reported data mean ± SE of duplicates, representative of two independent experiments. All collected data were submitted to ANOVA and the significance of differences among treatments was recorded at p < 0.05.

During the cell and microbes interaction, cells can be damaged, thereby affecting their viability. To further elucidate cell viability during the process of inoculation with B. cinerea and to study the impact of the presence of S. anulatus S37, grapevine cells were stained using fluorescein diacetate as a vital stain. Grapevine cells with intact plasma membranes and respiration revealed a green fluorescence while those without FDA-fluorescence were considered dead. Control cells showed strong FDA signals (Figure 2A), with high viability levels. While the presence of S. anulatus S37 in contact with cells has no impact on the viability, the FDA signal was weaker in cells infected with B. cinerea, indicating disturbed cell membranes and an inhibited respiratory activity. However, when cells were inoculated with S37 then with B. cinerea, the FDA signal was weak than the control but significantly higher than in cells infected solely with B. cinerea.

The multi-caspase activity was measured as described in “Materials and Methods.” The data in Figure 2B showed that this activity was not impacted significantly when grapevine cells suspension was brought into contact with S37. However, in the presence of B. cinerea, the percentage of cells with caspase activity increased significantly compared to the previous two terms (9.90%). When grapevine cells were previously inoculated with S37 before their infection with B. cinerea, this activity decreased significantly (2.55%).

Expression profiles of number defense-related genes were analyzed by qRT-PCR in grapevine cells suspension after their inoculation with S. anulatus S37 and/or B. cinerea. Genes used correspond to those reported to be up-regulated during grapevine-microbe/MAMP interactions (Aziz et al., 2003; Varnier et al., 2009; Bordiec et al., 2011) and include those encoding a lipoxygenase (LOX9), a phenylalanine ammonia-lyase (PAL), a stilbene synthase (STS), a basic glucanase (Gluc), and a glutathione S-transferase (GST). All defense genes were up-regulated when the cells were inoculated with S37 or B. cinerea starting after 9 h except for LOX9 (Figure 3).

The level of gene expression was lower in S37-treated cells compared to those infected with B. cinerea. Interestingly, inoculation with S37 + B. cinerea results in the priming of LOX9 gene expression, which was almost 20 times higher than control. A priming effect was also seen for PAL, STS, and GST. The basic glucanase (Gluc) was the only gene, which was induced but not primed by S37 (Figure 3).

The resveratrol (trans-3,4′,5-trihydroxystilbene) and its dehydrodimer viniferin have been reported as the major phytoalexins produced by grapevine in response to the microbial infection and was associated with plant resistance to fungal pathogens (Adrian et al., 1997; Aziz et al., 2016). As shown in Figure 4, induction of resveratrol and trans-δ-viniferin was observed following treatment of grapevine cells with S37. A slight induction of both stilbenes was also observed after challenge with B. cinerea. Amounts of δ-viniferin accumulated were generally greater than resveratrol. However, with both microbes, the level of the glycosylated resveratrol, piceid, remained comparable to that of the control. With S37 + B. cinerea, the production of resveratrol and piceid showed similar levels than with S37, while δ-viniferin accumulation was primed.

One of the characteristics of cells is the occurrence of the refractory state, which often occurs after the perception of MAMPs/PAMPs at high-affinity protein receptors in the plasma membrane. As S. anulatus S37 and B. cinerea induce the same pattern of early events, refractory assays (i.e., the inability of the cells to react to a second application of the same elicitor), using extracellular pH were performed by successive additions of S37 and/or B. cinerea. Grape cells pretreated with the S37 were shown to be refractory to the second application of S37. However, they were not refractory to an application of B. cinerea (Figure 5A). Similarly, cells pretreated with B. cinerea were shown to be refractory to the second application of B. cinerea, while they were not refractory to an application of S37 (Figure 5B).

Our results showed that S. anulatus S37 triggers the oxidative burst, one of the key events, in grapevine cell signaling. ROS play a crucial role either as toxic substances to halt the growth of the pathogen or as second messengers in the defense mechanism, leading to efficient plant protection (Torres, 2010; Zipfel and Robatzek, 2010). Nevertheless, only a few examples have described the involvement of ROS as a signal during the grapevine plants-beneficial bacteria interaction. For instance, P. fluorescens PTA-CT2, P. aeruginosa 7NSK2, and B. subtilis PTA-271 trigger a strong oxidative burst in grapevine cell suspensions (Verhagen et al., 2010, 2011). Authors suggested that the H₂O₂ production in grape cell suspensions could contribute as a signaling molecule to induce a disease resistance by these strains in planta. Indeed, although the accumulation of H₂O₂ is often associated with a characteristic plant early response following perception of pathogen avirulence signals (Lamb and Dixon, 1997), it was also shown that, in the symbiotic interaction with rhizobia, bacteria are initially recognized as intruders but then prevent or overcome plant defense responses (Santos et al., 2001). However, in contrast with our results, no significant variation in H₂O₂ levels was induced in grapevine cells by B. phytofirmans PsJN (Bordiec et al., 2011). Further, Van Loon et al. (2008) have reported that the production of H₂O₂ induced by P. fluorescens WCS417 was not matched with the expression of ISR in the whole tobacco against Erwinia carotovora.

The S. anulatus S37 also induces a strong extracellular alkalinization, which suggests an activation of plasma membrane H⁺-ATPases, to restore the pH gradient between the apoplast and the cytosol. This typical signature has been previously demonstrated during the microbial or MAMP perception (Aziz et al., 2004; Bordiec et al., 2011; Farace et al., 2015). The perception of S37 by grapevine cells was also associated with the phosphorylation of two mitogen-activated protein (MAP) kinases with relative molecular masses of 49 and 45 kDa. These effects resembled those achieved by the MAMPs in tobacco (Zhang et al., 1998) and in grapevine (Aziz et al., 2004; Trouvelot et al., 2008). MAP kinase cascades are major pathways downstream of sensors/receptors that transduce extracellular stimuli into intracellular responses in plants (Zhang et al., 1998). Their activation is known as an early physiological response to microbial recognition and plays a pivotal role in the plant innate immune system (Asai et al., 2002; Aziz et al., 2004; Meng and Zhang, 2013). In tobacco, two MAP kinases, designated SA-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) are activated in a disease resistance-specific manner following pathogen infection or elicitor treatment (Kishi-Kaboshi et al., 2010). Similarly, it has been shown that lack of MPK3 increases the basal susceptibility of Arabidopsis to B. cinerea, while the lack of MPK6 suppresses flg22-induced resistance to B. cinerea (Galletti et al., 2011). It is noteworthy that these early responses induced by the S. anulatus S37 were not accompanied by a programmed cell death of grapevine, as shown by the FDA signal and the multicaspase activity. This result is in line with previous reports who indicated that oxidative burst or the activation of MAP kinases induced by beneficial bacteria are independent of hypersensitive cell death (Bargabus et al., 2003; Van Loon et al., 2008; Verhagen et al., 2010).

The Ca²⁺ influx is also involved in the microbial signal perception and can regulate many early responses in plant cells. In this context, we investigated whether extracellular alkalinization, H₂O₂ production, and MAPK activity depend on the calcium influx in grapevine cell suspensions. Using the EGTA, a Ca²⁺ influx inhibitor, the extracellular medium alkalinization was not impacted for all treatment, suggesting that this event does not depend on Ca²⁺-mediated pathways (Supplementary Figure S1). However, the high oxidative burst and MAPK activity induced by S. anulatus S37 and/or B. cinerea were completely abolished by EGTA in grapevine cells, suggesting that both microorganisms trigger H₂O₂ production and MAPK activity through Ca²⁺ dependent pathways. Similar results have been reported by Vandelle et al. (2006), using elicitors derived from B. cinerea. The possible regulation of the oxidative burst and the MAPK activation by the Ca²⁺ influx could result from a stimulation of NADPH-oxidase, the major enzymatic source of ROS, together with activation of Ca²⁺-dependent MAPK.

The treatment with S37 induced the upregulation of genes that are responsive to B. cinerea (Bézier et al., 2002), MAMPs (Aziz et al., 2004), and beneficial bacteria (Gruau et al., 2015). These include genes encoding secondary metabolism (PAL, STS, LOX9, GST), and the glucanase (PR2). However, we observed that the expression of all selected genes was lower in S37-treated cells compared to those infected with B. cinerea. This suggests that S37, as for other beneficial bacteria, can initially be perceived as a potential invader, resulting in the activation of the plant immune system (Zamioudis and Pieterse, 2012). These results are consistent with the hypothesis that plants respond weakly to beneficial microbes or derivative MAMPs to avoid a strong activation of defense responses that could be detrimental to fitness (Van der Ent et al., 2009). However, even the level of gene expression was low during the S37-grapevine cell interaction, it emphasizes the involvement of JA and SA signaling pathways upon the S37 perception. This was supported by an enhanced expression of LOX9, as a key element of the oxylipin synthesis, and an induced expression of PR-2 (Gluc) and GST genes, which were found to be induced by SA in grapevine leaves (Dufour et al., 2013; Gauthier et al., 2014). The expression of these genes as JA- or SA-dependent responses was also responsive to Pseudomonas fluorescens PTA-CT2 and Burkholderia phytofirmans PsJN and as beneficial bacteria in grapevine tissues (Gruau et al., 2015; Miotto-Vilanova et al., 2016).

Our results show that grapevine cells infected with the B. cinerea exhibited similar early responses to those induced by the beneficial bacterium S. anulatus S37, except that the pathogen triggered the programmed cell death. This suggests the existence of some similarities and differences in signaling pathways involved in the recognition of beneficial and pathogenic microbes. However, the triggered cell death by B. cinerea might be a capital mechanism in the infection process with this necrotrophic pathogen (Gruau et al., 2015). As S37 and B. cinerea induced the same pattern for early events, we further performed desensitization assays using extracellular pH. We demonstrated that once increased by S. anulatus S37, the grapevine cells became refractory to further stimulation by B. cinerea, and inversely. This suggests a different mode of perception of the beneficial and pathogenic microbes by grapevine cells. Interestingly, upon a challenge with B. cinerea, S. anulatus S37 primed grapevine cells for enhanced defense reactions with a decline in the cell death. A higher production of H₂O₂ and an enhanced extracellular alkalinization were observed in S37-treated grapevine cells once challenged with B. cinerea. Since in this study S37 or B. cinerea do not produce H₂O₂ (data not shown), we might suggest that S37 primes the oxidative burst in grapevine cells as evident by the enhanced accumulation of H₂O₂ after pathogen challenge. B. cinerea itself has been shown to produce H₂O₂ in germinating conidia during the early steps of tissue infection (Heller and Tudzynski, 2011; Viefhues et al., 2014), or in response to CaCl₂ exposure (Marschall and Tudzynski, 2016). However, the priming state of grapevine cells to produce more H₂O₂ without direct contribution of S37 or B. cinerea is consistent with our previous study showing that the bacterium S37 exerts an antifungal effect on B. cinerea by destructing its mycelium (Couillerot et al., 2014). In meanwhile, it cannot be excluded that the magnitude of the burst of H₂O₂ could be ascribed to different PAMP/MAMP compounds released by both the bacterium (Van Loon et al., 2008) and B. cinerea (Vandelle et al., 2006) in the medium. Furthermore, the time course of H₂O₂ burst is more in agreement with the timeline observed in grapevine cell cultures, but not in B. cinerea, after treatment with different MAMPs (Aziz et al., 2007), in which this response was linked to the expression of defense-related genes and development of leaf resistance against B. cinerea.

Alternatively, the low contribution of B. cinerea, if any, to the enhanced oxidative burst, could be due the low virulence of the strain used in this study. Indeed, it has been shown that ROS production by B. cinerea is an important component of its virulence, and increased levels of ROS in plant cells may contribute to host cell death and favors fungal infection (Heller and Tudzynski, 2011; Marschall and Tudzynski, 2016).

The level of activation of the 45 and 49 kDa MAP kinases was also maintained at high level in S37-treated cells after B. cinerea challenge. We may suggest, the MAP kinase activity is already at it maximum and no more activation was observed after subsequent exposure to pathogen. In accordance with our results, it has been reported that, in Arabidopsis, MP3 kinase was not primed, but rather linked to direct responses to pathogen infection and MAMP signaling (Nakagami et al., 2005). This is in line with the fact that both oxidative burst and activation of MAPK were considered as possible regulators of defense responses (Aziz et al., 2004; Beckers et al., 2009).

Several reports have suggested that most beneficial bacteria primed plants for the activation of various cellular defense responses upon the pathogen attack (Conrath et al., 2006; Van der Ent et al., 2009; Gruau et al., 2015). In this study, we further showed that the relative expression of some defense-related genes was upregulated by S37 after B. cinerea challenge. The expression of LOX9 gene was almost 20 times higher in primed cells than in B. cinerea-infected ones. A priming effect was also seen with the expression of PAL, STS, and GST genes, while the basic glucanase (PR2) was not primed by the S37. Interestingly, the primed PAL and STS expressions were correlated with the S37-enhanced phytoalexin accumulation after B. cinerea challenge. These results are consistent with other previous researches showing that beneficial bacteria prime grapevine cells and leaves for an accelerated and an enhanced capacity to activate defense responses (Verhagen et al., 2011; Gruau et al., 2015), such as the rapid accumulation of hydrogen peroxide and phytoalexins as well as the activation of some defense-related genes upon the B. cinerea infection. Engineering the STS into plants of interest resulted in resveratrol accumulation and elevated pathogen resistance (Jeandet et al., 2013). A slight induction of stilbenic phytoalexins, resveratrol and its metabolic products, the glycoside piceid and the oxidized dimer δ-viniferin was observed after perception of S37, while only amount of δ-viniferin accumulated was greater after B. cinerea challenge. Similar results were observed in grapevine plants treated with P. fluorescens PTA-CT2 and Pantoea agglomerans PTA-AF2, which showed enhanced resistance to B. cinerea (Aziz et al., 2016). The accumulation of δ-viniferin in S37-treated cells indicated that this oligomer could be a possible marker for induced resistance to gray mold.

Overall, this study demonstrated for the first time that S. anulatus S37 induced a rapid and transient generation of H₂O₂, extracellular alkalinization and an activation of two MAPKs followed by a differential expression of some defense-related genes and a phytoalexin accumulation to lesser amounts, but not the programmed cell death. Interestingly, most of these defense responses were primed by the S37 after the pathogen challenge, with a decline in the cell death. Desensitization assays using the extracellular pH showed that once increased by the S. anulatus S37, cells became refractory to further stimulation by the B. cinerea, suggesting that grapevine cells distinctly perceived beneficial and pathogenic microbes.