Calcium- and ROS-mediated defence responses in BY2 tobacco cells by nonpathogenic Streptomyces sp

Authors


François Bouteau, LEM, Institut de Biologie des Plantes, Bât. 630, 91405 Orsay Cedex, France. E-mail: francois.bouteau@univ-paris-diderot.fr

Abstract

Aims:  The early molecular events underlying the elicitation of plant defence reactions by Gram-positive bacteria are relatively unknown. In plants, calcium and reactive oxygen species are commonly involved as cellular messengers of a wide range of biotic stimuli from pathogenic to symbiotic bacteria. In the present work, we checked whether nonpathogenic Streptomyces sp. strains could induce early signalling events leading to defence responses in BY2 tobacco cell suspensions.

Methods and Results:  We have demonstrated that nonpathogenic Streptomyces sp. OE7 strain induced a cytosolic Ca2+ increase and a biphasic oxidative burst in the upstream signalling events, leading to defence responses in BY2 tobacco cell suspensions. Streptomyces sp. OE7 also elicited delayed intracellular free scopoletin production and programmed cell death. In agreement with scopoletin production, OE7 induced accumulation of PAL transcripts and increased accumulation of transcripts of EREBP1 and AOX genes that are known to be regulated by the jasmonate/ethylene pathway. Transcript levels of PR1b and NIMIN2α, both salicylic acid pathway–linked genes, were not modified. Moreover, Streptomyces sp. OE7 culture filtrates could reduce Pectobacterium carotovorum- and Pectobacterium atrosepticum-induced death of BY2 cells and soft rot on potato slices.

Conclusions:  New insights are thus provided into the interaction mechanisms between Streptomyces sp. and plants; Streptomyces sp. could be sensed by plant cells, and through cytosolic Ca2+ changes and the generation of reactive oxygen species, defence responses were induced.

Significance and Impact of the Study:  These induced defence responses appeared to participate in attenuating Pectobacterium-induced diseases in plants. Thus, Streptomyces sp. OE7 could be a biocontrol agent against Pectobacterium sp.

Introduction

Streptomyces is one of the most important and versatile genera of actinobacteria that live in a wide spectrum of ecological niches (Karlovsky et al. 2008). In soil, Streptomyces spp. are able to colonize the rhizosphere and to interact with other micro-organisms and plants (Barakate et al. 2002; Coombs and Franco 2003), and they can even penetrate plant tissues (Franco et al. 2007). They produce various molecules, including proteins, peptides, oligosaccharides and antibiotics, that are secreted and play an important role in the Streptomyces life cycle and their interaction with other organisms (Chater et al. 2010). Different studies have aimed to test the efficiency of Streptomyces sp. in the biological control against phytopathogens (El-Tarabily 2006; Errakhi et al. 2007). The impact of antagonistic Streptomyces sp. depends on multiple synergistic strategies, including a direct interaction with the pathogenic partner (antibiosis), as well as indirect mechanisms like the degradation of quorum-sensing signal molecules (Park et al. 2005). Streptomyces sp. can also induce systemic and localized resistance to pathogens and improve plant growth and metabolism (Conn et al. 2008; Lehr et al. 2008). The presence in the exudates of many molecules potentially acting as elicitors may explain the ability of these bacteria to activate systemic defence responses in plants (Lehr et al. 2008). However, there are only a few reports on the mechanisms by which plant cells perceive the secreted bacterial metabolites. During plant–microbe interactions, the exchange of molecular messages probably induces signalling pathways. Changes in cytosolic free calcium concentration ([Ca2+]cyt) and the production of reactive oxygen species (ROS) are well-known components of signal transduction pathways leading to the activation of plant defence responses like the hypersensitive response (HR), a form of programmed cell death (PCD) and phytoalexin synthesis (Dodd et al. 2010). To date, the mechanisms that allow plant cells to perceive the secreted metabolites of nonpathogenic Streptomyces sp. have not been investigated despite their putative importance for biocontrol and/or growth stimulation. We have used tobacco BY2 cell suspensions as a simplified model system offering several advantages to carry out the dissection of complex cellular responses (Navazio et al. 2007; Terta et al. 2010; Verhagen et al. 2010). Our results indicate that plant cells can selectively perceive secreted components of Streptomyces sp. and that they trigger early and late responses including specific [Ca2+]cyt changes, oxidative bursts, induction of defence response genes, scopoletin synthesis and PCD. These responses appear to participate in limiting the virulence of Pectobacterium carotovorum or Pectobacterium atrosepticum in suspension cells and the development of soft rot on potato tuber slices.

Materials and methods

Bacteria strains, growth conditions and preparation of culture filtrates

Streptomyces sp. strains OE7 and OE53 were isolated from Moroccan rhizospheric soils. The analysis of 16S rDNA sequences indicated that OE7 isolate shared 99–100% maximal sequence identity with Streptomyces griseus and Streptomyces ornatus and that OE53 exhibited 99% maximal sequence identity to Streptomyces puniceus and Streptomyces californicus (Baz et al. 2012). Phylogenetic trees also indicate that these strains do not belong to Streptomyces scabiei, the pathogenic agent of common scab in potato (Baz et al. 2012). Their antimicrobial activity was evaluated by growing 107 CFU of Pectobacterium strains in 100 ml of liquid nutritive broth supplemented with 1 ml of Streptomyces sp. culture filtrates (CF) on a rotary shaker (200 rev min−1) at 28°C for 14 h (Fig. 1). Actinobacteria were maintained as a spore suspension in 20% glycerol at −20°C. Each isolate was grown separately in 100 ml Bennett’s liquid medium (beef extract, 1 g l−1; glucose, 10 g l−1; peptone, 2 g l−1; and yeast extract, 1 g l−1) (Jones 1949) on a rotary shaker (250 rev min−1) at 30°C for 7 days. This medium was shown to promote antibiotic production by some actinobacteria (Badji et al. 2007).

Figure 1.

In vitro antimicrobial activity of culture filtrates from Streptomyces sp. strain OE7 and OE53 against (a) Pectobacterium atrosepticum (CFBP 5889) and (b) Pectobacterium carotovorum (CFBP 5890). (a): (inline image) OE53+P. atrosepticum; (inline image) OE7+P. atrosepticum and (inline image) P. atrosepticum. (b): (inline image) OE53+P. carotovorum; (inline image) OE7+P. carotovorum and (inline image) P. carotovorum.

Streptomyces species were co-cultured with the referenced strains (INRA Angers, France) P. carotovorum (CFBP 5890) and P. atrosepticum (CFBP 5889) by inoculation of 106 CFU ml−1 of each Pectobacterium strain in the end of exponential phase of Streptomyces sp. growth culture (5 days old) (Badji et al. 2007) to avoid the inhibition of the actinobacteria growth by a rapid growth of the pectobacteria using the nutrients. The co-cultures in Bennett’s liquid medium were grown at 30°C, at 250 rev min−1, in the dark, for 2 more days.

CFs were obtained from the different cultures after filtration (0·2 μm) and concentration to approximately 20-fold by roto-evaporation. The final dose of CF applied to cells corresponded to 10% of the cell culture volume used, thus never exceed a twofold concentration of the molecules secreted by the strains in the Bennet medium.

Plant cell culture conditions

Nicotiana tabacum BY2 cell suspensions were grown in Murashige and Skoog medium (Pauly et al. 2001) and maintained by weekly dilution (2/80). The cell suspension was agitated on a rotary shaker at 120 rev min−1 at 22 ± 2°C in the dark. All experiments were performed at 22 ± 2°C using the cells in log phase (6 days after subculturing).

Aequorin luminescence measurements

Cytoplasmic Ca2+ variations were recorded from BY2 cell suspension expressing the apoaequorin gene (Pauly et al. 2001). For Ca2+ measurement, aequorin was reconstituted by an overnight incubation of the cell suspension in Murashige and Skoog medium supplemented with 2·5 μmol l−1 native coelenterazine. Cell culture aliquots (450 μl in Murashige and Skoog medium) were transferred carefully to a luminometer glass tube, and luminescence was recorded continuously at 0·2-s intervals using a FB12-Berthold luminometer (Berthold Technologies, Bad Wildbad, Germany). Treatments were given by 50-μl injections containing the CF. At the end of each experiment, residual aequorin was discharged by the addition of 500 μl of a 1 mol l−1 CaCl2 solution dissolved in 100% methanol. The resulting luminescence was used to estimate the total amount of aequorin in each experiment. Calcium measurement was calibrated using the equation pCa = 0·332588(−logk) + 5·5593, where k is a rate constant equal to luminescence counts per second divided by total remaining counts (Pauly et al. 2001). To test the effects of each different pharmacological treatment, cell suspensions were pretreated for 15 min before the application of CF.

Quantification of H2O2 in the culture medium

H2O2 release into the culture medium was quantified by measuring the chemiluminescence of luminol reacting with H2O2 (Jabs et al. 1997; Bouizgarne et al. 2006). Briefly, 6 ml of the BY2 cell suspension was inoculated with CF alone or with the appropriate chemical effectors. Before each measurement, 200 μl of the cell culture was added prior to 5 μl luminol 1·1 mmol l−1 addition. Chemiluminescence measurements were taken at 30-min intervals using a FB12-Berthold luminometer (signal-integrating time, 0·2 s).

Cell viability

Cell viability was assayed using the vital dye Evans Blue (EB) (Errakhi et al. 2008). Five hundred microlitres of BY2 cell suspension treated with the different filtrates during different time periods was incubated for 10 min in EB to a final concentration of 0·001% in 0·1 mol l−1 phosphate buffer, pH 7. Cells that accumulated EB were considered dead. Five hundred cells were counted for each infection/treatment. All experiments were repeated at least three times.

Dosage of scopoletin

Cells (0·5 g) were ground to a fine powder in liquid nitrogen and extracted twice with 1 ml 90% methanol. Aqueous methanol was removed by Speed-Vac and the dried residue dissolved in 120 μl of sodium acetate/acetonitrile (90 : 10; v/v) before HPLC analysis. 4-Methylumbelliferone (1 nmol) was added to each sample before extraction as an internal standard. HPLC analysis was performed on a C18 Nova Pak column (Waters, Oshawa, Ontario, Canada), using a gradient of CH3CN (A) in 20 mmol l−1 sodium acetate, pH 5, at a flow rate of 1 ml min−1. The gradient was 5–22% (A) for 35 min and then 22–80% (A) for 1 min. Scopoletin was detected by fluorescence (Vex 290 nm, Vem 402 nm). Identification of scopoletin was based on co-chromatography with an authentic standard coupled to a photodiode array detector (maxplot between 230 and 400 nm; Waters Millenium software). Scopoletin was quantified by comparison with the reference compounds.

The direct action of scopoletin on Pectobacterium viability was evaluated on microwells containing 4 μl of scopoletin dissolved in DMSO and inoculated with 196 μl of bacteria suspension in LB broth. The inoculated plates were incubated at 28°C for 24 h. All the tests were performed in triplicate.

RT-PCR analysis of gene expression

Six-day-old cells were treated with OE7 CF, harvested and frozen in liquid nitrogen. Total RNA was prepared using Trizol reagent. Total RNA was quantified by spectrophotometry, and their integrity checked on denaturing 1·2% agarose gels. Total RNA (2 μg) was converted into first-strand cDNA with the SuperscriptTM II Rnase H Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) and oligo(dT) primers. One microlitre of cDNA was amplified in a 25-μl PCR mixture with gene-specific primers for PR1b (Menard et al. 2004), PAL, AOX, EREBP1, NIMIN2α and ACT (Koshiba et al. 2010) (Table 1). Control PCR was performed using the housekeeping gene ACT. Thermal cycling conditions were as follows: an initial denaturation step at 94°C for 2 min, followed by 34 cycles (or by 35 cycles for ACT), of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, and ending with a single step at 72°C for 10 min. PCR products were separated by gel electrophoresis and visualized by ethidium bromide fluorescence. Representative results from two independent experiments are shown.

Table 1.   Primers used for semi-quantitative RT-PCR analyses
Gene nameAccession no.Primers used
PALAB0081995′-GCACAAAATGGTCACCAAGAAA-3′
5′-AAGCCATTGGGGCGACGTTCTA-3′
NIMIN2αAF0573795′-AGGCATGAGAGAAAAAGTGC-3′
5′-TAACCTCCAGGATCTTCACC-3′
EREBP1AF0573735′-GGAAATTGTGGTTTCTCCAG-3′
5′-AGCTGAACTATTTTCCGACG-3′
AOXS713355′-CACCAATGATGTTGGAAACAGTG-3′
5′-ATACCCAATTGGTGCTGGAG-3′
PR1bD901975′-GATGCCCATAACACAGCTCG-3′
5′-TTTACAGATCCAGTTCTTCAGAGG-3′
ACTAB1586125′-AAGGTTACGCCCTTCCTCAT-3′
5′-GCCACCACCTTGATCTTCAT-3′

Post-OE7 phytobacterial challenge on tobacco BY2 cells

Tobacco BY2 cells were pretreated with OE7 CF for 0, 2 and 6 h. After pretreatment, cells were rinsed three times with culture medium to eliminate OE7 CF before inoculation with 106 CFU ml−1 of P. carotovorum CFBP 5890 or P. atrosepticum CFBP 5889. Cell viability was monitored as described earlier.

Post-OE7 phytobacterial challenge on potato slices

Fresh potato (cv. Desirée) tubers were washed and chopped into equal slices with a sterile borer. The inoculation of potato slices with either P. carotovorum (106 CFU ml−1) or P. atrosepticum (106 CFU ml−1) was carried out 24 h after application of Streptomyces sp. CF. Experiments were repeated five times.

Statistical analysis

Data were analysed by analysis of variance (anova), and the mean separation was achieved by Newman and Keuls multiple range test. All numerical differences in the data were considered significantly different at the probability level of ≤ 0·05.

Results

Streptomyces sp. can activate Ca2+-mediated signalling in BY2 cells

Variations in [Ca2+]cyt are a well-known early component of signal transduction pathways involved in plant–microbe interactions (Lecourieux et al. 2006). To measure [Ca2+]cyt changes, we used transgenic BY2 cells containing the bioluminescent Ca2+ indicator aequorin protein in the cytosol (Pauly et al. 2001). Two Streptomyces sp. strains OE7 and OE53, isolated from Moroccan rhizospheric soils, were chosen as (i) they are nonpathogenic (Baz et al. 2012) and (ii) they secrete different metabolite profiles as judged by their high (strain OE7) and low (strain OE53) antimicrobial activities against P. atrosepticum development in vitro (Fig. 1a) (Baz et al. 2012). Both strains also counteracted P. carotovorum development but to a lower extent (Fig. 1b). OE7 and OE53 CF were directly applied to BY2 cells as previously described for in vivo bioassays of physiological effects of Trichoderma metabolite mixtures on plant cells (Navazio et al. 2007). OE7 and OE53 CF triggered an immediate slight increase in [Ca2+]cyt as did the Bennet’s medium used as a control (Fig. 2a). Interestingly, after 5 min, only OE7 CF had induced a significant increase in [Ca2+]cyt (Fig. 2a–d) that was sustained during at least 25 min (data not shown). This [Ca2+]cyt increase could be abolished by either La3+, a plasma membrane Ca2+ channel inhibitor, or the Ca2+ chelator BAPTA (Fig. 2b,d), suggesting that OE7 CF induced an influx of Ca2+ through the plasma membrane. To determine whether the Ca2+ increase induced by OE7 CF could be modified when the CF came from the strain grown in the presence of the pathogens P. carotovorum or P. atrosepticum, we compared CFs produced by the OE7 strain grown alone or in co-culture with these pathogenic bacteria. Culture supernatants of Pectobacteria alone did not induce a significant increase in [Ca2+]cyt (Fig. 2c,d), while the CF produced during the co-cultures of OE7 and Pectobacteria could induce a [Ca2+]cyt increase in the BY2 cells, albeit to a slightly decreased extent to OE7 alone (Fig. 2c,d). Such observations indicate that the synthesis of the OE7 CF component(s) that induces the [Ca2+]cyt increase was not avoided in the presence of the Pectobacteria.

Figure 2.

 Time course of [Ca2+]cyt changes in BY2 cells challenged with Streptomyces sp. culture filtrates. (a) Cells transformed by the apoaequorin gene were treated with CFs of Streptomyces sp. strains OE7 and OE53. Noninoculated Bennet’s culture medium was used as a control. (b) Effect of pretreatment with the Ca2+ channel inhibitor La3+ (500 μmol l−1) or the Ca2+ chelator BAPTA (1 mmol l−1) on OE7 CF-induced [Ca2+]cyt changes. (c) [Ca2+]cyt changes induced by CF from OE7 strain cultured alone or in the presence of Pectobacterium carotovorum CFBP 5890 (Pc) or Pectobacterium atrosepticum CFBP 5889 (Pa). CFs of Pc and Pa are given as controls. (d) Means values of [Ca2+]cyt 15 min after the different treatments. Data are representative of at least five independent repetitions. Bars with different superscript letters are significantly different.

Streptomyces sp. can elicit oxidative bursts in BY2 cells

One of the most common plant responses to micro-organisms is an increased production of ROS (Shetty et al. 2008). Therefore, the ability of Streptomyces CFs to elicit H2O2 production in BY2 cells was investigated. The BY2 cells treated with OE7 CF showed a biphasic increase in luminol-mediated chemiluminescence caused by H2O2 release into the culture medium (Fig. 3a). A first oxidative burst peaked after 30 min, followed by a larger one that reached a maximum at around 3 h. Both returned to the control level after 1·5 and 5 h, respectively. This H2O2 production was blocked by the inline image scavenger tiron (5 mmol l−1) and the NADPH oxidase inhibitor DPI (10 μmol l−1), suggesting that plasma membrane NADPH oxidases were involved in the H2O2 production (Fig. 3a,c). The attenuation of the oxidative bursts by either La3+ or BAPTA, which limit Ca2+ influx, suggested that H2O2 generation occurred downstream from the CF-induced Ca2+ changes (Fig. 3b,c). Cell suspensions treated with the OE53 CF showed no significant increase in external H2O2 levels during the time course of the experiments (Fig. 3b,c), in accordance with the nonsignificant [Ca2+]cyt variations attributed to the OE53 CF treatment (Fig. 2a).

Figure 3.

 Time course of H2O2 accumulation in BY2 cells challenged with Streptomyces sp. culture filtrates. (a) H2O2 accumulation in the cell culture medium treated with OE7 CF with or without the NADPH oxidase inhibitor DPI (10 μmol l−1) or the inline image scavenger tiron (5 mmol l−1). (b) H2O2 accumulation in the cell culture medium treated with OE7 CF with or without the Ca2+ channel inhibitor La3+ (500 μmol l−1) or the calcium chelator BAPTA (1 mmol l−1), and H2O2 accumulation in the cell culture medium treated with OE53 CF. (c) Means values of H2O2 generation after 30 min and 3 h for the different treatments. Noninoculated Bennet’s culture medium was used as a control. The data correspond to the means of five replicates, and error bars correspond to SD. (□) 30 min and (inline image) 3 h.

OE7-induced programmed cell death of BY2

Both intracellular Ca2+ overload and H2O2 release may cause PCD (Hoeberichts and Woltering 2003). Because OE7 CF induced [Ca2+]cyt and ROS increases, its effect on cell viability was determined. Based on EB staining, OE7 CF significantly increased the percentage of dead cells after 24 h. On the other hand, while CFs of P. carotovorum and P. atrosepticum did not induce cell death (Fig. 4a), CF produced by the OE7 strain grown in co-culture with these bacteria also induced cell death, confirming that the synthesis of the CF component(s) required to induce cell death was secreted even in the presence of the pathogenic bacteria (Fig. 4a). This cell death was accompanied by a strong cell plasmolysis (Fig. 4b), a hallmark of PCD (van Doorn et al. 2011). To check whether the induction of cell death was an active mechanism requiring gene expression and cellular metabolism as expected during PCD (Lam 2004), BY2 cell suspensions were pretreated either with actinomycin D (AD), an inhibitor of RNA synthesis, or with cycloheximide (Chx), an inhibitor of protein synthesis. Although the pretreatment of BY2 cells with these inhibitors resulted in a slight increase of cell death compared with the control, AD and Chx reduced OE7 CF-induced cell death by about 50% (Fig. 4c), thus indicating that active cell metabolism, namely gene transcription and de novo protein synthesis, was required, suggesting a PCD process (Vacca et al. 2004; Kadono et al. 2010). In addition, the effect of OE7 on cell death in the presence or the absence of La3+ or BAPTA was tested. The pretreatment of BY2 cells with either La3+ or BAPTA caused a slight increase in cell death compared with the control. However, both treatments reduced the cell death induced by the CF of OE7 (Fig. 4d). In the same way, OE7-induced cell death was also decreased in the presence of tiron (5 mmol l−1) (Fig. 4d). Taken together, these data strongly suggest that OE7 induces PCD as a defence response and that Ca2+ influx and ROS generation are upstream events in the signalling pathway leading to this process.

Figure 4.

 Changes in BY2 cell viability in response to Streptomyces sp. culture filtrates (CFs). Exponentially growing cells were incubated with Streptomyces sp. CFs for 24 h and stained with Evans Blue (EB). (a) Extent of cell death for cells treated with CF from OE7 and OE53 cultured alone or co-cultured with P. carotovorum (Pc) or P. atrospeticum (Pa). Control cells were incubated with Bennet’s culture medium only (b). Light micrographs of BY2 cells stained with EB 24 h after treatment with Streptomyces sp. strain OE7 CF. (c) Effect of pretreatment with actinomycin D (AD, 20 mg ml−1) or cycloheximide (Chx, 20 mg ml−1) on OE7-induced cell death. (d) Effect of a pretreatment with La3+ (500 μmol l−1), BAPTA (1 mmol l−1) or tiron (5 mmol l−1) on OE7-induced cell death. The data correspond to the means of at least three replicates, and error bars correspond to standard errors. Bars labelled with a different letter are significantly different. (inline image) 5 h and (inline image) 24 h.

OE7 induced an accumulation of scopoletin in BY2 cells

The generation of ROS and [Ca2+]cyt variations is also known to play a role in the HR, a form of PCD associated with the induction of phytoalexins like scopoletin (Greenberg 1997). Therefore, the induction of scopoletin synthesis in response to OE7 was tested using the BY2 cell system. The levels of free intracellular scopoletin in BY2 cells were determined after 0, 2, 4, 6 and 14 h of OE7 CF treatment. Scopoletin levels were low during the first 6 h post-treatment, but significantly increased to about 0·5 nmol g−1 of fresh cells after 14 h (Fig. 5a). Coumarins, like scopoletin, are known to be effective against Gram-negative bacteria (Cespedes et al. 2006); therefore, the action of scopoletin on P. carotovorum and P. atrosepticum growth was checked. Scopoletin effectively inhibited the growth of these Pectobacteria in a dose-dependent manner after 24 h (Fig. 5b).

Figure 5.

 (a) Levels of free intracellular scopoletin in BY2 cells elicited by OE7 CF. The data correspond to the means of 3 replicates, and standard errors are given. Bars labelled with a different letter are significantly different. (b) Effect of scopoletin on the growth of Pectobacterium carotovorum CFBP 5890 (Pc) or Pectobacterium atrosepticum CFBP 5889 (Pa) after 24 h. The culture was inoculated with 106 CFU bacteria before treatment. (□) P. carotovorum and (inline image) P. atrosepticum.

OE7-induced expression of defence response genes in BY2 cells

Defence responses are often concomitant with an increase in the expression of defence-related genes. We analysed by RT-PCR the accumulation of several gene transcripts in response to OE7 CF: PAL, which encodes phenyl ammonia-lyase, a key enzyme of the phenylpropanoid biosynthetic pathway; PR1b (pathogenesis-related) and NIMIN2α involved in the salicylic acid (SA) defence signalling pathways (Glocova et al. 2005); and EREBP1 and AOX involved in the ethylene (ET) defence signalling pathways (Chang and Shockey 1999; Ederli et al. 2006). As expected from the production of scopoletin by the BY2 cells, an increase in PAL mRNA levels was observed after a 14-h OE7 treatment. An increase in mRNA levels was also detected for EREBP1 and AOX (Fig. 6). A constitutive expression was observed for PR1b and NIMIN2α because OE7 treatment did not increase the transcript levels of these genes (Fig. 6).

Figure 6.

 Effect of a 14-h treatment with OE7 CF on the expression of defence-related genes (PAL, PR1b, NIMIN2α, EREBP1 and AOX). ACT, actin was used as a constitutive control.

OE7 CF protected BY2 cells against Pectobacterium spp.

We recently demonstrated that suspension cells could be an alternative tool to evaluate rapidly and efficiently the virulence of different P. carotovorum strains (Terta et al. 2010). We thus checked whether the OE7-induced defence responses protected cells from a P. carotovorum or P. atrosepticum postchallenge. To avoid any direct effect of OE7 on Pectobacterium growth, BY2 cells were rinsed three times with culture medium to remove the OE7 CF before performing the postchallenge. In these conditions, BY2 cell death caused by P. carotovorum and P. atrosepticum was significantly reduced when the cells were pretreated with OE7 CF (Fig. 7a), suggesting that the OE7-induced defence responses participated in limiting Pectobacterium virulence.

Figure 7.

 (a) Effect of pretreatment with OE7 CF on cell death induced by Pectobacterium carotovorum CFBP 5890 (Pc) or Pectobacterium atrosepticum CFBP 5889 (Pa) on BY2 cells after 24 h. (b) Mean rot weight 48h after inoculation of tubers slices of potato cv. Desirée with P. carotovorum (Pc) or P. atrosepticum (Pa) treated or not with OE7 CF. Control tubers treated with OE7 CF alone displayed no rot symptoms. Bars with different superscript letters are significantly different. (inline image) P. atrosepticum and (inline image) P. carotovorum.

OE7 CF protected potatoes from Pectobacterium-induced soft rot symptoms

To determine whether the elicitation of defence responses also had an effect on Pectobacterium-induced soft rot symptoms on potato, tuber slices were pretreated with OE7 CF before challenging them with the potato rot pathogens, P. carotovorum and P. atrosepticum. Tissue rot in tubers inoculated with P. carotovorum and P. atrosepticum was lower in OE7 CF-treated tubers compared with those treated only with either P. carotovorum or P. atrosepticum (Fig. 7b).

Discussion

The aim of this study was to investigate the metabolic events induced when plant cells perceive rhizospheric nonpathogenic Streptomyces sp. One of the most direct ways to have an overview of the complex reactions and effects caused by Streptomyces sp. in plants is to assay the impact of the metabolic mixture contained in the CF on markers linked to plant defence such as variations in [Ca2+]cyt, ROS generation, phytolaexin production and changes in defence-related gene expression. Because a simplified model offers several advantages in the dissection of such complex cellular responses (Navazio et al. 2007; Terta et al. 2010), tobacco BY2 cell suspensions challenged with actinobacteria CFs were used as a simplified experimental model. Possible modifications in the pattern of the secreted molecules were analysed by testing the effects of CFs from Streptomyces sp. strains co-cultured with the phytopathogenic P. carotovorum and P. atrosepticum. Our approach allowed to highlight a sustained influx of Ca2+ as an early step during the interaction of BY2 cells with OE7 CF. Although the role of Ca2+ in plant defence signalling is firmly established (Lecourieux et al. 2006), to our knowledge, this is the first report of Ca2+ influx in plant cells induced by nonpathogenic Streptomyces sp. In the same way, a biphasic oxidative burst reminiscent of the oxidative burst associated with incompatible plant–pathogen interactions (Levine et al. 1994) and induced in sugar beet by the biological control agent Bacillus mycoides (Bargabus et al. 2003) was also observed in response to the OE7 CF. The attenuation of this oxidative burst by the inline image scavenger tiron and the NADPH oxidase inhibitor DPI suggested that inline image was generated via an NADPH oxidase activity. It should be noted that OE7-induced ROS generation was dependent on Ca2+ influx and that the CF from the less-efficient OE53 strain failed to induce [Ca2+]cyt variations and ROS generation. Calcium influx and ROS generation are frequently linked to plant–pathogen responses (Ishihara et al. 1996; Jabs et al. 1997; Able et al. 2001; Errakhi et al. 2008), and they are required to induce defence-related genes, to stimulate phytoalexin biosynthesis and to promote hypersensitive cell death (Low and Merida 1996). Indeed, it was found that OE7 induced the accumulation of transcripts coding for PAL, a key enzyme of the phenylpropanoid pathway, and the synthesis of the scopoletin, a phytoalexin derived from l-phenylalanine via this pathway (Dorey et al. 1997; Chong et al. 2002). Scopoletin displays well-recognized antimicrobial activities against various plant pathogens (Chong et al. 2002; Lerat et al. 2009), and we found that scopoletin, known to be efficient against Gram-negative bacteria (Cespedes et al. 2006), inhibited Pectobacterium growth in vitro. OE7 CF also induced BY2 cell death, which fulfilled the widely accepted criteria for PCD, that is, cell plasmolysis and the requirement of active transcription and translation processes (Lam 2004; van Doorn et al. 2011). This PCD, dependent on Ca2+ influx and ROS generation, could consist in a defence response. In accordance with the Ca2+ and ROS dependence of the OE7 CF-induced cell death, the CF from the weakly efficient OE53 strain failed to induce cell death in BY2 cells. However, the OE7 CF-induced cell death concerns <30% of the cells, suggesting that the signalling pathway leading to this PCD was not induced in all cells. Our BY2 cell cultures being not synchronized and in log phase, the PCD induced by OE7 CF could be due to a cell cycle phase–dependent sensitivity as described for BY2 cell death induced by cryptogein (Kadota et al. 2004). It is noteworthy that in BY2 cells, the ROS generation was independent of the cell cycle (Kadota et al. 2004), indicating that defence response occurs in all cells independently of PCD. This suggests that cell death was only a part of cell response to cryptogein. The PCD induced by OE7 CF could reflect the ability of some BY2 cells to engage a specific signalling pathway in the network of defence responses.

In the control experiment, the genes PR1b and NIMIN2α, involved in the SA defence signalling pathways (Glocova et al. 2005), as well as the genes EREBP1 and AOX, known to be related to the defence signalling pathways induced by ET (Chang and Shockey 1999; Ederli et al. 2006), were already induced. This indicates that in our model system these pathways, known to interact in response to biotic but also abiotic stress (Wang et al. 2002), could be constitutively activated, at least to a basal level, probably due to culture conditions. However, no increase in the transcript levels of PR1b and NIMIN2α was observed in response to OE7 in BY2 cells, when accumulation of EREBP1 and AOX transcripts could be observed. Although it is thus difficult to conclude on the involvement of SA signalling pathways in response to OE7 CF in these conditions, our data suggest that OE7-secreted molecules could reinforce a pathway related to induced systemic resistance (ISR), because signal transduction leading to ISR requires responsiveness to ET and jasmonate rather than to SA (Yan et al. 2002; Choudhary and Johri 2009). This interpretation should be considered with caution and requires further investigation because, in addition to a possible basal activation of SA-related and ET/JA-related pathways, the OE7-induced responses were local reactions measured in suspension cells, whereas ISR is a systemic response. However, taken together, our data suggest that the OE7 strain can induce early and delayed defence responses in BY2.

To check whether these defence responses participate in protecting the cell against a pathogen attack, the impact of OE7 CF pretreatments on P. carotovorum- and P. atrosepticum-induced BY2 cell death was tested. Recently, we showed that cell death induced by these pathogens in cultured suspensions was reminiscent of the pathogen-induced effects on potatoes because this cell death involved pectinolytic enzymes that induced a complete degradation of the cell structures (Terta et al. 2010). Indeed, pretreatments with OE7 CF were efficient in reducing the extent of cell death induced by such pathogenic bacteria in BY2 cells. Thus, despite the fact that OE7 CF could induce a slight PCD in BY2 cell population, the global effect on pathogen challenge was positive because P. carotovorum- and P. atrosepticum-induced BY2 cell death was reduced by pretreatment with this CF. These data further indicate that suspension cells are a good model to study ‘in vitro biocontrol processes’. Resistance to pathogen attacks by crops is the ultimate objective of biocontrol experiments, and therefore, the reliability of our observations using suspension cells was evaluated by testing the ability of OE7 CF to limit the extent of soft rot symptoms on potato slices. As observed for suspension cells, OE7 CF could reduce the extent of P. carotovorum- and P. atrosepticum-induced symptoms on potato tubers. Thus, our data provide good evidence that, in addition to direct antagonism, OE7 CF participate in attenuating disease by elicitation of plant defence responses, thereby highlighting the potential of the nonpathogenic OE7 strain for biological control against Pectobacterium species. Although the Bennet medium used to grow the OE7 strain is supposed to promote the antibiotic production (Badji et al. 2007), further studies will be needed to determine (i) which compound(s) of the CF is responsible for the effects seen in the tobacco cell culture, (ii) whether the same compound(s) could reduce pathogen development and induce plant defence responses and (iii) finally, whether such compound(s) could effectively be produced when the Streptomyces strain colonizes the rhizosphere of the plant.

Acknowledgements

This work was supported by the Agronomic Research for Development Project PRAD no. 07-07 and the Moroccan Excellence grant no. E3/003. BY2 cells were kindly provided by C. Mazars (UMR CNRS-UPS, Toulouse). Authors thank M. Hodges and K. Massoud (IBP, Orsay) for critical reading of the manuscript.

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