# Deciphering the dual effect of lipopolysaccharides from plant pathogenic Pectobacterium

Mohamed Kettani-Halabi, Tran Daniel, Aurélien Dauphin, Hayat

El-Maarouf-Bouteau, Errakhi Rafik, Delphine Arbelet-Bonnin, Bernadette

Biligui, Florence Val, Ennaji Moulay Mustapha, François Bouteau

► To cite this version:

Mohamed Kettani-Halabi, Tran Daniel, Aurélien Dauphin, Hayat El-Maarouf-Bouteau, Errakhi Rafik, et al.. Deciphering the dual effect of lipopolysaccharides from plant pathogenic Pectobacterium. Plant Signaling and Behavior, 2015, 10 (3), pp.e1000160. 10.1080/15592324.2014.1000160. hal-03419719

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# Deciphering the dual effect of lipopolysaccharides from plant pathogenic *Pectobacterium*

Kettani-Halabi Mohamed<fnref n="1" />,², Tran Daniel<fnref n="1" />, Dauphin Aurélien³, Hayat El-Maarouf-Bouteau⁴, Errakhi Rafik<fnref n="1" />, Delphine Arbelet-Bonnin<fnref n="1" />, Bernadette Biligui<fnref n="1" />, Val Florence⁵, Ennaji Moulay Mustapha², and Bouteau François<fnref n="1" />,*

**Keywords:** defense responses, lipopolysaccharides, *Pectobacterium* spp, programmed cell death, reactive oxygen species

**Abbreviations:** AD, actinomycin D; Chx, cycloheximide; DPI, diphenylene iodonium; EB, Evans Blue; ETI, effector-triggered immunity; HR, hypersensitive response; LPS, lipopolysaccharides; MAMP, microbe associated molecular pattern; OPS, O-polysaccharide part; Pa, *Pectobacterium atrosepticum*; PAMP, pathogen- associated molecular pattern; Pcc, *Pectobacterium carotovorum carotovorum*; PCD, programmed cell death; PTI, PAMP triggered immunity; ROS, reactive oxygen species; Tiron, sodium 4, 5-dihydroxybenzene-1,3-disulfonate.

Lipopolysaccharides (LPS) are a component of the outer cell surface of almost all Gram-negative bacteria and play an essential role for bacterial growth and survival. Lipopolysaccharides represent typical microbe-associated molecular pattern (MAMP) molecules and have been reported to induce defense-related responses, including the expression of defense genes and the suppression of the hypersensitive response in plants. However, depending on their origin and the challenged plant, LPS were shown to have complex and different roles. In this study we showed that LPS from plant pathogens *Pectobacterium atrosepticum* and *Pectobacterium carotovorum* subsp. carotovorum induce common and different responses in *A. thaliana* cells when compared to those induced by LPS from non-phytopathogens *Escherichia coli* and *Pseudomonas aeruginosa*. Among common responses to both types of LPS are the transcription of defense genes and their ability to limit of cell death induced by *Pectobacterium carotovorum* subsp. carotovorum. However, the differential kinetics and amplitude in reactive oxygen species (ROS) generation seemed to regulate defense gene transcription and be determinant to induce programmed cell death in response to LPS from the plant pathogenic *Pectobacterium*. These data suggest that different signaling pathways could be activated by LPS in *A. thaliana* cells.

## Introduction

In the environment, potential pathogenic microorganisms constantly challenge plants. The phytopathogenic bacteria can either lead to diseases, which may cause great economic losses in crops or to a rapid activation of plant defense responses that contain or eliminate the pathogen.<fnref n="1" /> Activation of plant cell defenses occurs after perception of conserved microbe-specific molecular motifs, referred to microbe or pathogen-associated molecular patterns (MAMPs or PAMPs), which induce the innate immune system in plants. PAMPs that are similar to those activating innate immune responses in animals have also been shown to mediate the activation of plant defense.² These plant reactions constitute PAMP-triggered immunity (PTI).

MAMPs such as LPS, peptidoglycans, lipoteichoic acid, eubacterial flagellin, elongation factor Ef-Tu and unmethylated bacterial DNA fragments, are able to trigger PTI in various plant species.³⁻⁷

Among MAMPs, LPS is a major amphiphilic cell surface component of the outer cell surface of Gram-negative bacteria. It plays a major role for bacterial growth and survival.⁸,⁹ Structurally, LPS is a glycoconjugate, tripartite molecule, consisting of a hydrophobic lipid part (lipid A), a core oligosaccharide and an O-polysaccharide part (OPS).¹⁰,¹¹ Although, recognition of some MAMPs seemed to occur through pattern recognition receptors,¹²,¹³ LPS recognitions in plants remains little-known.¹⁴ Nevertheless, LPS from different bacterial species can trigger or potentiate PTI and several studies show their implication into the mechanism of plant defense activation.¹⁵⁻¹⁷ Lipopolysaccharides have been reported to induce an oxidative burst, NO production, calcium influx, alkalization of the extracellular medium, pathogenesis-related (PR) gene expression and cell wall alterations that include the deposition of callose and phenolics.¹⁴ In *Burkholderia cepacia* the lipid A and OPS moieties of the LPS were able to up-regulate sub-sets of defense-associated genes over the same spectrum of categories as intact LPS, the up-regulation

*Correspondence to: Bouteau F; Email: francois.bouteau@univ-paris-diderot.fr
Submitted: 12/10/2014; Accepted: 12/12/2014
http://dx.doi.org/10.1080/15592324.2014.1000160

<footnote n="1">¹Université Paris Diderot-Paris 7; Institut des Energies de Demain (UMR8236); Paris, France; ²LVHM-Université Hassan II Mohammed media - Casabalanca; FSTM, Maroc; ³ UPMC UMR-S975; Inserm U975; CNRS UMR 7225 Plateforme d'lmagerie cellulaire Pitié-Salpetrière; GH Pitié-Salpetrière; Paris, France; ⁴UPMC UMR7622-IBPS; Paris, France;
⁵AGROCAMPUS OUEST; UMR1349 IGEPP; Rennes, France</footnote>
observed with intact LPS was the more comprehensive, suggesting that the lipid A and glycan molecular patterns of the molecule act as partial agonists, but that the intact LPS structure is required for full agonist activity.¹⁸ These molecular and cellular events are frequently associated with the initiation of the hypersensitive response (HR), a form of programmed cell death (PCD).¹⁹ Programmed cell death is an active cellular process that facilitates the removal of unwanted or damaged cells. It is essential for tissue differentiation and plant development.²⁰ However, one of the effects of LPS described on plants is their ability to prevent HR in leaves.²¹ As discussed by Erbs and Newman¹⁴, these findings present a conundrum since both, basal resistance responses and the HR, are supposed to contribute to plant defense. Moreover, although LPS-induced PCD was never reported in dicots, it could induce PCD in monocots such as rice cells.²²

The LPS from the plant pathogen *Pectobacterium atrosepticum* (*Pa*) seems to be crucial not only for optimal growth and survival of the bacteria but for its virulence as well.<sup>9,23-25</sup> It was shown to induce different early responses such as acidification of the medium.<sup>26</sup> The fact that LPS induced variable cellular responses can be attributed to LPS preparations, plants and cell types used and reflect the ability of particular plants to recognize structural features within LPS that are not widely conserved.<sup>14,27</sup> This is reminiscent of what was observed in animals, since LPS administration could stimulate immune activation, inflammation and PCD (Nolan *et al.*, 2003; Aballay *et al.*, 2003; Becerra *et al.*, 2011).<sup>28-30</sup> Also, depending on animal model and cell type, LPS could acts as mechanism of protection and preventing cell death,<sup>31</sup> or virulence factors by favoring the escape from the innate immune system.<sup>32</sup> Thus, LPS could have complex and different roles in plants as well as in animals.

Among Gram-negative bacteria, LPS are effectively similar although not identical. Slight differences were observed for the structures of lipid A between *Pectobacterium spp* and *E. coli*<sup>33,34</sup> or those of the core oligosaccharide, such as phosphorylation or substitution of a disaccharide.<sup>35,36</sup> Heterogeneity ascribed to LPS structure diversity was also observed at the species level for *P. carotovorum*.<sup>25</sup> Variations in sugar composition of the OPS from *Pectobacterium* species was used for differentiation into serogroup<sup>37</sup> while the OPS from *P. atrosepticum* present a specific higher branched monosaccharide.<sup>38</sup>

In this study, using *A. thaliana* cell suspensions as a host model system,<sup>39-40</sup> we compared the effects of LPS from phytopathogenic bacteria, *i.e.* *Pectobacterium carotovorum subsp carotovorum (Pcc)* and *Pa*, and non-phytopathogenic bacteria *i.e.*, *Pseudomonas aeruginosa* and *Escherichia coli*, on early cellular responses classically associated with defense responses and PCD. LPS from plant pathogenic and non-pathogenic bacteria share some defense responses, but only LPS from plant pathogenic bacteria could induce a PCD depending on an early ROS production.

# Materials and Methods

## Plant cell culture conditions

*Arabidopsis thaliana* L. (ecotype Columbia) cells were cultured at 24 ± 2°C, under continuous white light (40 µE.m⁻².s⁻¹) with continuous rotation shaking (gyratory shaker) at 120 rpm,

in a 1 L round bottom flask containing 300 mL Gamborg culture medium at pH 5.8 supplemented with 30 g.L⁻¹ sucrose and with 200 µg.L⁻¹ α-naphthalene acetic acid.<sup>41,42</sup> Cells were transferred to fresh medium every 7 d by a 1/10 dilution. The experiments were conducted using the cells in log-phase (4 d after sub-culturing).

## Bacterial strains and culture conditions

*Pectobacterium atrosepticum* (*Pa*, CFPB 5889) and *Pectobacterium carotovorum subsp carotovorum* (*Pcc*, CFBP 5990) were grown for 24 h at 20°C in the dark, in agitated liquid King B medium. Cells were collected by centrifugation (15 min, 10 000 g), washed 3 times in PBS (phosphate buffer saline buffer), and lyophilized before LPS extraction.

## LPS extraction and purification from Pa and Pcc

LPS preparations from *Pa* (LPS<sub>Pa</sub>) and *Pcc* (LPS<sub>Pcc</sub>) were extracted using the hot phenol-water method of Westphal and Jann<sup>43</sup> with some modifications.<sup>26</sup> One gram of lyophilized bacteria was suspended in 100 ml of cold distilled water and sonicated for 20 min in hot water at 65°C, before adding 100 ml of cold phenol 90 % (w/v). After a centrifugation (16 000 g, 45 min), the water phase was collected. The phenol phase was treated again 2 times with 100 ml of cold distilled water, as described above. The three water phase extracts were combined, dialyzed against water and lyophilized. Proteins and nucleic acids were removed from 250 mg of crude LPS in 20 mL of distilled water by enzymatic treatment with DNase I, RNase, and proteinase K, each at 100 µg.mL⁻¹ final concentrations, before the extract was dialyzed and lyophilized again. The LPS were purified by gel filtration chromatography through a Sephacryl S 400 HR column (600 × 16 mm, 120 ml volume; Amersham Bioscience), with 30 mM triethylamine/10 mM EDTA (pH 7) as eluent. The chromatography products were collected as successive fractions of 5 mL. Concentrations of total carbohydrates<sup>44</sup> and nucleic acids were obtained from reading optical densities at 260 nm on a spectrophotometer. An assessment of total proteins<sup>45</sup> in the final preparation confirmed that this preparation contained no protein. The presence of LPS was identified by determining the amount of KDO.<sup>46</sup> All LPS-containing fractions were pooled, dialyzed against distilled water, lyophilized, and tested for purity using the carbocyanine assay.<sup>47</sup> To confirm the presence of purified LPS a carbocyanine assay and SDS-PAGE electrophoresis was performed on the pooled fraction as described by Desender et al.<sup>26</sup> LPS preparations were analyzed by SDS-PAGE in slab gels (4% polyacrylamide in stacking gel and 14% polyacrylamide in resolving gel) with the buffer system described by Laemmli.<sup>48</sup> After electrophoresis LPS were stained with silver nitrate<sup>49</sup> typical bands indicate the repeating units of oligosaccharide in the O antigen structure and the lipid A linked to the oligosaccharidic core (Fig. S1).

## Commercially available LPS

Two types of LPS from *E. coli* (*LPS*<sub>*E. coli*</sub>) serotype O111:B4 purchased from Sigma were tested: a preparation purified by gel filtration and chromatography after phenol extraction (product
number L 3012) and a preparation obtained by simple phenol
extraction (product number L 2630). The LPS from *Pseudomonas
aeruginosa* (*LPS*<sub>P. aeruginosa</sub>) serotype 10 purchased from Sigma
was purified by phenol extraction (product number L9143).

Cell death quantification

Cell viability was assayed using the vital dye Evans Blue.⁴⁰ An *A. thaliana* cell suspension was incubated (24 h) in their culture medium in presence of different concentrations of LPS with or without the appropriate pharmacological effectors. Then, cells (500 µL) were incubated for 10 min in 1 mL phosphate buffer (0.1 M - pH 7) supplemented with Evans Blue to a final concentration of 0.001%. Cells accumulating Evans Blue were considered dead. At least 500 cells were counted for each independent treatment and repeated at least 3 times for each condition.

Detection and quantification of reactive oxygen species
in the culture medium

Quantification of ROS in the culture medium was performed by measuring the chemiluminescence of luminol reacting with ROS.⁴¹ *A. thaliana* cell suspension was inoculated with LPS alone or with the appropriate chemical effectors. For each measurement, 2 µL of luminol (500 µM) were added in 200 µL of the cell culture. Chemiluminescence measurements were carried out at 30 min intervals using a FB12-Berthold luminometer (sig- nal integrating time 0.2 s).

Confocal analysis

For confocal laser-scanning microscopy, 500 µL of cell sus-
pension was treated or not with 100 µg.mL⁻¹ LPS and 15 min
before observation with 5 µM 5-(and-6)-chloromethyl-2',7'-
dichlorodihydrofluorescein diacetate, acetyl ester (CM-
H₂DCFDA – Invitrogen) a probe allowing ROS detection.⁵⁰
For nucleus staining we used 4',6-diamino-2-phenylindole
(DAPI) at 300 nM. All samples were placed between slide and
cover slip and imaged using an oil immersion objective (40X, N.
A. One.25) on a Leica SP2 AOBS AOTF confocal microscope.
Images were analyzed using ImageJ (NIH, Bethesda, Maryland,
USA).

Extracellular pH measurements

Extracellular pH was measured directly in the medium.⁵¹ The experiments were run simultaneously in 7 × 5 mL flasks (control and tests) each containing 2 mL of suspension cells under continuous shaking. Simultaneous changes in pH were measured by using ELIT 808 ionometer with pH sensitive combined electrodes functioning in parallel. Data are means of 3 experiments ± SE.

Real-time quantitative RT-PCR

Total RNA (2 µg) was treated with DNase I (Sigma), reverse
transcribed with Revertaid Reverse Transcriptase (Fermentas) in
a 25 µl reaction volume and amplified with Mastercycler ep
Realplex (Eppendorf) using 5 µl of 50-fold diluted cDNA solu-
tion. Primers were designed with primer3 software. They were
obtained from Eurogentec and the primer sequences are listed in

supplemental **Table 1**. Real-time PCRs were performed with the
MaximaTM SYBR Green qPCR Master Mix (Fermentas) and
0.23 µM of each primer in a 15 µl reaction. Cycle thresholds
(Cts) were calculated using the Realplex 2.0 software (Eppen-
dorf). For each plate and each gene, a standard curve made with
dilutions of cDNA pools was used to calculate the reaction effi-
ciencies, and the relative expression was calculated according to
Hellemans et al.⁵² with EF1α4 and UBQ5 as reference genes.⁴²
An arbitrary value of 100 was assigned to control samples for nor-
malization according to Pfaffl.⁵³ The mean of 3 biological repeti-
tions are shown.

Post-LPS *Pcc* challenge on *A. thaliana* cells

*A. thaliana* cells (1 mL) were pretreated or not with 100 µg. mL⁻¹ of LPS<sub>Pcc</sub> or *LPS*<sub>P. aeruginosa</sub> for 14 h and then inoculated with 100 µL of *Pectobacterium carotovorum* (CFBP 5890) at 10⁸ cfu.mL⁻¹. *A. thaliana* cell viability was monitored after 24 h as described above.

Statistical analysis

Data were analyzed by variance analysis (ANOVA), and the
mean separation was achieved by Newman and Keuls multiple
range test. All numeric differences in the data were considered
significantly different at the probability level of p ≤ 0.05.

Results

LPS from *Pectobacterium* sp induces cell death in *A. thaliana*
cells

The LPS from the non-phytopathogenic bacteria *E. coli* and *P. aeruginosa* (up to 100 µg. mL⁻¹) did not induce cell death of *A. thaliana* cultured cells (Fig. 1A). These results are in accordance with previous data obtained by Desaki *et al.*²² Conversely, LPS from the plant pathogenic *Pcc* and *Pa* induced a dose-dependent cell death, evaluated by Evans blue staining of the cells (Fig. 1A). About fifteen and 30 percent of cells were dead 24 h after addition of 100 µg.mL⁻¹ of LPS*<sub>Pcc</sub>* or LPS*<sub>Pa</sub>* respectively, thus in the range of LPS concentrations known to elicit defense responses in different plants.¹⁴

This cell death was accompanied by cell shrinkage (Fig. 1B), a
hallmark of programmed cell death.²⁰ In order to check whether
LPS<sub>Pcc</sub>-induced cell death was due to an active mechanism requir-
ing gene expression and active cellular metabolism, *A. thaliana*
cells were pretreated with actinomycin D (AD), an inhibitor of
RNA synthesis, or with cycloheximide (Chx), an inhibitor of pro-
tein synthesis, at 20 mg.mL<sup>-1</sup> each. Although AD and Chx
induced a slight cell death by themselves, these both inhibitors
reduced the LPS<sub>Pcc</sub>-induced cell death (Fig. 1C). These results
suggest that the LPS from *Pectobaterium* could induce a PCD in
*A. thaliana* cells. This type of cell death resembles the one
detected during the early phase of infection of *A. thaliana* suspen-
sion cells with *P. carotovorum*.⁴⁰ Upon co-culture of plant cells
with 10⁸ cfu.mL<sup>-1</sup> Pcc an early small increase in plant cell death
occurred during the first 3 h before pectate-lyase activities were
detectable,⁴⁰ with dead cells displaying plasmolysis. After 12 h
<figure><img src="image_1.png" /><figcaption>**Figure 1.** Effect of LPS preparations from various bacteria on cell viability of *A. thaliana* cultured cells. Effect of increasing concentrations (0 to 100 µg.mL⁻¹) of LPS from non-phytopathogenic, or phytopathogenic bacteria (A) on *A. thaliana* cell death after 24 h of treatment. (B). LPS<sub>Pcc</sub> induced cell shrinkage of *A. thaliana* cells treated with 100 µg.mL⁻¹ during 24 h. (C). Effect of pre-treatment with, actinomycin D (AD, 20 mg.mL⁻¹) or cycloheximide (Chx, 20 mg.mL⁻¹) on cell death induced by 100 µg.mL⁻¹ LPS<sub>Pcc</sub>. C corresponds to the control cells without treatment. Data represent the mean and standard errors (SE) of at least 5 independent experiments. \*significantly different from controls, *P* < 0.05 and \*\*significantly different from the LPS treated cells, *P* < 0.05.</figcaption></figure>

and pectate lyase activity increase, plant cell death largely increased and cell integrity was not preserved (⁴⁰ and Fig. 1B).

## LPS induced generation of reactive oxygen species in *A. thaliana* cells

Various LPS were shown to induce ROS in plant.<sup>14,22</sup> In accordance, LPS<sub>Pcc</sub> and LPS<sub>Pa</sub> induced an increase in luminol-mediated chemiluminescence caused by ROS release into the culture medium (Fig. 2A) within 30 min after the addition of 100 µg.mL<sup>-1</sup> LPS. The ROS concentration of the culture medium reached a maximum at 1 h, and then decreased till 3 h (Fig. 2A). This kinetics was compared in timing and magnitude with those induced by LPS from plant non-pathogens, LPS<sub>E. coli</sub> and LPS<sub>P. aeruginosa</sub>. A strong production of ROS started after approximately 2 h, reached a peak around 3 h and decreased to a level similar to that of the control in 2 h (Fig. 2A). The LPS from plant non-pathogenic bacteria induced more important ROS increases at 3 h than those induced by LPS from *Pectobaterium* at 1 h (Fig. 2A). It is noteworthy that despite different modes of purification (phenol extraction plus gel filtration and chromatography

versus phenol extraction), LPS from the plant non-pathogenic *E. coli* and *P. aeruginosa* induced ROS generations, presenting the same kinetics and extent. In the same way, despite comparable modes of purification, the LPS from *E. coli* showed a delayed and larger ROS generation when compared to the one induced by the LPS from pathogenic *Pcc* and *Pa*. These data indicate that these ROS generations were more likely due to LPS than to putative contamination in LPS preparations.

The CM-H₂DCFDA fluorescence was used to confirm the accumulation of ROS in *A. thaliana* cells in response to the different type of LPS (Fig. 2B). LPS<sub>Pcc</sub> and LPS<sub>Pa</sub> induced strong ROS generation after 1 h and lower ones at 3 h while for LPS<sub>E. coli</sub> and LPS<sub>P. aeruginosa</sub> the ROS level was maximal at 3 h (Fig. 2B). The LPS-induced increase in ROS could be detected in the cytosol and putatively in the nucleus as shown by the overlay of staining with DAPI and CM-H₂DCFDA (Fig. 2C).

Pre-treatment of the cells with Tiron (5 mM), a scavenger of O₂⁻,⁵⁴ completely abolished the LPS-induced luminol chemiluminescence, for LPS from *Pectobaterium* at 1 h and non-pathogenic bacteria at 3 h (Fig. 2D). Also to a lesser extent, diphenyleneiodonium chloride (DPI, 50 µM), an inhibitor of the NADPH-oxidase,⁵⁵ decrease ROS levels induced by both

type of LPS (Fig. 2D), suggesting that NADPH-oxidases participate in the ROS generation.

Since ROS release may cause PCD,<sup>56</sup> we further checked if the pre-treatment of *A. thaliana* cells with Tiron or with DPI could impact the LPS<sub>Pcc</sub>-induced cell death. Both Tiron and DPI alone induced a slight increase in cell death (Fig. 2E). However, these pretreatments decreased the LPS<sub>Pcc</sub>-induced cell death (Fig. 2E), indicating that the generation of ROS induced by LPS<sub>Pcc</sub> is probably an upstream event in the signaling pathway leading to cell death.

## LPS from plant non-pathogenic bacteria induced alkalinization of the medium

Alkalinization of the external medium is recognized as a classical response to MAMPs, and various LPS were shown to alkalinize the medium of cell cultures.<sup>57,16</sup> Time courses of the pH of the culture medium of *A. thaliana* cells were monitored in presence of the different type of LPS (Fig. 3). Both LPS, LPS<sub>P. aeruginosa</sub> and LPS<sub>E. coli</sub>, induced an alkalinization of medium of about 0.6 upH after 1 h then slightly decreased. LPS<sub>Pcc</sub> and LPS<sub>Pa</sub> did not
induced significant variations of medium pH. These data suggest differential perceptions of LPS from *Pectobacterium* and non-pathogenic bacteria by *A. thaliana* cells.

## LPS induced gene transcription in *A. thaliana* cells

Defense responses of plants to pathogens or pathogen-derived molecules comprising LPS becomes also apparent in induction of defense genes,¹⁶,⁵⁸ including *PAL* (phenylalanine ammonia-lyase) gene⁵⁹ and *PR* (pathogenesis-related) genes.⁶⁰ Elicitation of *A. thaliana* cells with LPS<sub>Pcc</sub>, induced a large increase in transcript level of *PAL1* after 1 h which decreased at 3 h (Fig. 4A). The expression of *PAL1* in response to LPS<sub>P. aeruginosa</sub> was increased after 1 h and more pronounced after 3 h (Fig. 4A). This increase in *PAL1* expression seemed to be synchronized with the ROS generation, since maximal level of *PAL1* expression occurred at the same time to the maximal ROS generation, i.e. One h after treatment by LPS<sub>Pcc</sub> and 3 h after LPS<sub>P. aeruginosa</sub> treatment respectively (Fig. 2A and B). Pre-treatments of the cells with Tiron (5 mM) or DPI (50 µM), efficient to decrease LPS-induced ROS generation (Fig. 2D), decreased the *PAL1* transcription observed with LPS<sub>Pcc</sub> or LPS<sub>P. aeruginosa</sub> (Fig. 4A). These data strongly suggest that ROS generation was involved in *PAL1* transcript upregulation. Concerning *PR1* gene, its expression was only induced 24 h after elicitation whatever the LPS preparations tested (Fig. 4B). These increases in *PR1* expression were also reduced upon pre-treatment of the cells with Tiron (5 mM) and DPI (50 µM) suggesting that ROS generation were also involved in the activation of pathway(s) leading to *PR1* transcript up-regulation.

## LPS from *Pectobacterium* and non-pathogenic bacteria could antagonize *Pcc*-induced death of *A. thaliana* cells

One of the effects of LPS described on plants is their ability to prevent HR, a form of PCD.²¹ LPS from *Pectobacterium* were also shown to decrease *Pcc*-induced soft rot symptoms on potato tubers.⁶¹ Since we have recently showed that *A. thaliana* cells

could be used as a model to analyze *Pcc* virulence,⁴⁰ we checked if pretreatment with LPS from *Pectobacterium* or non-pathogenic bacteria could counteract the *A. thaliana* cell death induced by *Pcc*. As we previously reported,⁴⁰ the rate of *A. thaliana* cell death

<figure><img src="image_2.png" /><figcaption>**Figure 2.** Production of reactive oxygen species (ROS) by *A. thaliana* cultured cells in response to LPS from various bacteria. (A) Kinetics of ROS generation in the medium of cells treated by 100 µg.mL⁻¹ LPS derived from *Pectobacterium carotovorum subsp. carotovorum* (Pcc) and *Pectobacterium atrosepticum* (Pa) for the plant pathogens, and *Escherichia coli* and *Pseudomonas aeruginosa* for the non-phytopathogenic bacteria. (B) Imaging of ROS accumulation in *A. thaliana* cells treated with LPS from pathogenic or non-pathogenic bacteria after 1 h (left) or 3 h (right). Green fluorescence is indicative for ROS (Scale bars, 10 µm). (C) DIC image, DAPI staining of the nuclei and ROS accumulation after 1 h with LPS<sub>Pa</sub>. (D) Effects of Tiron (5 mM), a scavenger of superoxide anions (O₂⁻); or diphenyleneiodonium (DPI, 50 µM), an inhibitor of the NADPH oxidase, after 1 h and 3 h on the production of ROS by *A. thaliana* cells. (E) Effect of pretreatment with Tiron (5 mM) or DPI (50 µM) on cell death induced by 100 µg.mL⁻¹ LPS<sub>Pcc</sub>. Data represent the mean and standard errors (SE) of at least 3 independent experiments. * significantly different from the LPS treated cells, *P* < 0.05.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 3.** Variations in the pH medium of *A. thaliana* cultured cells elicited by 100 µg.mL⁻¹ LPS from non-phytopathogenic or phytopathogenic bacteria. Data represent the mean and standard errors (SE) of 3 independent experiments. \*significantly different from control, *P* < 0.05.</figcaption></figure>

after 24 h treatment by of a 10⁸ cfu.mL⁻¹ *Pcc* was 100% (Fig. 5). This cell death caused by *Pcc* was reduced by more than half upon pretreatment with 100 µg.mL⁻¹ *LPS*<sub>*P. aeruginosa*</sub> (14 h)

<figure><img src="image_4.png" /><figcaption>**Figure 5.** Effect of 14 h of pre-treatment with 100 µg.mL⁻¹ *LPS*<sub>*Pcc*</sub> and *LPS*<sub>*P. aeruginosa*</sub> on cell death induced by *Pectobacterium carotovorum* CFBP 5890 on *A. thaliana* cells after 24 h. Data represent the mean and standard errors (SE) of 3 independent experiments. \*significantly different from controls, *P* < 0.05.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Effect of LPS on defense-related gene expression in *A. thaliana* cells. (A) Effect of LPS<sub>Pcc</sub> and LPS<sub>aeruginosa</sub> at 100 µg.mL<sup>-1</sup> on expression of PAL1 (encoding phenylalanine ammonia-lyase), and (B) PR1 (pathogenesis- related protein 1) with or without Tiron (5 mM) or DPI (50 µM). Data represent the mean and standard errors (SE) of 3 experiments. \*significantly different from controls, *P* < 0.05 and \*\*significantly different from the LPS treated cells, *P* < 0.05.</figcaption></figure>

before inoculation (Fig. 5). Pre-treatment of cells with 100 µg. mL⁻¹ *LPS*<sub>*Pcc*</sub> during 14 h before inoculation with *Pcc* also decreased cell death but was less efficient (only 40% of decrease, Fig. 5).

## Discussion

From these data, it seems that purified LPS from plant pathogenic *Pa* and *Pcc* as well as LPS from plant non-pathogenic *P. aeruginosa* and *E. coli* could induce, in *A. thaliana* cell suspensions, cellular events classically associated with defense responses as previously reported.<sup>15-17</sup> However, some of the responses seemed to be specific to LPS of the pathogen *Pectobaterium* (e.g. PCD), or non-pathogenic bacteria (e.g., medium alkalization), when other shared responses (e.g. ROS generation, upregulation of gene transcription) could present variable kinetics and amplitudes depending on the origin of the LPS used. These differences could not be ascribed to the purification mode since LPS from *Pectobaterium* were highly purified like the one from *E. coli* and that LPS from *P. aeruginosa* was only purified by phenol extraction.

Among the shared responses, ROS generation was recorded in *A. thaliana* cells upon treatment with LPS from *Pectobacterium* and LPS from plant non-pathogenic bacteria. Our data are in accordance with previous studies showing that various LPS could induce ROS generation in different plants.<sup>14,22,57</sup> However
depending on the plant species and the origin of the LPS, all did not induce an oxidative burst.²⁶,⁶²,⁶³ In our model, addition of DPI before elicitation altered the ROS generation by all LPS we tested, pointing out to a NADPH-oxidase-dependent pathway. Among the LPS preparations, those derived from *Pectobaterium* showed weaker ROS-generations compared to LPS from plant non-pathogenic bacteria, as previously described in rice cell culture.²² The kinetics of ROS generation were different, LPS<sub>Pa</sub> and LPS<sub>Pcc</sub>, induce a peak 1 h after treatment, thus 2 h before the one induced by LPS<sub>E.coli</sub> and LPS<sub>P. aeruginosa</sub>.

Another shared defense response between tested LPS was the induction of *PAL1* transcripts, as already observed with other LPS preparations⁶⁰ and in accordance with induction of PAL activity by LPS<sub>Pa</sub>.²⁷ Interestingly, the induction of *PAL1* transcript seemed to be correlated with the LPS-induced ROS generation, since *PAL1* transcription after treatment with LPS<sub>Pcc</sub> was larger at 1 h than at 3 h on the opposite of what observed with LPS<sub>P. aeruginosa</sub>. Tiron, a scavenger of ROS, as well as DPI were able to decrease ROS generation and the LPS-induced *PAL1* transcription. This suggests a common pathway involving ROS generation leading to *PAL1* transcription, in accordance with recent reports showing ROS-dependent increase in *PAL* transcription and activity.⁶⁴,⁶⁵ Generation of ROS and its effect on nuclear gene expression are well described and accepted in the literature⁶⁶ and *in silico* analysis of sequenced promoters from *PAL1* and *PR1* genes (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) effectively revealed putative ARE (Antioxidant Response Element) domains known to be ROS responsive. However, the different kinetics of ROS generation and *PAL1* transcription suggest the ability of *A. thaliana* cells to display specific responses depending on the origin of LPS. The *PR1* transcription was up-regulated also in a ROS dependent manner by LPS from *Pectobaterium* and non-pathogenic bacteria, but only 24 h after, as already observed by Silipo *et al*.⁸ with the lipid A part of LPS preparation from *X. campestris*. Although earlier transcription of *PR1* by LPS was reported,¹⁶ delayed ROS-induced *PR1* expression compared to *PAL* expression was recently reported in *Capsicum annuum*.⁶⁵

Ion fluxes, especially early Ca²⁺ influx, are frequently associated with response to microbe-derived molecules⁶⁵ and notably LPS.⁵⁷,⁶⁸ Using *A. thaliana* cell expressing aequorin, we failed to detect any modification in cytosolic Ca²⁺ level in response to all tested LPS (not shown). We further could not provide direct electrophysiological evidence that these LPS could induce differential early ion current regulations (not shown). On the contrary, addition of LPS<sub>E.coli</sub> or LPS<sub>P. aeruginosa</sub> caused a strong alkalization of extracellular medium within 30 min when LPS<sub>Pa</sub> and LPS<sub>Pcc</sub> did not induced significant variation of pH. This alkalization could not be related to consumption of H⁺ for production of ROS since this last occurred only after 90 min, thus largely after the alkalization observed. Such alkalization in response to LPS from *B. cepacia* was already reported in tobacco cell culture.⁵⁷ However, the LPS<sub>Pa</sub> was reported to induce acidification of the extracellular medium of tomato culture cells when it failed to acidify potato extracellular medium.²⁶ The differential response to LPS from *Pectobaterium* and non-pathogenic bacteria

is an additional indication on the ability of *A. thaliana* cells to display specific outcomes in response to the type of LPS origins. This difference is probably mediated by recognition of structural features within LPS that are not necessarily conserved between *Pectobaterium* and non-pathogenic bacteria.

As a matter of fact, only LPS<sub>Pcc</sub> and LPS<sub>Pa</sub> were able to induce a dose-dependent death of a part of the cell population, when LPS<sub>E.coli</sub> and LPS<sub>P. aeruginosa</sub> did not. The extent of this cell death was in the same order of magnitude to those observed in response to various harpins, considered by some authors as another type of MAMP,³ in *A. thaliana* cultured cells.⁶⁹,⁷⁰ This cell death was accompanied by cell shrinkage and needs an active metabolism as indicated by its reduction by inhibitors of transcription and translation; thus displaying widely accepted hallmarks of PCD.²⁰ Although the HR is supposed to contribute to plant defense,¹⁴ LPS-induced PCD was never reported in dicots⁶⁰ but it was reported in rice cultured cells.²² Our data are also reminiscent of the LPS-induced ROS-mediated endothelial cell death observed in animal,³¹ since in our model, inhibition of ROS generation by DPI or Tiron allowed a significant decrease of the LPS<sub>Pcc</sub>-induced cell death. The early ROS generation observed in response to LPS from *Pectobacterium* could participate in, at least, 2 signaling pathways, one leading to PCD and another one leading to regulation of gene transcription. Despite H₂O₂ having been reported to induce PCD in cultured cultures of *A. thaliana*,⁷¹ MAMP-induced oxidative stress alone is not necessarily sufficient to elicit cell death.⁵⁸ Here, the time course of ROS generation seemed to be a key difference to induced PCD, since delayed larger ROS generation induced by LPS from non-pathogenic bacteria did not lead to cell death.

Pre-treatment of the *A. thaliana* cells with LPS<sub>Pcc</sub> as LPS<sub>P. aeruginosa</sub> decreased the cell death induced by Pcc during the co-infection, probably due to the elicitation of defense responses by LPS (e.g., *PAL1* transcription). Thus these induced defense responses could participate in attenuating *Pectobacterium*-induced diseases in plants.²⁶,⁷² However, the weaker decrease, in Pcc-induced cell death, caused by LPS<sub>Pcc</sub> when compared to the one induced by LPS<sub>P. aeruginosa</sub> could reflect the dual effects of LPS<sub>Pcc</sub>: on the one hand, it induces defense responses allowing to decrease Pcc-induced cell death like LPS<sub>P. aeruginosa</sub> on the other hand, LPS<sub>Pcc</sub> mediate a direct PCD. These data reinforce the hypothesis that a LPS preparation could induce several signaling pathways in *A. thaliana* cells perhaps through recognition of structural features within LPS that are not necessarily widely conserved. Indeed, the core structure of the LPS from *P. carotovorum* slightly differs from that of *E. coli*.³⁴,³⁶ Although in previous studies on animal cells, the main biological activities was described to be exerted by lipid A,³⁴,⁷³ the lipid A of *P. carotovorum* and *E. coli* LPS exhibit the same biological activity.³⁴ However other studies point out that in plant the 3 parts of the LPS, the lipid A, the core oligosaccharide and the OPS could be active in virulence,⁹,⁷⁴ in defense-related gene induction¹⁶,¹⁸,⁷⁵,⁷⁶ and HR suppression.⁷⁵⁻⁷⁷ In *A. thaliana* leaves, the defense responses occurred in 2 phases, the core oligosaccharide inducing an early phase and the lipid A moiety only a later phase⁷⁵ suggesting a differential recognition of these molecular motifs. Moreover, in
animals as well as in plants, modifications in the structure of the
lipid A, such as palmitoylation, acylation and phosphorylation
could influence biological activity of LPS,⁷⁸⁻⁸⁰ notably in trigger-
ing innate immune responses.⁸ Alterations in lipid A or other
structures within LPS were shown to occur during interactions
with plants.⁸¹,⁸² The isolation of pagP mutants from animal
pathogenic Gram-negative bacteria has revealed that palmitoy-
lated lipid A can both protect the bacterium from certain host
immune defenses and attenuate the ability of lipid A to activate
those same defenses through the TLR4 signal transduction path-
way.⁸⁰ In *Pcc*, a homolog pagP has been cloned,¹⁴ and it thus
will be of interest to know if LPS<sub>Pcc</sub> palmitoylation could be
responsible for the different responses we observed and if such
modification could have a role in virulence.

In conclusion, this study provides evidence on the ability of *A. thaliana* cells to display specific responses to LPS from *Pectobacterium* when compared to those from LPS from plant non-pathogenic bacteria. Although part of the responses to both types of LPS could overlap, different signaling pathways could be

activated, and among the LPS-induced responses the differential
kinetics and amplitude in ROS generation seems to be determi-
nants to induce PCD in *A. thaliana* cells, allowing putative role
of LPS from *Pectobaterium* in virulence, as previously reported
for LPS<sub>pA</sub>.⁹,²³ Further studies will be needed to determine the
roles of the different parts of the LPS molecules in inducing the
different signaling pathways activated in plant cells.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Agronomic Research for
Development Project PRAD N° 07-07 and the Arimnet project
PoHMed. MKH was supported by funds from the “Agence Uni-
versitaire de la Francophonie” (AUF).

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