# Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein

Jean-Luc Cacas, Patricia Gerbeau-Pissot, Jérôme Fromentin, Catherine Cantrel, Dominique Thomas, Emmanuelle Jeannette, Tetiana Kalachova, Sébastien Mongrand, Françoise Simon-Plas, Eric Ruelland

► To cite this version:

Jean-Luc Cacas, Patricia Gerbeau-Pissot, Jérôme Fromentin, Catherine Cantrel, Dominique Thomas, et al.. Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein. Plant, Cell and Environment, 2016, 40 (4), pp.585-598. 10.1111/pce.12771. hal-01328631

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<figure><img src="image_1.png" /></figure>
1 Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in

2 response to cryptogein

3

4 Running head: Control of RBOHD activity by DGK

5 Jean-Luc Cacas¹,*,§, Patricia Gerbeau-Pissot¹,§, Jérôme Fromentin¹, Catherine Cantrel²,

6 Dominique Thomas¹, Emmanuelle Jeannette², Tetiana Kalachova³,⁴, Sébastien Mongrand⁴,

7 Françoise Simon-Plas¹, Eric Ruelland²,³,⁴

8 Author Affiliation

9 1 Agroécologie, AgroSup Dijon, CNRS, INRA, Univ. Bourgogne Franche-Comté, F-21000
10 Dijon, France

11 2 UPMC UnivParis06, UR5, Physiologie Cellulaire et Moléculaire des Plantes, 4 place
12 Jussieu, 75252 Paris cedex 05, France

13 3 UPE, UPEC, Institut d'Ecologie et des Sciences de l'Environnement de Paris, 61 avenue du
14 général de Gaulle, 94010 Créteil, France

15 4 CNRS, UMR7618, Institut d'Ecologie et des Sciences de l'Environnement de Paris, 61
16 avenue du général de Gaulle, 94010 Créteil, France

17 5 CNRS University of Bordeaux, UMR 5200 Laboratoire de Biogenèse Membranaire, INRA
18 Bordeaux Aquitaine, BP81, F-33883 Villenave d'Ornon, France

19 * Present address: Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Centre
20 INRA de Versailles-Grignon, Route de St. Cyr, 78026 Versailles Cedex France

21
22 § These authors contributed equally to this work.

23 Corresponding author: Ruelland Eric

24 Université Paris-Est, Institute of Ecology and Environmental Sciences of Paris, CNRS/UMR

25 7618 61, avenue du général de Gaulle, Créteil - France

26 Telephone number: + 33 1 45 17 14 67. E-mail address: eric.ruelland@u-pec.fr
27 Summary statement

28 Cryptogein is a protein secreted by the oomycete *Phytophthora cryptogea* that activates
29 defence mechanisms in tobacco. We show here in BY-2 tobacco suspension cells that
30 phosphatidic acid rapidly accumulates in response to cryptogein due to the coordinated onset
31 of phosphoinositide-dependent phospholipase C and diacylglycerol kinase activities. Both
32 enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK
33 family were found to reduce PA production upon elicitation and to strongly decrease the
34 RBOHD-mediated oxidative burst. This establishes that phosphatidic acid production by
35 diacylglycerol kinases is upstream of the oxidative burst in response to cryptogein.

36

37
38 **ABSTRACT**

39 Cryptogein is a 10 kDa-protein secreted by the oomycete *Phytophthora cryptogea* that
40 activates defence mechanisms in tobacco plants. Among early signalling events triggered by
41 this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is
42 dependent on the NADPH oxidase activity of the RESPIRATORY BURST OXIDASE
43 HOMOLOG (RBOH) isoform D. Using radioactive [³³P]-orthophosphate labelling of tobacco
44 Bright Yellow-2 suspension cells, we here provide *in vivo* evidence for a rapid accumulation
45 of phosphatidic acid (PA) in response to cryptogein due to the coordinated onset of
46 phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities.

47 Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco
48 DGK family were found to reduce PA production upon elicitation and to strongly decrease the
49 RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK
50 controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of *DGK5-like*
51 was up-regulated in response to cryptogein. Besides *DGK5-like* is likely to be the main cluster
52 III DGK isoform silenced in one of our mutant line, making it a strong candidate for the
53 observed response to cryptogein. The relevance of these results is discussed with regard to
54 early signalling lipid-mediated events in plant immunity.

55

56 **KEYWORDS:** cryptogein, microbial-associated molecular pattern, diacylglycerol kinase;  
57 phospholipase C, phosphatidic acid, reactive oxygen species, RBOHD, NADPH oxidase

58

59
60 INTRODUCTION

Many studies have suggested that membrane lipids act as reservoirs for signalling molecules in response to developmental and environmental cues in both animal and plant cells. Indeed, under biotic or abiotic stresses, a regulated production of phospholipid-derived molecules was reported to occur, and to launch intracellular signalling cascades. Phospholipases, lipid kinases and lipid-phosphate phosphatases are considered as the main activities involved in such early signalling events (Janda et al. 2013, Sheng et al. 2015, Hawkins & Stephens 2015, Ruelland et al. 2015).

Phosphatidic acid (PA) is the most basic phosphoglycerolipids, being composed of a diacylglycerol with a phosphoryl group esterified on the *sn*-3 hydroxyl of the glycerol backbone. Besides being an intermediate of phospholipid synthesis (Petroutsos et al. 2014), PA also transduces stress signals in eukaryotic cells. In serum-withdrawn fibroblast cultures for example, reverse genetic approaches have established a marked correlation between elevated endogenous PA levels and apoptosis inhibition; the latter probably being partially responsible for cancer establishment in tissues (Bruntz et al. 2014). In plants, upon pathogen attack and situations mimicking infection by the addition of elicitors, as well as under many abiotic (including drought, cold, osmotic) stresses, PA accumulation was either inferred from pharmacological strategies or directly shown by biochemical methods (Pinosa et al. 2013, Ruelland et al. 2015). In Eukaryotic cells, the synthesis of PA as a signalling molecule relies on two distinct enzymatic systems. Phospholipases D (PLD) can hydrolyse structural phospholipids within membranes, directly releasing PA (Wang et al. 2012) while, diacylglycerol kinases (DGK) can catalyse the phosphorylation of diacylglycerol (DAG) to produce PA (Arisz et al. 2009). DAG can be provided to DGK either by phosphoinositide-dependent phospholipases C (PI-PLCs) that act on phosphorylated phosphoinositides or by non-specific phospholipases C (NPCs) that use structural phospholipids (Pokotylo et al. 2013,
2014). In contrast to animal systems where both DAG and PA have been shown to act as secondary messengers, it is commonly assumed that PA plays the central role in plant cells.

Indeed, no plant proteins orthologous to mammalian protein kinases C (which are activated through a direct interaction with DAG) have been identified from plant genomes (Dong et al. 2012), even though some proteins bearing the C1 domain responsible for DAG binding to PKCs have been found (Janda et al. 2013). On the other hand, up to 35 plant proteins targeted by PA have been identified on the basis of their direct interaction with the lipid (Hou et al. 2015). However, no consensus sequence to define a canonical PA binding domain was identified from these proteins.

Upon pathogen attack, two lines of defence can successively occur. The first relies on the recognition of common pathogen motifs, named *microbial-associated molecular patterns* (MAMPs), at the plasma membrane and this can result in basal resistance to a broad range of pathogens. The second depends on the specific perception of pathogen effectors within host cells by resistance proteins (Dangl & Jones 2001, Jones & Dangl 2006, Stotz et al. 2014, Bigeard et al. 2015). This latter defence response often leads to a rapid and highly localized hypersensitive cell death that prevents biotrophic pathogens from propagating (Cacas 2015).

Interestingly, PA has been shown to accumulate in response to many MAMPs (xylanase, flagellin, N-acetylchitooligosaccharide, chitosan) in several independent plant models (van der Luit et al. 2000, den Hartog 2003, Bargmann et al. 2006). PLD-, PI-PLC-, NPC- and DGK-encoding genes and/or corresponding enzymatic activities were also reported to be rapidly up-regulated in numerous studies dealing with either MAMP- or Effector-Triggered Immunity (MTI or ETI, respectively), suggesting the involvement of these lipid-modifying enzymes in early defence events (Zhang & Xiao 2015, Ruelland et al. 2015). With the notable exception of DGK, this assumption was corroborated by several works using loss-of-function mutants. While Arabidopsis PLDδ isoform was found to be involved in MTI, multiple PLD
110

isoforms were proposed to act redundantly in ETI (Pinosa et al. 2013, Johansson et al. 2014).

111

In tomato (*Solanum lycopersicum*) cell suspensions, MTI involved PLDβ1 (Bargmann et al.

112

2006) whereas full establishment of ETI required the PI-PLC-encoding gene *slPLC4* (Vossen et al. 2010).

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The small, basic and hydrophilic 10-kDa protein named cryptogein is a MAMP secreted by the oomycete *Phytophthora cryptogea* (Ricci et al. 1989). More than two decades after its discovery, extensive investigations have provided a broad picture of cryptogein-induced physiological responses. Although this protein exhibits both cell death-promoting and systemic acquired resistance-inducing activities in tobacco (*Nicotiana tabacum*) plants (Keller et al. 1996, Rustérucci et al. 1999, Cacas et al. 2005), it is also able to activate many characteristic hallmarks of MTI. The interaction of cryptogein with a yet-to-be-identified protein receptor at the plasma membrane results in calcium influx (Tavernier et al. 1995, Pugin et al. 1997), potassium and chloride effluxes associated with apoplasm alkalization (Blein et al. 1991), NADPH oxidase-dependent oxidative burst (Simon-Plas et al. 2002), modification of plasma membrane order (Gerbeau-Pissot et al. 2014), activation of a MAPK cascade (Dahan et al. 2009) and clathrin-dependent endocytosis (Leborgne-Castel et al. 2008).

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Reactive oxygen species (ROS) are assumed to be the main downstream actors to PA in pathogen-induced signalling cascades (Zhang & Xiao 2015). Although it was demonstrated that PLD-produced PA directly regulates the NADPH oxidase-dependent oxidative burst during a drought stress (Zhang et al. 2009), this link has not been clearly established with respect to pathogen-response. Firstly, is PA production a common signature of MTI, irrespective of the MAMP and the cell model? Secondly, can PA produced by DGK activities act upstream of the NADPH oxidase during plant-pathogen interactions? In this present work, we report on the regulatory function of DGK in controlling the cryptogein-induced oxidative burst in Bright Yellow-2 (BY-2) cell suspensions. Using biochemical, pharmacological and
reverse genetic approaches, we show that PA, originating from a DGK coupled to PI-PLC, is
an upstream positive regulator of the plasma membrane-localized NADPH oxidase isoform
RBOHD in response to cryptogein.

138
139 **MATERIALS & METHODS**

140 **Culture conditions and cryptogein elicitation of tobacco cells**

141 Bright Yellow-2 (*Nicotiana tabacum*) cells were grown in modified liquid Murashige and Skoog (MS, M0221, Duchefa-Kalys, Bernin, France) medium at pH 5.6 (M0221, Duchefa-Kalys, Bernin, France) supplemented with 1 mg.L⁻¹ thiamine-HCl, 0.2 mg.L⁻¹ 2,4-dichlorophenylacetic acid, 100 mg.L⁻¹ myo-inositol, 30 g.L⁻¹ sucrose, 200 mg.L⁻¹ KH₂PO₄ and 2 g.L⁻¹ MES. Cells were maintained under continuous light (200 µE m⁻² s⁻¹) on a rotary shaker (140 rpm) and sub-cultured to fresh medium (4:80, (v/v)) on a weekly basis.

147 For radiolabeling experiments, ROS quantification and pharmacological treatments, cells were harvested from 7-day-old suspensions using a vacuum-system and resuspended at 0.1g/mL in either equilibration medium I2 (2 mM MES, 175 mM mannitol, 0.5 mM CaCl₂, 0.5 mM K₂SO₄, pH 5.9) or I20 (20 mM MES, 175 mM mannitol, 0.5 mM CaCl₂, 0.5 mM K₂SO₄, pH 5.9). After a 3 h incubation period under mild shaking at 24°C, cells were elicited by adding 50 nM cryptogein (final concentration) from a solution prepared according to (Ricci et al. 1989).

154 **Radiolabelling experiments**

155 BY-2 cells were aliquoted (7 mL) in wide neck flasks (capacity 50 mL) for equilibration. Cells were then labelled by the addition of 53 MBq.L⁻¹ [³³P]-orthophosphate. Total lipids were extracted according to the procedure previously described by (Krinke et al. 2009). Lipids were separated by thin layer chromatography (TLC) and developed either in an acidic solvent system composed of chloroform:acetone:acetic acid:methanol:water (10:4:2:2:1, (v/v), Lepage 1967) or in a solvent system composed of an upper phase of ethyl acetate:isooctane:acetic acid: water (12:2:3:10 (v/v), Munnik et al. 1995). Radiolabelled spots
162 were quantified by autoradiography using a Storm phosphorimager (Amersham Biosciences,
163 Buckinghamshire, UK). Separated phospholipids were identified by co-migration with
164 authentic non-labelled standards visualized by primuline staining (under UV light) or by
165 phosphate staining.

166 **Quantification of reactive oxygen species**

167 Reactive oxygen species were measured using a luminol-based method as described
168 previously (Simon-Plas et al. 2002) but with slight modifications. Briefly, after a 3 h-
169 equilibration period in I20 medium, cells were treated with cryptogein. Before elicitation,
170 samples corresponding to zero time were harvested and the luminescence background of the
171 assay was measured. Samples (250 µL) were regularly harvested over the first 90 min for
172 ROS determination using a luminometer (Lumat LB9507, Berthold Technologies, Bad
173 Wilbad, Germany). Fifty microliters of a 0.3 mM luminol solution and 300 µL of the assay
174 buffer (50 mM MES, 175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, pH 6.5) were
175 automatically added to cell aliquots before luminescence was measured (delay in injection of
176 1.2 s, data acquisition time of 10 s). For each biological and technical replicates, 2 h after
177 elicitation (when the oxidative burst was over), a calibration curve was made in order to
178 convert relative luminescence units (RLU) into H2O2 equivalents by recording cell
179 luminescence upon the addition of increasing H2O2 concentrations during the assay. Results
180 were normalized to BY-2-cell weight and expressed as nmoles H2O2 equivalents per gram of
181 cells.

182 **Reverse transcription-quantitative polymerase chain reaction**

183 RNA extraction, reverse transcription and quantitative measurement of gene expression by Q-
184 PCR were carried out as described by (Anca et al. 2014). Two reference genes were used for
185 expression normalization: *ELONGATION FACTOR 1α* (EF-1α, AF120093), forward, 5'-
186 TGAGATGCACCACGAAGCTC-3', and reverse, 5'-CCAACATTGTCACCAGGAAGTG-
187 3'; and L25 gene coding for a ribosomal subunit (L18908), forward, 5'-
188 CCCCTCACCACAGAGTCTGC-3', and reverse, 5'-
189 AAGGGTGTTGTTGTCCTCAATCTT-3'. Primer sequences for NtDGK5-like gene are as
190 follows: NtDGK5-like specific couple: forward (F85sq), 5'-
191 AGTCCGAGCTCAATGACAACA-3'; and reverse (R85sq2), 5'-
192 GTACGAAGAAGTTCTCCTCCAAGTT-3'; Cluster III gene-targeting couple: forward
193 (ClusterIII-F), 5'-TTCAGCATGGGGATGGATGCA-3'; and reverse (ClusterIII-R), 5'-
194 AACCATCCTTGYGTACATCC-3'

195 Cloning of *NtDGK5*-like coding sequence

196 *NtDGK5*-like coding sequence was amplified by PCR from cDNA obtained from untreated 7-
197 day-old BY-2 cells using high fidelity DNA polymerase (Phire Hot Start, ThermoFisher
198 SCIENTIFIC) and specific primers: forward (F1c85), 5'-ATGGCGAATTCTGAGTCCGA-
199 3'; and reverse (R1c85), 5'-CTAACTGAGACGAGAAACATCGA-3'. Primers were
200 designed according to the sequence of SGN-U439985 (Solanacea Genomic Network
201 database). Resulting PCR products were cloned into pSC-B Amp/Kan vector using the
202 topoisomerase-based StrataClone Blunt PCR cloning kit (Agilent). Seven clones were
203 sequenced among which one was selected for further work. The sequence was deposited in
204 GenBank (accession number KU934207). This cDNA was named *NtDGK5*-like because its
205 closest *Arabidopsis thaliana* ortholog is AtDGK5.

206 Production of overexpressing *NtDGK5*-like transgenic lines

207 The *NtDGK5*-like coding sequence was first amplified by PCR using high fidelity DNA
208 polymerase from *pSC-B::DGK5*-like using the following primer pairs: couple 1, forward
209 (CACC-F85), 5'-CACCATGGCGAATTCTGAGTCCGA-3'; and the reverse R1c85; couple
210 2: the forward (CACC-F85) and reverse (R85-c-myc), 5'-
211 CTAGAGGTCTTCTTCGCTGATGAGCTTTTGTTCACTGAGACGAGAAACATCGAC-
212 3'. Two independent intermediate constructs to express a C-terminally c-myc tagged protein
213 and an untagged version were generated by cloning the PCR products into pENTR/D-TOPO
214 (ThermoFisher SCIENTIFIC). Sequencing of both inserts revealed no mutations. Using
215 Gateway LR Clonase II enzyme mix (ThermoFisher SCIENTIFIC), MluI-digested
216 pENTR/D::DGK5-like-c-myc and MluI-digested pENTR/D::DGK5-like were then
217 independently recombined with pMDC32 (Karimi et al. 2007) to generate plant-expressing
218 vectors that were checked for correct recombination by restriction digestions before being
219 used to transform WT BY-2 cell suspensions following a previously described procedure
220 (Simon-Plas et al. 2002). Among the 6 hygromycin-resistant calli isolated, only one
221 overexpressed the untagged version of NtDGK5-like (named line OE.NtDGK5-A) while five
222 out of twenty hygromycin-resistant calli overexpressed the C-terminally tagged version of
223 NtDGK5-like. The two lines, named OE.NtDGK5myc-A and OE.NtDGK5myc-B, which
224 exhibited the strongest levels of transgene expression, were selected for further
225 characterization.

226 Generation of cluster III DGK-silenced cell lines

227 A conserved region of cluster III DGKs of the *NtDGK5-like* coding sequence, from
228 nucleotides 511 to 692 (see supporting information Fig. S2) was chosen to be inserted into the
229 post-transcriptional gene silencing vector (pH7GWIWG2(II), Karimi et al. 2007). This
230 fragment was PCR-amplified using high fidelity DNA polymerase and the following specific
231 primers: forward (Fc-ClusterIII), 5'-**CACCTCATTTTTGAAGCAAGTAATGAATGCA-3'**,
232 and reverse (Rc-ClusterIII), 5'-**TGGAAACCTTCCACATTCAGTTCA-3'**. The resulting PCR
233 product was directionally cloned into pENTR/D-TOPO and the *pENTR::DGK5-like* fragment
234 was sequenced before the plasmid was recombined with pH7GWIWG2(II). The final
construct was verified by restriction digestion and the fragment-intron loop-fragment was also
sequenced. To check that the construct was functional, a transient tobacco expression system
was designed (explained hereafter). Wild-type (WT) BY-2 cells were transformed as
previously described (Simon-Plas et al. 2002). Among the 30 hygromycin-resistant calli
isolated, 5 were screened and 2 independent transgenic lines (named *hp-clusterIII-A* and *hp-
clusterIII-B* lines) silenced for *NtDGK5-like* were identified and selected for further
characterization.

242 **Transient expression system in tobacco plants**

243 To test for functional miRNA constructs, *Agrobacterium tumefaciens* (GV3101) were
244 transformed with the Cluster III-targeting vector and selected clones were grown overnight
245 under mild shaking at 28°C in regular Luria-Bertani medium containing 50 µg.mL⁻¹
246 rifampicin, 50 µg.mL⁻¹ gentamycin and 50 µg.mL⁻¹ spectinomycin. Bacteria were collected
247 the following day by centrifugation and the pellets were resuspended in induction buffer (20
248 mM MES pH 5.5, 10 mM MgSO₄, 200 µM acetosyringone) so that the OD₆₀₀ nm was 0.5-0.6.
249 After incubation at room temperature for 3 h, the bacterial suspension was infiltrated into the
250 abaxial side of detached tobacco leaves (*Nicotiana tabaccum*, 4 to 6 week-old plants) using a
251 needleless syringe. Samples for RT-PCR were harvested 2 days-post inoculations. Each
252 experiment included non-infiltrated and infiltrated leaf material with bacterial clones
253 containing the empty vector as controls. Stem-loop reverse transcription reactions to show
254 specific pri-miRNA was performed on 2 independent biological replicates according to (Chen
255 2005) using the following primer: 5'-
256 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGGAAAC-3'; the
257 underscored sequence corresponding to the gene specific part of the primer whereas the other
258 part corresponds to the stem-loop backbone of the microRNA *miR172* (Martin et al. 2009).
259 Stem-loop RT controls were carried out using a 5S-rRNA-specific oligonucleotide 5'-
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACgcaaca-3'. The generated cDNA matrix was amplified by regular PCR with a gene specific-forward primer and a miR172 reverse primer (5'-GGGTCGACATCAAGTCATCAATTTGCCA-3'). For the 5S-rRNA template, the forward primer was as follows: 5S-rRNA-F, 5'-GGATGCGATCATACCAGCACT-3'; for the Cluster III-targeting miRNA, the primer used for generating the construct (Fc-ClusterIII, described above) was used. PCR products were then sequenced and proved to correspond to Cluster III pri-miRNA.

## 267 Western blot analysis

Seven-day old cells from the *OE.NtDGK5myc-A* and *OE.NtDGK5myc-B* lines and the corresponding empty vector line were harvested by filtration and protein extracts (cytosol, microsomes and plasma membrane) were prepared as described by (Mongrand et al. 2004). Proteins were solubilized in a buffer containing 40 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 1.5% SDS, 1 mM EDTA, 2M urea, 1M thiourea, 1% n-octyl-glucoside, 10% glycerol and bromophenol blue for 2h at room temperature, separated by SDS-PAGE (8% (w/v) acrylamide-bisacrylamide) and transferred onto nitrocellulose membrane (Hybond ECL; GE Healthcare, Chalfont St Giles, UK). The membrane was blocked overnight in a 5% (w/v) milk solution and probed with an anti-c-myc antibody directly coupled to horseradish peroxidase (GenScript, Piscataway, New Jersey, USA). Specific signals were revealed using an ECL kit (Perkin Elmer, Waltham, MA, USA) according to the manufacturer's instructions.

## 279 Assessment of cell suspension density

The packed cell volume (PCV) and dry weight (DW) were used as proxies of cell density. PCV was determined upon centrifugation of 15mL cell suspension samples (700 x g, 3 min). It represents the ratio of pelleted cell volume to the total sample volume, expressed in percent (Cacas et al. 2005). Dry weight was measured upon drying 12mL cell suspension aliquots at
284 55°C for 3 days; a condition that allows the culture medium to fully evaporate. It is expressed
285 in percent with reference to WT BY-2 conditions set to 100.

286 Phylogenetic analysis

287 Sequence alignment and phylogenetic tree building were carried out as described previously
288 (Cacas et al. 2011).

289
291 The MAMP cryptogein triggers rapid accumulation of phosphatidic acid in tobacco BY-
292 2 cell suspensions

293 The potential production of PA following elicitation of BY-2 tobacco cell suspensions with
294 cryptogein was investigated by treating cells with the peptide and extracting lipids for up to
295 one hour after elicitor addition. Cells were incubated with [³³Pi]-orthophosphate for 20 min
296 before lipid extraction and [³³P]-labelled PA levels were measured after lipid separation by
297 thin layer chromatography (TLC). Marked changes in labelled PA levels were observed after
298 the addition of cryptogein (Fig. 1a) and quantification of the signal showed that after a 3 min
299 lag, radiolabelled PA increased and reached a maximum within the first 10 min and then
300 plateaued (Fig. 1b).

301 PLC and DGK inhibitors reduce the accumulation of phosphatidic acid in response to
302 cryptogein

303 Under our experimental conditions, i.e. short-time radioisotopic labelling, the main pool of
304 radiolabel is incorporated into ATP whereas structural phospholipids are only weakly
305 labelled. This experimental design is commonly used to detect *in vivo* activity of lipid kinases,
306 including DGKs, because these enzymes use the γ-phosphate group of ATP as their substrate.
307 Hence, it could be postulated from our data that, in response to cryptogein, a phospholipase C
308 activity produced DAG that was then processed by DGK into PA. These two hypotheses were
309 tested by a pharmacological approach relying on the use of edelfosine, an inhibitor of
310 phosphoinositide-dependent PLC (PI-PLC; Wong *et al.* 2007; Kelm *et al.* 2010; Djafi *et al.*
311 2013) and R59022, an inhibitor of DGK (Gómez-Merino *et al.* 2004). Wild-type cell
312 suspensions were preincubated with inhibitors and radioactivity incorporated into PA was
313 quantified 10 min after cryptogein addition, i.e. when PA levels are maximal. Edelfosine was
314

found to significantly alter cryptogein-induced PA accumulation in a dose-dependent manner,
leading to a maximum inhibition of radioactive PA production of around 70% (Fig. 2a).
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Furthermore, 75 µM R59022 provoked a similar decrease of radioactive PA accumulation
317
(Fig. 2b).

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318

It was shown in *Arabidopsis thaliana* that phosphoinositides, substrates of PI-PLCs, are
provided by type III-phosphatidylinositol-4 kinases (PI4K) that are sensitive to micromolar
concentrations of wortmannin (Delage et al. 2012, Djafi et al. 2013). Pre-treatment of BY-2
cells with 30 µM wortmannin resulted in a 46% inhibition of cryptogein-induced PA
accumulation whereas 1 µM of the inhibitor, a concentration that represses
phosphatidylinositol-3 kinase activity (Krinke et al. 2007) but not that of PI4K, did not exhibit
any effect (Fig. 2c). Furthermore, since PI-PLC activity is highly dependent on calcium (Hunt
et al. 2004, Pokotylo et al. 2014), lanthanides that can act as inhibitors of plasma membrane
calcium channels (Tester 1990, Knight et al. 1997) were tested for their ability to impact PA
production upon elicitation. As expected, increasing concentrations of La³⁺ led to decreasing
PA accumulation in response to cryptogein (Fig. 2d).

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Taken together, these findings clearly point to a role for a PI-PLC/DGK pathway in
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cryptogein-induced PA production in BY-2 tobacco cell cultures.

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PLC and DGK inhibitors diminish RBOHD-mediated oxidative burst intensity in
response to cryptogein

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Among early signalling events triggered by cryptogein in BY-2 cells is the RBOHD-mediated
oxidative burst which takes place within the first 10-15 min following elicitation and is,
therefore, concomitant to the onset of PA production. To examine the hypothesis that a PI-
PLC/DGK pathway could control the oxidative burst, a pharmacological approach was
undertaken. The exogenous application of the PI-PLC inhibitor edelfosine was found to

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338 drastically alter the cryptogein-induced oxidative burst (Fig. 3a), resulting in a 70% decrease
339 of the total amount of ROS when compared to untreated WT cells (Fig. 3b). Similarly,
340 treatment with the DGK inhibitor R59022 also promoted a significant decrease in ROS levels
341 (Fig. 3c,d). Interestingly, neither PI-PLC nor DGK inhibitors led to the inhibition of
342 extracellular pH alkalization, another early signalling event associated with cryptogein
343 elicitation (Supporting information Fig. S3). As the results of both PA production and ROS
344 burst supported a working scenario where a PI-PLC/DGK pathway controls the cryptogein-
345 induced signalling cascade, a reverse genetic strategy was developed to identify DGK
346 candidates involved in the regulation of RBOHD.

347 Phylogenetic clustering of the tobacco diacylglycerol kinase family

348 Tobacco DGK nucleotide sequences were retrieved by both a keywords and a sequence
349 homology search with coding sequences of the seven identified *Arabidopsis thaliana* DGK
350 genes and those published for rice (*Oryza sativa*) and tomato (*Solanum lycopersicum*)
351 (Snedden & Blumwald 2000, Zhang et al. 2008) using the BLAST algorithm of the *Solanacea*
352 *Genomics Network* (SGN) server. No unigene could be identified, but 16 partial contigs were
353 isolated (Supporting information Fig. S4). Manual EST and contig assembly led to the
354 identification of two potential full-length mRNAs corresponding to the accessions SGN-
355 U440091 and SGN-U439985. A phylogenetic tree was built using the longest *in silico*
356 translated tobacco primary amino acid sequences, including SGN-U440091, SGN-U439985
357 and 7 additional partial coding sequences, in addition to Arabidopsis, rice and tomato
358 sequences (Fig. 4a).

359 DGKs are bipartite proteins composed of one catalytic domain and one accessory domain,
360 assumed to be necessary for the kinase activity. While the animal DGK family can be
361 subdivided into 5 classes (Goto & Kondo 2004), plant DGKs only form three phylogenetic
clusters characterized by distinct protein structures (Fig. 4a). Cluster I encompasses the closest sequence orthologous of metazoan enzymes with proteins that display in their N-terminus one or two DAG-binding domain(s) (noted C1) and either one or no transmembrane helix (Vaultier et al. 2008). Contrary to Cluster II DGKs that have a minimal DGK structure, cluster III DGKs can exhibit a C-terminal calmodulin binding region (referred to as CBD) depending on splicing variants (Arisz et al. 2009). SGN-U440091 and SGN-U439985 appear to belong to clusters II and III, respectively. Furthermore, amongst the 7 partial tobacco DGK coding sequences that were retrieved, four are present in cluster I, one in cluster II and two in cluster III.

**Overexpressing a member of the phylogenetic cluster III, *NtDGK5-like*, does not result in a gain-of-function**

Among tobacco cluster III members, the full-length cDNA corresponding to the unigene SGN-U439985 (hereafter referred to as *NtDGK5-like* after its closest *Arabidopsis thaliana* ortholog) was cloned and deposited in GenBank (Accession number KU934207). The isolated *NtDGK5-like* cDNA corresponds to a 1482 bp-long coding sequence that encodes a 493-amino acid protein displaying a molecular weight of 55.24 kDa and a theoretical pI of 6.75, with no predicted signal peptide. The *NtDGK5-like* gene is constitutively expressed in BY-2 cells but its expression is significantly up-regulated in response to cryptogein (Fig. 4b). Using transgenic lines overexpressing a C-terminal c-myc-tagged NtDGK5-like protein (named *OE.NtDGK5myc-A* and *OE.NtDGK5myc-B* lines), a cell fractioning approach was developed to identify where the protein was located. Cytosolic, microsomal and plasma membrane fractions were isolated and their proteins analysed by western blotting. The tagged protein was found in both the endomembrane and plasma membrane fractions (Fig. 4c).
To study the role of *NtDGK5-like*, the two *OE.NtDGK5myc-A* and B lines were analysed further along with a third line (named *OE.NtDGK5-A*) allowing the expression of an untagged *NtDGK5-like* protein under the control of the CaMV35S promoter. Despite a constitutive 10-to-15-fold increase in steady-state levels of *NtDGK5-like* transcripts with respect to endogenous gene expression (Fig. 5a), all three overexpressor lines accumulated PA levels comparable to those of WT BY-2 and an empty vector-containing cell line in response to cryptogein (Fig. 5b). Accordingly, no differences in ROS production could be seen between overexpressing lines and WT or empty vector-control genotypes (Fig. 5c), with the notable exception of the *OE.NtDGK5myc-B* line. This line displays a significant difference with the WT line, but not with the empty vector control line. Because cryptogein-induced oxidative burst intensity was assessed on 7-day-old cell suspensions in our experimental system, the impact of overexpressing *NtDGK5-like* on cell density was estimated at this time-point. Packed cell volume (PCV) and dry weight (DW) of overexpressing lines were measured as two independent proxies of biomass and compared to those of WT cell culture. Only the *OE.NtDGK5myc-B* line had a slight defect in cell size (Fig. 5d) and this could explain the small but significant decrease in ROS levels produced upon elicitation.

Silencing the phylogenetic cluster III DGK family of tobacco strongly inhibits cryptogein-induced oxidative burst

In an attempt to specifically silence *NtDGK5-like* gene expression, a miRNA transgenic cell line strategy was developed but it was unsuccessful due to the early death of transformed calli. However, to get insights into the function of cluster III DGKs, a construct targeting a conserved DNA region of cluster III members was engineered. A 180 bp-long portion of the *NtDGK5-like* coding sequence was cloned into the hairpin vector pH7GWIWIG2(II) (Fig. 6a).

Using an *Agrobacterium*-mediated transient expression system, this construct was shown to produce DGK specific miRNA *in planta* (Fig. 6b) and it was therefore used to generate stable
transgenic cell lines in the WT BY-2 genetic background. Amongst the 30 transformed clones obtained, two independent lines (named *hp-clusterIII-A* and *hp-clusterIII-B* lines) were identified and selected on the basis of their reduced constitutive expression of *NtDGK5-like*. While an empty vector-containing line displayed slightly higher levels of expression compared to the WT cell line, miRNA cell lines exhibited similar lower steady state-levels of *NtDGK5-like* transcripts, averaging 30 to 40% of that of WT cells (Fig. 6c). To test whether the expression of other cluster III DGK genes were affected in the two transgenic lines, Q-PCR was carried out using primers that allowed to simultaneously amplify the coding sequences of each cluster III genes. In this way, it was found that the bulk expression of cluster III genes was not altered in *hp-clusterIII-A* line whereas it was silenced approximately 3-fold in line *hp-clusterIII-B* line (Fig. 6c). A 3-fold down-regulation of *NtDGK5-like* is probably not enough to induce changes in the bulk expression of cluster III genes (see *hp-clusterIII-A* cell line). This implies that that *NtDGK5-like* is not the only cluster III isoform to be silenced in the *hp-clusterIII-B* line. On the contrary, this gene is likely to be the main cluster III-DGK isoform to be silenced in the *hp-clusterIII-A* cell line *NtDGK5-like*.

The impact of silencing DGK cluster III on cell density was estimated. No evidence for statistical differences between the four analysed genotypes (BY-2, empty vector line, *hp-clusterIII-A* and *hp-clusterIII-B* lines) on PCV and DW at day 7 after sub-culturing was found (Fig. 6d). Hence, potential effects of cluster III silencing on both PA and ROS production was determined upon cryptogein elicitation. The *DGK*-silenced lines were shown to no longer over-accumulate PA, 10 min after cryptogein addition (Fig. 7a). Remarkably, the absence of early PA accumulation in these elicited transgenics could be correlated with a marked decrease in oxidative burst intensity. Wild-type and empty-vector lines showed a similar stereotyped behaviour characterized by a rapid and transient peak of ROS, being maximum within 10-15 min post-treatment before decreasing and stabilizing for the last 45min, whereas
435 ROS profiles of the two *hp-clusterIII-* lines were devoid of a well-defined peak, exhibiting
436 rather weak and stable ROS levels over 90 min (Fig. 7b). Indeed, total levels of extracellular
437 ROS generated by the miRNA lines accounted for approximately one third to one half of
438 those recorded in WT and empty vector-containing lines upon cryptogein application (Fig.
439 7c).
440 DISCUSSION

441 In the present work, radio-labelling experiments clearly showed that *in vivo* levels of PA
442 rapidly increased when BY-2 cell suspensions were challenged with cryptogein, reaching a
443 maximum after 10 min and then staying stable during 1 h. Under our experimental conditions,
444 [$^{33}$Pi]-orthophosphate was added to cell cultures 20 min before lipid extraction. In contrast to
445 long time periods of isotope incubation that lead to labelled structural phospholipids, short
446 time labelling results in the incorporation of isotopic phosphorus into ATP. Hence, this setup
447 is optimized to detect products of lipid kinases using ATP as substrate (Vaultier et al. 2006)
448 and being active within the duration of labelling. These kinase activities include DGK and
449 others, such as PI4-kinase and PI4P-5-kinase. Indeed, the radioactivity incorporated into PI4P
450 is typically 4- to 5-fold that of PI, in control cells, although PIP (not labelled) is much less
451 abundant than PI (Furt et al. 2010). However, this experimental design is not optimal for
452 detecting the possible occurrence of PLD activity. The radioactivity associated with PA upon
453 cryptogein treatment represented 3- to 4-fold of that monitored in phosphatidylcholine,
454 phosphatidylethanolamine and phosphatidylglycerol, putative substrates of PLDs. This
455 strongly supports the idea that radiolabelled PA found after a 20 min labelling cannot arise
456 from PLD action. Yet, this does not mean a PLD is not activated upon cryptogein addition. A
457 long time labelling with [$^{33}$Pi]-orthophosphate (16 hours) designed to reveal PLD activity was
458 also carried out. In the presence of primary alcohol *n*-butanol, PLD catalyses a trans-
459 phosphatidylation reaction transferring the phosphatidyl moiety from structural lipids onto the
460 hydroxyl group of the primary alcohol. In our hands, no radiolabeled phosphatidylbutanol
461 accumulated in response to cryptogein (data not shown). However, since the signal-to-noise
462 ratio was low in these experiments, we cannot rule out the possible activation of PLD by
463 cryptogein.
The fact that the increase in radiolabelled PA in our experimental design was due to DGK was indicated by the pharmacological approach where DGK inhibitors led to an inhibition of radiolabelled PA accumulation. Moreover, the inhibiting effects of wortmannin and edelfosine strongly suggest that the DAG substrate of DGK was produced by a PI-PLC activity, the substrate of which is provided by a type III PI4K. In *Arabidopsis thaliana*, it has been shown that PI-PLC substrates are provided by such PI4Ks, either for basal PI-PLC activity (i.e. active in control cell, Djafi et al. 2013) or during PI-PLC activation by chilling (Delage et al. 2012). Therefore it seems that the role of type III PI4Ks to supply substrates to PI-PLC is likely to be the case for other plant species, including tobacco. Besides, PI-PLCs are strictly dependent on calcium and therefore it was not surprising that a blocker of plasmalemma calcium channels, lanthanides, inhibited radioactive PA accumulation.

Since inhibitors of different chemical nature acting on different enzymes (PI4K, PI-PLC, DGK) of a given pathway led to a similar inhibition of cryptogein-induced accumulation of radiolabelled PA, it is unlikely that this is due to hypothetical side effects of each inhibitor (Djafi et al. 2013, Ruelland et al. 2014). Moreover, silencing the phylogenetic cluster III of tobacco DGKs resulted in the loss of the cryptogein-induced PA increase thus demonstrating unambiguously the involvement of such enzymes in this response to cryptogein. Our knowledge concerning plant DGKs is still limited. Prokaryotic DGKs exhibit a peculiar multimeric membrane-embedded structure with three transmembrane spanning-regions per subunit (Van Horn & Sanders 2012). Metazoan counterparts display a conserved bidomain architecture associated with catalytic activity and decorated by a huge diversity of additional functional domains that are responsible for membrane or protein interactions and subcellular localization (Goto & Kondo 2004). Predictions of plant DGK structures using the Conserved Domain Architecture Retrieval Tool (Geer et al. 2002) reveal a simpler organization that falls into three evolutionary phyla (Vaultier et al. 2008). In this work, we focused our efforts on
489 DGKs of phylogenetic cluster III. Two members of this cluster have been either
490 biochemically or functionally characterized. The rice DGK1 was stably expressed in tobacco
491 plants and found to confer enhanced disease resistance to tobacco mosaic virus (TMV) and to
492 the oomycete *Phytophthora infestans* (Zhang et al. 2008). Two distinct spliced mRNA
493 variants were reported for the tomato LeDGK1/CBK1-encoded gene and this transcriptional
494 regulatory mechanism was demonstrated to regulate protein docking at the plasma membrane
495 (Snedden & Blumwald 2000) In addition, the *AtDGK5* gene expression was found to be
496 upregulated in response to flagellin 22, a bacterial MAMP, as well as several avirulent
497 pathogens (GENEVESTIGATOR database, Hruz et al. 2008).

498 In this work, one member of cluster III was cloned and named *NtDGK5-like* after its closest
499 Arabidopsis ortholog. Independent transgenic cell lines were generated that allowed
500 constitutive overexpression of either native *NtDGK5-like* (*OE.NtDGK5-A*) or a C-terminally
501 c-myc tagged version of the protein (*OE.NtDGK5myc-A* and *OE.NtDGK5myc-B*). Despite the
502 absence of any predicted signal peptide and transmembrane domain, western blotting
503 established that the protein was localized to the plasma membrane, as already reported for a
504 LeDGK1/CBK1 variant from tomato (Snedden & Blumwald 2000). In all *NtDGK5-like*
505 overexpressing lines the accumulation of radioactive PA after 10 min of cryptogein addition
506 was similar to that of control and WT lines. Thus it is tempting to speculate that DGK is not a
507 limiting step in the coupled reaction leading to PA in response to cryptogein.

508 Previously, we had established that early cryptogein-induced ROS synthesis was fully
509 dependent on the isoform D of the RBOH family in BY-2 cell suspensions (Simon-Plas et al.
510 2002). In addition, Zhang et al. (2009) demonstrated that PA could directly interact with the
511 Arabidopsis RBOHD isoform *via* an N-terminally-localized PA-binding domain and,
512 subsequently, stimulate its activity. Alignment of the highly homologous tobacco RBOHD
513 isoform with its Arabidopsis counterpart pinpointed that the location and sequence of the PA-
binding domain, as well as the basic nature of the two amino acid residues involved in the interaction with PA, are conserved between the two plant species (Supporting information Fig S5). Both PI-PLC and DGK inhibitors reduced the RBOHD-dependent oxidative burst. Furthermore, transgenic lines deficient for the tobacco cluster III DGKs displayed a significantly reduced oxidative burst upon cryptogein addition. Such findings strongly point to a direct regulation of RBOHD activity by PA, and place the PI-PLC/DGK pathway upstream from the enzyme in a signalling cascade induced by the MAMP cryptogein. It can be postulated that at least one DGK member of cluster III is responsible for the required DGK activity. Even though our genetic strategy concerning *NtDGK5-like* was partly unsuccessful, this gene still remains a good candidate since it was the only cluster III DGK gene suppressed in the *hp-clusterIII-A* cell line.

Distinct regulatory mechanisms have been described for plant RBOH proteins. These include post-translational modifications like phosphorylation (Kobayashi et al. 2007, Ogasawara et al. 2008) and S-nitrosylation (Yun et al. 2011), association with RacGTPase (Wong et al. 2007b, Oda et al. 2010) as well as regulation by extracellular ATP level and calcium influx (Song et al. 2006, Demidchik et al. 2009). Zhang *et al.* (2009) also elegantly demonstrated that direct binding of PA activates AtRBOHD, *in fine* controlling stomatal aperture upon drought stress. Contrary to our model, where a PI-PLC/DGK pathway is involved, the authors showed that PLDα1 was the main source of PA under their experimental conditions. In the context of plant defence, whether it is MTI or ETI, to date there is no genetic evidence for a role of DGKs in the positive regulation of RBOH enzymes, even though it has been suggested previously by several works (de Jong et al. 2004, Andersson et al. 2006, Vossen et al. 2010). Instead, many reports indicate PLDs as the main suppliers of PA in response to MAMP/pathogen effectors, irrespective of the downstream protein target (Young et al. 1996, van der Luit et al. 2000, de Torres Zabela et al. 2002, Bargmann et al. 2006, Laxalt et al. 2007, Krinke et al. 2009,
Yamaguchi et al. 2009, Kalachova et al. 2013, Zhao et al. 2013, Pinosa et al. 2013). Thus, our work sheds light on new insights into regulatory mechanisms that control early signalling events associated with NADPH oxidase since PA produced by a PI-PLC/DGK pathway can also control NADH oxidase activity. As underlined by (Zhang & Xiao 2015), this raises interesting questions as to how the spatiotemporal regulation of PA neo-synthesis and the engaged molecular species of PA could influence the specificity of downstream responses.
546 ACKNOWLEDGMENTS

547 This work was supported by the French National Research Agency (ANR, Programme Blanc
548 PANACEA NT09_517917), the Bordeaux Metabolome Facility-MetaboHUB (grant no.
549 ANR-11-INBS-0010 to S.M.) and platform Métabolome-Lipidome-Fluxome of Bordeaux
550 (contribution toward lipid analysis equipment). We would like to thank Dr. Olivier Lamotte
551 and Pr. Nathalie Leborgne-Castel for their support and stimulating discussions, and Dr
552 Michael Hodges, IPS2, Orsay France for carefully reading the manuscript. The authors have
553 no conflict of interest to declare.

554 AUTHOR'S CONTRIBUTION: JLC, PGP, JF, ER, CC, DT, TK & KM carried out
555 experiments; FSP, SM, EJ, PGP, JLC & ER designed experiments and discussed results; JLC,
556 PGP & ER wrote the manuscript; SM & FSP coordinated the project and retrieved ANR
557 funds.

558
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798
799 FIGURE LEGENDS

800
**Figure 1:** Early accumulation of phosphatidic acid in response to cryptogein. Lipids were extracted at different time points after cryptogein addition. Cells were radiolabeled for 20 min before lipid extraction. (a) TLC plates were developed in an isooctane based solvent system. (b) The radioactivity associated with PA was quantified relative to the radioactivity associated with all others phospholipids (PL) and then expressed as % of the highest value. For each point of the kinetics, 3 flasks of tobacco cells were treated independently with cryptogein. Data are mean ± SD, *n*=3. Note that the control error bars are within the symbol.

808
**Figure 2: Decrease in cryptogein-induced phosphatidic acid accumulation by PLC and DGK inhibitors.** Edelfosine (a) and R50022 (b) were used as PI-PLC and DGK inhibitors, respectively. Wortmannin (c) and lanthanum (d) were used as type III-PI4K and calcium channel inhibitors, respectively. Cells were radiolabeled for 10 min and then cryptogein was added. Lipids were extracted at 10 min after cryptogein addition. When necessary, inhibitors were added 15 min before the cryptogein. Labelling was carried out during 20 min for each point. Extracted lipids were separated using TLC plates and developed in an acidic solvent system. Radioactivity in PA spots was quantified relative to PE and results are expressed as % of that in cryptogein treated cells. For each treatment, 3 flasks of tobacco cells were used to be elicited or not with cryptogein in presence or not of inhibitors. The data are mean ± SD, n=3. Results were analysed using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison *post hoc* test. Different letters indicate a significant difference (Tukey HSD, P<0.05).

822
823

**Figure 3: Inhibition of RBOHD-dependent oxidative burst by both DGK and PLC inhibitors.** Time course of ROS production of WT BY-2 cells pretreated with either R59022, an inhibitor of DGK activity (a), or edelfosine, an inhibitor of PLC activity (c). Total amounts of ROS produced over a 90 min period following elicitation were calculated for cell suspensions treated with R59022 (b) or edelfosine (d). Cells were preincubated in I20 medium for 30 min with the inhibitor at the indicated concentrations before cryptogein was added.

824

825

826

827

828

829

830

831

832

Extracellular ROS synthesis was regularly quantified using a luminol-based method. Data are expressed as nmoles of H₂O₂ equivalents per gram of cells. Means and SD for 3 independent replicates. Statistical difference for the total ROS level was assessed by a Student T-test, where * indicates P<0.05 and ** refers to P<0.01.

833
**Figure 4: Phylogenetic clustering of tobacco DGKs and characterization of one member of cluster III.** (a) Phylogenetic cluster of plant DGKs. Nucleic acid sequences were retrieved for DGKs from the *Solanacea Genomics Network* (SGN) database (https://solgenomics.net/) using the BLAST algorithm and keyword search tool. Known Arabidopsis, rice and tomato DGKs were used as nucleic acid sequence queries. The phylogenetic analysis was carried out using identified nucleic acid sequences (indicated by their SGN references) as previously described (Cacas et al. 2011). Plant DGKs were grouped into 3 phyla, each showing slightly different protein domain structures. **Abbreviations:** C1, C1 DAG binding domain; CBD, calmodulin-binding domain; DGKcat, DGK catalytic domain; DGKacc, DGK accessory domain. For plant species, At refers to *Arabidopsis thaliana*, Le to *Solanum esculentum* (previously *Lycopersicum esculentum*), Nt to *Nicotiana tabacum* and Os to *Oryza sativa* (b) Time course of *NtDGK5-like* gene expression in response to cryptogein. Gene expression was determined by Q-PCR as described in the *Material and Methods*. Data are means and SD for 3 independent biological replicates (n=3 samples per cell line). (c) Subcellular localization of *NtDGK5-like* protein. Cells from overexpressing *NtDGK5-like-c-myc* lines (*OE.NtDGK5myc-A* and *OE.NtDGK5myc-B*) and the corresponding empty vector-containing line (i.e. line transformed with an empty pMDC32) were harvested 7 days after subculture. Proteins were extracted from cytosolic (lanes 1), microsomal (lanes 2) and plasma membrane (lanes 3) fractions. After western blotting using anti-c-myc antibodies, a specific signal was only found in membrane fractions. Two independent biological replicates were carried out (n=2 samples per cell line).
**Figure 5: Characterization of *NtDGK5-like* overexpressing transgenic lines.** (a) Expression levels of *NtDGK5-like* overexpressing lines. Gene expression was determined by Q-PCR using primers that amplify both the endogenous and transgene-encoding transcripts. Data are means and SD for 4 independent experiments. These experiments were carried out on material obtained from untreated 7-day-old cells. BY-2, WT cells; the empty vector-containing line is a BY-2 line transformed with an empty pMDC32; *OE.NtDGK5-A*, line overexpressing an untagged version of NtDGK5-like protein; *OE.NtDGK5myc-A* and *OE.NtDGK5myc-B* are independent lines overexpressing a C-terminally tagged version of NtDGK5-like protein. (b) Radioactivity associated with PA in cryptogein-elicited overexpressors. Cells were radiolabeled for 20 min before lipid extraction. Lipids were extracted 10 min after cryptogein addition. TLC plates were developed in an acid solvent system. The radioactivity associated with PA was normalized to that of PE and PC. The data are expressed as % of the value in the empty vector control line elicited with cryptogein. Data are means and SD, n= 3. (c) Total ROS levels produced during a 90 min period (same experimental design as that described in the legend of figure 4). Data are expressed as percent compared to the ROS level of BY-2 cells being set to 100 (corresponding to 500 nmoles H₂O₂ eq./g of cells). Means and SD for 3 independent experiments. Statistical differences for the total ROS level was assessed using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison *post hoc* test. Different letters indicate a significant difference, Tukey HSD, P<0.05. (d) Cell density of untreated transgenic lines. Packed cell volume (PCV) and dry weight (DW), were expressed in percent. Presented data are means and SD for 6 independent experiments (n=6 samples for each cell line). Control (empty vector) and overexpressors were compared to WT BY-2 suspensions using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison *post hoc* test. For PCV, different letters indicate a significant difference, Tukey HSD, P<0.05. No
881 significant difference could be detected for DW (Tukey HSD, P<0.05.) significant differences

882 (Tukey HSD, P<0.01) in PCV and DW between genotypes were detected. Letters marked

883 with an apostrophe (') are used for DW data.

884
<caption>**Figure 6: Characterization of DGK Cluster III-targeted miRNA transgenic cell lines. (a)**</caption>

Scheme of the nucleic region targeted by the miRNA construct (see also Supporting information Fig. S2). (b) Evidence for miRNA production *in planta*. The cluster III-specific miRNA construct was expressed in tobacco leaves using *Agrobacterium tumefaciens* (GV3101). Foliar samples were harvested 2 day-post inoculation. Total RNAs were extracted, the cluster III pri-miRNA was specifically reverse transcribed using a stem-loop system and generated cDNA used as template for PCR amplification (expected size of PCR product=181bp). Sequencing showed PCR products to be the hairpin construct. (c) Expression levels of cluster III and *NtDGK5-like* genes in miRNA cell lines. Expression levels were quantified by Q-PCR on samples obtained from untreated 7-day-old cell suspensions. Means and SD are from 4 independent biological replicates (n=4 samples for each cell line). The empty vector line corresponds to a line transformed with an empty pH7GWIWG2(II). (d) Cell suspension density of 7 day-old transgenic lines. Packed cell volume (PCV) and dry weight (DW), are expressed as a %. Presented data are means and SD for 6 independent biological replicates (n=6 samples for each cell line). The empty vector line and the *hp-clusterIII-A* and *hp-clusterIII-B* cell lines were compared to WT BY-2 suspensions using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison *post hoc* test. No significant differences (Tukey HSD, P<0.01) in PCV and DW between genotypes were detected. Letters marked with an apostrophe (') are used for DW data.
**Figure 7:** Silencing DGK Cluster III of tobacco abolishes PA production and strongly inhibits the oxidative burst in response to cryptogein. (a) Levels of PA in response to cryptogein. Cells were radiolabeled for 20 min before lipid extraction. Lipids were extracted 10 min after cryptogein addition. TLC plates were developed using an acid solvent system. The radioactivity associated with PA was normalized to that of PE and PC. For each line, the data are expressed as % of the value of the non-elicited cells. For each treatment, 3 flasks of cells were used and the data are mean ± SD, n=3. Statistical difference between treated and non-treated conditions was assessed by a Student T-test, where * indicates P<0.05. (b) Time-course of ROS levels following elicitation with cryptogein. Data are means and SD for 8 independent biological replicates (n=8 samples per kinetic point for each cell line). (c) Total amounts of ROS produced over a 90 min period after cryptogein addition (same data as in panel b). Statistical differences was assessed using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P<0.01.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>921 **Figure 1**</figcaption></figure>

Cryptogein treatment (min)
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 3</figcaption></figure>

925

926
<figure><img src="image_12.png" /><figcaption>(a)</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_14.png" /></figure>

927

928
<figure><img src="image_15.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_18.png" /></figure>

Figure 5

929

930
<figure><img src="image_19.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_22.png" /></figure>

Figure 6

931

932
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

Figure 7

933

934
935 SUPPORTING INFORMATION

936 **Supporting information figure S1:** The translated coding sequence of the tobacco NtDGK5-
937 like gene. The identified nucleic acid sequence was deposited in GenBank under the reference
938 KU934207. It codes for a 493-amino acid-long protein (55.24kDa), having a predicted pI of
939 6.75. *In silico* translation was performed using the freeware ORF Finder available at the
940 NCBI website (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).

941 **Supporting information figure S2:** Partial alignment of plant DGK coding sequences from
942 clusters II and III. This figure shows the nucleic acid coding region used for miRNA design
943 (squared in red), which is highly conserved among cluster III members and divergent with
944 cluster II counterparts. Full-length nucleic acid sequences were aligned using version 2.0.10
945 of Clustal X (Larkin et al. 2007) and the resulting alignment was processed online at the
946 BoxShade server (http://www.ch.embnet.org/software/BOX_form.html). Roman numbers (II
947 and III) on the right of the alignment indicate DGK cluster.

948 **Supporting information figure S3:** Effects of inhibitors on the alkalization triggered by
949 cryptogein. Cells were preincubated 15 min with inhibitors (or DMSO, solvent of the
950 inhibitors) before cryptogein (50 nM) elicitation. Final concentrations of edelfosine, R59022
951 and R59949 were 25 µM, 75 µM and 75 µM, respectively. R59949 is another DGK inhibitor.
952 Extracellular pH alkalinization was measured and reported as the difference between initial
953 and final (1 hour of cryptogein treatment) pH values. Means +/- SD.

954 **Supporting information figure S4:** Topology of the sixteen identified tobacco DGK
955 proteins. Protein structure is depicted for the 3 phylogenetic clusters. DGK nucleic acid
956 sequences were retrieved from the *Solanacea Genomics Network* (SGN) database
957 (https://solgenomics.net/) using the BLAST algorithm and keyword search tool. Known
958 Arabidopsis, rice and tomato DGKs were used as nucleic acid sequence queries. Upon *in*
silico translation using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), the
resulting polypeptides were compared to their Arabidopsis counterparts and the Conserved
Domain Architecture Retrieval Tool (CDART, (Geer et al. 2002)) was used to predict domain
location. Within a panel (corresponding to one protein cluster), the closest Arabidopsis
relative is followed by orthologous tobacco translated sequences. The latter are referenced by
their SGN numbers and the identified part of the protein is presented. The length of the
corresponding contigs is also indicated in green. Abbreviations: AtDGK5lv, AtDGK5 long
splice variant; AtDGK5sv, AtDGK5 short splice variant; C1, DAG binding domain; CBD,
calmodulin-binding domain; DGKcat, DGK catalytic domain; DGKacc, DGK accessory
domain. The grey rectangle localized to the N-terminal part of AtDGK1 represents a
transmembrane domain.

**Supporting information figure S5:** Identification of the PA binding domain of NtRBOHD by alignment with the cognate domain of Arabidopsis isoform D. Full length primary amino-acid sequences of RBOHD from *Arabidopsis thaliana* (SwissProt:Q9FIJ0.1) and *Nicotiana tabacum* (GenBank:ABN58915) were aligned using version 2.0.10 of Clustal X (Larkin et al. 2007) and the resulting alignment was then processed online at the BoxShade server (http://www.ch.embnet.org/software/BOX_form.html). Stars indicate the two basic amino-acid residues experimentally proven to be critical for interaction with PA (Zhang *et al.* 2009). Note the conservation of these residues between the two proteins.