1 Running head: Sphingolipids in plant defense responses 2 ...€¦ · 16/9/2015 · 164 in response to B. cinerea infection and in comparison to Pst infection. For this 165 purpose,

Running head Sphingolipids in plant defense responses 1

Corresponding author Sandrine Dhondt-Cordelier 3

Uniteacute de Recherche Vigne et Vin de Champagne 4

(URVVC-EA 4707) Laboratoire Stress Deacutefenses et 5

Reproduction des Plantes Universiteacute de Reims 6

Champagne-Ardenne BP 1039 F-51687 Reims 7

cedex 2 France 8

Telephone +33 326 918 587 10

Email sandrinecordelieruniv-reimsfr 11

Research area Signaling and Response 14

Plant Physiology Preview Published on September 16 2015 as DOI101104pp1501126

Modifications of sphingolipid content affect tolerance to 18

hemibiotrophic and necrotrophic pathogens by modulating 19

plant defense responses in Arabidopsis 20

Magnin-Robert Maryline1 Le Bourse Doriane2 Markham Jonathan2 Steacutephan 22

Dorey1 Cleacutement Christophe1 Baillieul Fabienne1 and Dhondt-Cordelier 23

Sandrine1 24

25 1 Uniteacute de Recherche Vigne et Vin de Champagne (URVVC-EA 4707) 26

Laboratoire Stress Deacutefenses et Reproduction des Plantes SFR Condorcet FR 27

CNRS 3417 Universiteacute de Reims Champagne-Ardenne BP 1039 F-51687 28

Reims cedex 2 France 29 2 Center for Plant Science Innovation and Department of Biochemistry 30

University of Nebraska-Lincoln Beadle Center 1901 Vine Street Lincoln NE 31

68588 USA 32

One-sentence summary 35

Sphingolipids play a key role in plant defense towards different lifestyle 36

pathogens by modulating cell death ROS accumulation and jasmonate 37

signaling pathway 38 39

Footnotes 41 42 This work was supported in part by a grant (EliDeRham project ndash A2101-03) 43 from the Region Champagne-Ardenne 44

45 46 Corresponding author Sandrine Dhondt-Cordelier 47

Email sandrinecordelieruniv-reimsfr 48 49 50

ABSTRACT 52

Sphingolipids are emerging as second messengers in programmed cell death 54

and plant defense mechanisms However their role in plant defense is far from 55

being understood especially against necrotrophic pathogens 56

Sphingolipidomics and plant defense responses during pathogenic infection 57

were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase 58

encoded by the AtDPL1 gene and regulating LCBLCB-P homeostasis Atdpl1 59

mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but 60

susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv 61

tomato (Pst) Here a direct comparison of sphingolipid profiles during infection 62

with pathogen differing in lifestyles is described In contrast to LCBs (d180 and 63

d182) hydroxyceramide and LCB-P (t180-P and t181-P) levels are higher in 64

Atdpl1-1 than in WT plants in response to B cinerea Following Pst infection 65

t180-P accumulates more strongly in Atdpl1-1 than in WT plants Moreover 66

d180 and t180-P appears as key players in Pst- and B cinerea-induced cell 67

death and reactive oxygen species accumulation Salicylic acid (SA) levels are 68

similar in both types of plants independently of the pathogen In addition SA-69

dependent gene expression is similar in both types of B cinerea-infected plants 70

but is repressed in Atdpl1-1 after treatment with Pst Both pathogen infection 71

triggers higher jasmonic acid (JA) JA-Ile accumulation and JA-dependent gene 72

expression in Atdpl1-1 mutants Our results demonstrate that sphingolipids play 73

an important role in plant defense especially towards necrotrophic pathogen 74

and highlight a novel connection between jasmonate signaling pathway cell 75

death and sphingolipids 76

INTRODUCTION 79

Plants have evolved a complex array of defenses when attacked by 81

microbial pathogens The success of plant resistance firstly relies on the 82

capacity of the plant to recognize its invader Among early events a transient 83

production of reactive oxygen species (ROS) known as oxidative burst is 84

characteristic of successful pathogen recognition (Torres 2010) Perception of 85

pathogen attack then initiates a large array of immune responses including 86

modification of cell walls as well as the production of anti-microbial proteins and 87

metabolites like pathogenesis-related (PR) proteins and phytoalexins 88

respectively (Schwessinger and Ronald 2012) The plant hormones salicylic 89

acid (SA) jasmonic acid (JA) and ethylene (ET) are key players in the signaling 90

networks involved in plant resistance (Bari and Jones 2009 Tsuda and 91

Katagiri 2010 Robert-Seilaniantz et al 2011) Interactions between these 92

signal molecules allow the plant to activate andor modulate an appropriate 93

array of defense responses depending on the pathogen lifestyle necrotroph or 94

biotroph (Glazebrook 2005 Koornneef and Pieterse 2008) Whereas SA is 95

considered as essential for resistance to (hemi)biotrophic pathogens it is 96

assumed that JA and ET signaling pathways are important for resistance to 97

necrotrophic pathogens in Arabidopsis (Thomma et al 2001 Glazebrook 98

2005) A successful innate immune response often includes the so-called 99

hypersensitive response (HR) a form of rapid programmed cell death (PCD) 100

occurring in a limited area at the site of infection This suicide of infected cells is 101

thought to limit the spread of biotrophic pathogens including viruses bacteria 102

fungi and oomycetes (Mur et al 2008) 103

During the past decade significant progress has been made in our 104

understanding of the cellular function of plant sphingolipids Besides being 105

structural components of cell membranes sphingolipids are bioactive 106

metabolites that regulate important cellular processes such as cell survival and 107

PCD occurring during either plant development or plant defense (Dunn et al 108

2004 Berkey et al 2012 Markham et al 2013) First evidence of the role of 109

sphingolipids in these processes came from the use of the fungal toxins 110

fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111

alternata f sp lycopersici These toxins are structural sphingosine (d181) 112

analogs and function as ceramide synthase inhibitors They triggered PCD 113

when exogenously applied to plants Mutant strains in which production of such 114

toxin is abrogated failed to infect the host plant implying that toxin 115

accumulation is required for pathogenicity and that induction of plant PCD could 116

be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117

2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118

bases (LCBs) are also potent inducers of PCD in plants For example 119

exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120

in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121

al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122

Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123

PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124

t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125

Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126

phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127

accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128

participation in induction of HR and associated PCD also came from studies on 129

accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130

mutants which displayed over-accumulation of Cers These mutants exhibited 131

spontaneous cell death and resistance to biotrophic pathogen which seemed to 132

be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133

2008) Altogether these data provide evidence of a link between PCD defense 134

and sphingolipid metabolism However the fatty acid hydroxylase 12 135

(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136

to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137

display a PCD-like phenotype suggesting that Cers alone are not involved in 138

the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139

(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140

primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141

double mutant completely lacking trihydroxy-LCBs showed enhanced 142

expression of PCD marker genes (Chen et al 2008) On the contrary increase 143

in t180 was specifically sustained in plant interaction with the avirulent Pst 144

strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145

Thus the nature of sphingolipids able to induce PCD is still under debate and 146

may evolve depending on plants and their environment Phosphorylated form of 147

LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148

dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149

blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150

PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151

rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152

FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153

AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154

treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155

existence of a rheostat between LCBs and their phosphorylated forms that 156

controls plant cell fate toward cell death or survival 157

Data on plant sphingolipid functions are still fragmentary Only few reports 158

described interconnections between sphingolipids cell death and plant defense 159

responses almost exclusively in response to (hemi)biotrophic pathogens 160

Knowledge about such relation in response to necrotrophic pathogen is still in 161

its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162

the link between sphingolipids cell death and plant defense has been explored 163

in response to B cinerea infection and in comparison to Pst infection For this 164

purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165

displaying any phenotype under standard growth conditions (Tsegaye et al 166

2007) have been analyzed after pathogen infection Our results revealed that 167

modification of sphingolipid contents not only impacted plant tolerance to 168

hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169

signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170

plants JA signaling pathway is significantly enhanced Cell death and ROS 171

accumulation are markedly modified in Atdpl1-1 mutant plants We further 172

demonstrated that t180-P and d180 are key players in pathogen-induced cell 173

death and ROS generation Here we thus established a link between JA 174

signaling PCD and sphingolipid metabolism 175

RESULTS 178

Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179

plant response 180

In order to assess the role of sphingolipids in the plant immune responses to 181

necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182

affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183

2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184

to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185

(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186

defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187

Arabidopsis (Glazebrook 2005) To get some information about the 188

susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189

or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190

thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191

similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192

plants disease symptoms showing chlorosis and necrosis increased more 193

rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194

symptoms developed in response to Pst infection were more pronounced in 195

mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196

and 60 h after drop-inoculation with B cinerea and classified in size categories 197

(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198

largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199

of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200

plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201

percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202

approximately 45 and 65 of small lesions whereas WT showed only 17 203

and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204

lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205

1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206

than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207

with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208

were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209

strain growth was less important compared to virulent strain in WT plants (Fig 210

1 C and D) Interestingly infection with both bacterial strains revealed an 211

increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212

growth as compared with WT plants (Fig 1 C and D) These results were also 213

correlated by fungal and bacterial population quantification in infected leaves by 214

qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215

either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216

AtDPL1 transcript accumulation could be observed AtDPL1 expression 217

significantly increased between 12 and 24 hpi and continuously rose until the 218

later stages of infection Symptoms due to either B cinerea invasion as well as 219

infection with virulent or avirulent strain of Pst visually appeared between 24 220

and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221

expression Deregulation of photosynthesis is considered as a tool for 222

evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223

2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224

15-bisphosphate carboxylase) after pathogen infection occurred at the same 225

time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226

symptom appearance (Supplemental Fig S1) suggesting that an immediate 227

consequence of pathogen perception includes induction of AtDPL1 gene 228

expression Collectively these data indicate that lack of AtDPL1 activity in 229

mutant plants significantly delays the development of lesions triggered by B 230

cinerea infection but renders plants more susceptible to Pst infection 231

Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233

upon pathogen infection 234

To determine whether changes in the level of certain sphingolipids are 235

responsible for the delayed development of B cinerea infection in Atdpl1 236

mutant sphingolipid profiles were analyzed The main sphingolipid species in 237

Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238

(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239

glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240

both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241

Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242

than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243

As previously described the only significant alteration in sphingolipid basal 244

levels observed in Atdpl1-1 mutant compared to WT under typical growth 245

conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246

2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247

infection on the sphingolipid profile in WT plants B cinerea infection triggered 248

LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249

moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250

compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251

and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252

and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253

than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254

(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255

also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256

4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257

g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258

presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259

modified (Supplemental Fig S4) however significant accumulation of saturated 260

α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261

observed after challenge with B cinerea (Fig 5 A and C) No change could be 262

noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263

better understand the role of sphingolipids in plant resistance to the 264

necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265

infected Atdpl1-1 mutant and WT plants was then performed With respect to 266

the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267

especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268

accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269

t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270

and F) The amount of total GIPCs and more precisely saturated α-271

hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272

was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273

cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274

S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275

Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276

contents especially saturated and mono-unsaturated VLCFA-containing hCers 277

(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278

DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279

accumulated three times in the mutant compared to WT plants in response to 280

the fungus (Fig 5 C and D) No significant change was observed in total 281

GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282

In order to compare sphingolipid profile in response to an hemibiotrophic 283

pathogen analyses were performed 48 h after WT plant inoculation with 284

avirulent or virulent strains of Pst Our data confirmed previous results showing 285

that sphingolipid increase was more sustained during the incompatible than 286

compatible interaction (Peer et al 2010) Increase in t180 was observed in 287

response to both types of bacteria but infection with only Pst AvrRPM1 288

triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289

AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290

only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291

levels were also not significantly modified in response to both types of bacteria 292

(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293

t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294

E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295

Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296

of the incompatible interaction than in the case of the compatible one (40 vs 24 297

nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298

also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299

only C16-Cer accumulation was more pronounced in the case of interaction with 300

Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301

(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302

to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303

strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304

Similarly to B cinerea infection no regulation of GclCer content could be 305

noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306

sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307

revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308

infiltration since it was also observed in control plants An increase in t180-P 309

level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310

in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311

Cer hCer or GclCer pools was observed in response to either virulent or 312

avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313

Changes in sphingolipid profiles affect pathogen-induced cell death 315

Recently several reports have revealed that some sphingolipids are important 316

players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317

HR is an effective strategy of plants to protect themselves against 318

(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319

promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320

Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321

differences in tolerance upon B cinerea or Pst infection prompted us to 322

examine cell death response upon pathogen attack We thus measured 323

electrolyte leakage to detect changes in loss of ions caused by plasma 324

membrane damage characteristic of plant cell death (Dellagi et al 1998 325

Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326

plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327

B) These results suggested that modification in sphingolipid content could play 328

a role in modulating cell death processes in response to pathogen infection 329

Expression levels of PCD marker genes such as flavin-containing 330

monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331

(Brodersen et al 2002) were also evaluated in order to verify if cell death 332

responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333

were induced in both types of plants with increasing infection spread of B 334

cinerea Interestingly these inductions occurred earlier and stronger in WT 335

(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336

7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337

and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338

than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339

As expected in the case of Pst infection SAG13 and FMO gene expressions 340

were induced earlier and stronger during the incompatible interaction than 341

during the compatible interaction (Fig 7 B and D) WT and mutant plants 342

displayed similar expression profiles with both types of bacteria however 343

induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344

infection SAG12 transcript accumulation occurred only at the later stages of the 345

infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346

followed a similar pattern than AtDPL1 gene expression in WT plants in 347

response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348

SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349

are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350

al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351

after either B cinerea or Pst infection could result from a HR-like PCD whereas 352

a senescence program is activated later This could also explain the tolerance 353

of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354

towards Pst 355

Modification of sphingolipid contents affect ROS production in response 357

to pathogen infection 358

Transient production of ROS is a hallmark of successful pathogen recognition 359

(Torres 2010) To investigate whether sphingolipid content perturbation in 360

Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361

the mutant versus WT plants WT plants displayed a transient oxidative burst 362

peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363

or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364

times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365

On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366

plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367

demonstrated that signaling events linked to pathogen recognition are affected 368

by sphingolipid perturbation in Atdpl1-1 plants 369

Exogenous t180-P and d180 differently modifies pathogen-induced cell 371

death and ROS production 372

Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373

plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374

We thus tested the ability of these sphingolipids to modulate pathogen-induced 375

cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376

exogenous t180-P or d180 alone did not affect cell death or ROS production a 377

finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378

WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379

was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380

not modify disease symptoms and electrolyte leakage in WT-infected plants by 381

B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382

strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383

leakage were strongly reduced when WT plants were co-infiltrated with d180 384

and Pst AvrRPM1 (Fig 9 B and F) 385

Whereas addition of t180-P increased and delayed ROS production upon 386

challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387

C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388

(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389

(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390

d180 modify signaling event and cell death triggered by infection with these two 391

pathogens 392

SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394

after pathogen challenge 395

Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396

in differential activation of defense responses after pathogen infection PR1 and 397

PR5 are well-known SA-dependent defense marker gene Nonexpressed 398

Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399

suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400

expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401

responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402

pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403

plants No significant difference in expression of these defense genes was 404

detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405

(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406

LCB-P lyase itself did not result in any defense response changes in plants 407

The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408

then compared to WT plants in response to B cinerea infection (Fig 11) 409

Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410

levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411

markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412

was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413

JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414

regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415

in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416

that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417

Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418

significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419

returned to a level comparable to the WT thereafter (Fig 11) In contrast 420

expression of OPR3 was similar in both genotypes JAR1 expression was not 421

affected by the fungus inoculation (Fig11) These results indicated that both JA 422

synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423

When infected with Pst WT plants displayed a strong induction of PR1 424

expression and as expected this induction was more pronounced (4-fold at 48 425

hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426

repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427

WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428

expression was still higher in incompatible compared to compatible interaction 429

Accumulation of PR5 transcripts was also slightly more important in WT plants 430

but expression levels were more important in the case of the compatible 431

interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432

difference between WT and Atdpl1-1 mutant was observed CHIT expression 433

was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434

(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435

plants (Fig 12) As already described inoculation with the bacterial pathogen 436

(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437

expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438

JAZ8 were induced during Pst infection and VSP1 expression was slightly 439

induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440

infection (Fig 12) Expression of these three genes was markedly enhanced in 441

Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442

ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443

than in WT plants after infection with either virulent or avirulent strains 444

respectively Similarly to B cinerea infection JAR1 expression was not affected 445

by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446

LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447

was slightly induced but not difference between the two genotypes was 448

observed (Fig 12) These data suggested that only JA signaling pathway is 449

positively affected in mutant plants upon challenge with Pst 450

To get further information on Atdpl1-1 mutant defense responses some 451

defense-related phytohormones were also quantified (Fig 13 A and B) No 452

change in phytohormone basal levels was observed between WT and Atdpl1-1 453

mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454

contrast to other mutants with modified sphingolipid contents does not display 455

high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456

Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457

phytohormone levels were enhanced SA accumulation was essentially 458

unchanged in the mutant compared to WT plants whatever the pathogen 459

considered Interestingly levels of JA and its biologically active conjugate JA-460

Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461

after B cinerea or Pst infection respectively However no difference in JA 462

levels between virulent and avirulent interaction was noticed but JA-Ile 463

accumulation was slightly higher in the case of the avirulent interaction in 464

Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465

pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466

infection 467

DISCUSSION 470

Only few papers described a connection between sphingolipid content PCD 472

and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473

most of them focused on responses against (hemi)biotrophic pathogen the role 474

of sphingolipid in plant defense against necrotrophs being largely unsolved 475

(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476

revealed basal sphingolipid levels and data of sphingolipid contents during 477

pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478

The present work described a comparison of sphingolipid content during 479

hemibiotrophic and necrotrophic infection In the present study we investigated 480

the consequences of the disruption of the sphingolipid profiles on plant 481

immunity responses such as cell death ROS production and signaling of plant 482

defense response during pathogen infection 483

Interplays between sphingolipids and PCD 485

Like in animal systems new emerging evidence showed that bioactive 486

sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487

2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488

showed that infection by B cinerea or Pst triggered accumulation of some 489

species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490

(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491

in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492

death and symptoms especially in the case of the incompatible interaction (Fig 493

9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494

our results suggested that a modification in d180 levels could impact plant cell 495

death and thus resistance to such pathogens Recently Coursol et al (2015) 496

showed that addition of d180 had no significant effect on viability of cryptogein-497

treated cells indicating that distinct mechanisms of regulation are involved in 498

cell death of cell culture or plant tissue or after treatment by an elicitor or a 499

pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500

cells PCD including HR can be beneficial to this kind of pathogens and could 501

thus facilitate their infection and spread of disease (Govrin and Levine 2000 502

Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503

HR or with reduced cell death present enhanced tolerance to B cinerea 504

infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505

Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506

transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507

pattern established that infection of Arabidopsis by B cinerea is promoted by 508

and requires an active cell death program in the host (van Kan 2006) and 509

resistance against this fungus depends on the balance between cell death and 510

survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511

mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512

species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513

a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514

sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515

lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516

metabolism is involved in cell death triggered by Botrytis species Cell death 517

activation could thus be disturbed in Atdpl1 plants leading to a higher 518

susceptibility towards (hemi)biotrophs and higher tolerance towards 519

necrotrophs In the present work B cinerea infection triggered Cer and LCB 520

accumulation in WT plants It is thus possible that the necrotrophic fungus 521

promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522

penetration and spread inside plant cells However exogenous d180 did not 523

modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524

not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525

infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526

t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527

showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528

(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529

thus plant resistance in response to pathogen infection Moreover it was 530

recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531

hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532

suppression (Nagano et al 2012) These results confirmed that sphingolipids 533

play important role in plant defense responses and plant is able to adjust its 534

response by regulating a dynamic balance between cell death (eg HR)- or cell 535

survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536

the fah1fah2 double mutant presented reduced amount of hCers and elevated 537

levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538

Thus it seems that the connection between sphingolipids and PCD is regulated 539

by a fine-tuned process and could thus be more complex than expected Other 540

parameters such as defense signaling pathways could be involved in such 541

mechanism 542

Interconnections between sphingolipids and defense mechanisms 544

Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545

plant cell death Moreover plant cell death processes such as HR are also 546

associated with plant defense or disease It is thus conceivable that some 547

sphingolipids play key role in plant innate immunity Recent studies brought to 548

light interconnections between sphingolipids and defense mechanisms 549

Resistance to biotrophic pathogen often required ROS production (Torres et al 550

2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551

accumulation of ROS and were more sensitive to the bacterial attack In 552

addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553

(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554

bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555

production of ROS (Fig 8) Several studies demonstrated that resistance 556

against B cinerea (and other necrotrophs) is accompanied by generation of 557

ROS and mutants impaired in ROS production failed to resist to the 558

necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559

LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560

2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561

ROS production (Peer et al 2011) In the present study exogenously applied 562

t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563

cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564

some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565

sphingolipids may differently interact with ROS production depending on the 566

presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567

accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568

treated WT plants indicated that phytosphingosine-1-phosphate and 569

dihydrosphingosine could have a key role in pathogen perception and thus in 570

plant resistance towards hemibiotrophic and necrotrophic pathogen 571

Several lines of evidence showed that plants disrupted in sphingolipid 572

metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573

al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574

et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575

2015) Recently it has been shown that SA and its analog BTH 576

(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577

AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578

pathway is effective against biotrophic and hemibiotrophic pathogens it has 579

been postulated that sphingolipids played a key role in defense against such 580

pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581

However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582

enhanced resistance to powdery mildew they displayed a similar phenotype to 583

WT plants upon infection with P syringae pv maculicola or Verticillium 584

longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585

sphingolipid-triggered cell death and plant resistance could be independent 586

regarding the plantpathogen pair Unfortunately only basal levels of 587

sphingolipid were described no sphingolipid quantification during pathogen 588

infection is available making difficult a direct link between sphingolipid 589

metabolism and plant defense In the present work infection with either 590

necrotrophic or hemibiotrophic pathogen induced production of all quantified 591

phytohormones It has been reported that several pathogens including B 592

cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593

Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594

2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595

the plant to adjust its response in favor of the most effective pathway 596

Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597

were more susceptible to A alternata infection However no information 598

concerning SA JA or sphingolipid levels in response to infection is available 599

especially as transgenic plants still displayed residual NbLCB2 gene expression 600

(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601

constitutive high SA levels and expression of PR1 gene This mutant was also 602

more susceptible to B cinerea and contained higher Cer levels but reduced 603

apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604

(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605

plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606

accumulation and CHIT up-regulation in response to infection but was more 607

resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608

SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609

Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610

In tomato B cinerea produces an exopolysaccharide that activates the SA 611

pathway which through NPR1 antagonizes the JA signaling pathway thereby 612

allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613

NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614

SA accumulated in WT plants and NPR1 was also stimulated upon infection 615

with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616

Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617

mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618

that perturbation in sphingolipid metabolism rendered either SA unable to 619

activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620

results highlighted that disturbance of sphingolipid metabolism could impact not 621

only cell death program but also JA signaling pathway leading to plant 622

tolerance towards necrotrophic pathogen such as B cinerea In that case the 623

relationship between sphingolipids and JA could be either indirect implying that 624

changes in sphingolipids operate in the crosstalk between SA and JA pathways 625

but in a NPR1 independent manner or direct as some key genes involved in JA 626

biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627

strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628

signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629

growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630

al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631

He 2013) The dramatic reduction in the expression of the SA-dependent 632

marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633

accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634

expression profile correlated JA and JA-Ile accumulation profile in response to 635

infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636

PDF12 and CHIT require both JA and ET signaling pathways but also the 637

function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638

et al 2000) Discrepancy between PDF12 expression and JA accumulation 639

has also been observed during the induced systemic resistance triggered by P 640

fluorescens and which is regulated through JA signaling pathway (van Wees et 641

al 1999) This suggested that a component in JA or ET signaling pathway 642

might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643

infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644

a pathway that does not include PDF12 or CHIT Collectively our results 645

suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646

and SA-regulated defense pathway respectively Whereas relationship 647

between SA signaling and sphingolipids was often described (Sanchez-Rangel 648

et al 2015) our results highlight for the first time that sphingolipids could also 649

play a key role in JA signaling pathway 650

In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652

the most appropriate response to defend themselves against pathogen attack 653

by acting on sphingolipid metabolism in order to modulate the cell 654

deathsurvival balance in close cooperation with JA andor SA signaling 655

pathways (Fig 14) Whereas SA involvement in PCD is well known the 656

relationship between JA and cell death is less understood Plants treated with 657

coronatine which shares structural similarities with JA-Ile and functional 658

similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659

2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660

associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661

has been reported that JA is also essential in FB1- and AAL-induced cell death 662

(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663

sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664

metabolism seemed to be intimately connected to defense processes to 665

regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666

involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667

recently been described as an important contributor to the LCB-mediated PCD 668

(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669

leading to sphingolipid-induced cell death is far from being totally elucidated 670

Further identification of target genes and their functions will provide new 671

insights into how sphingolipids could be linked to cell death and defense 672

processes 673

MATERIALS AND METHODS 677

Chemicals 679

Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680

purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681

prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682

to a final concentration of 100 microM Luminol and horseradish peroxidase were 683

obtained from Sigma-Aldrich (France) 684

Plant material and growth conditions 686

Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687

(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688

At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689

(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690

performed experiments since it exhibits same phenotype than other Atdpl1 691

mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692

accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693

wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694

conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695

Isolation of T-DNA insertion mutant and genotype characterization 697

The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698

SALK_078119 were isolated according to the published procedure SIGnAL 699

(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700

PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701

AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702

TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703

TGGTTCACGTAGTGGGCCATCG-3) 704

Sphingolipidomic analysis 706

Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707

LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708

with modifications using a Shimadzu Prominence UHPLC system and a 709

4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710

a 100mm Dionex Acclaim C18 column Data analysis was performed using 711

Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712

independent repeats were performed and a minimum of three technical 713

replicates was run from each sample 714

RNA extraction and real-time quantitative RT-PCR 716

Isolation of total RNA and real-time PCR was performed as described in Le 717

Henanff et al (2013) Gene specific primers are described in Supplemental 718

Table S1 For each experiment PCR reactions were performed in duplicate and 719

at least 3 independent experiments were analyzed Transcript levels were 720

normalized against those of the Actin gene as an internal control Fold induction 721

compared to mock treated sample was calculated using the ΔΔ Ct method 722

Pathogen growth and inoculation 724

B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725

(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726

resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727

002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728

150 rpm germinated spores were used for plant inoculation by spraying the 729

upper face of the leaves Control inoculations were performed with PDB Sylwett 730

L-77 002 731

The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732

AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733

medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735

resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736

mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737

the abaxial side into leaf using a 1-mL syringe without a needle Control 738

inoculations were performed with 10 mM MgCl2 739

Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740

at - 80degC until use 741

Pathogen assay in planta 743

B cinerea infections were performed as previously described (Le Heacutenanff et al 744

2013) Plants were placed in translucent boxes under high humidity at 150 microE 745

m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746

conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747

measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748

per genotype and experiments were independently repeated 4 times 749

Bacterial infections were performed as previously described (Sanchez et al 750

2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751

ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752

plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753

were counted Data are reported as means and SD of the log (cfu cm-2) of three 754

replicates Growth assays were performed four times with similar results 755

Electrolyte leakage 757

Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758

(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759

from the infected area and washed extensively with water for 50 min and then 760

eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761

effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762

t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763

treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764

conductivity meter 765

ROS production 767

Measurements of ROS production were performed as described previously 768

(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769

a 96-well plate containing 150 μL of distilled water and then incubated overnight 770

at room temperature Just before ROS quantification distilled water was 771

replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772

peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773

the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774

involving B cinerea germinated spores were added to the elicitation solution to 775

a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776

t180-P or d180 were added concomitantly with bacterium or fungus to the 777

elicitation solution 778

Phytohormone analysis 780

Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781

al (2014) 782

Supplemental material 784

Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785

cinerea or Pst infection 786

Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787

infection 788

Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789

Atdpl1-1 mutant plants before infection with B cinerea or Pst 790

Atdpl1-1 mutant plants after infection with B cinerea or Pst 792

Supplemental Table S1 Gene-specific primers used in real time reverse-793

transcription polymerase chain reaction 794

ACKNOWLEDGEMENTS 796

We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797

Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798

technical assistance in phytohormone quantification 799

FIGURE LEGENDS 801

Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803

susceptible to Pst than WT 804

B cinerea conidia suspension was deposited by using droplet-inoculation (A 805

and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806

solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807

Photographs represent disease symptoms observed 60 or 72 h after infection 808

by the fungus or Pst respectively B Symptoms due to B cinerea infection 809

were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810

dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811

differences of the mean of lesion diameter between WT and Atdpl1 plants were 812

calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813

virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814

and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815

h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816

indicate significant differences between WT-and Atdpl1-treated samples 817

according to Studentrsquos t test (Plt0005) Results are representative of three 818

independent experiments 819

Figure 2 Free LCB and LCB-P accumulation after challenge with 821

pathogen 822

Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823

suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824

AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825

LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826

differences between the pathogen-treated WT sample and the control sample 827

and asterisks on Atdpl1-1 bars indicate significant differences between the 828

pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829

according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830

mean of four to five independent biological experiments plusmn SD Notice the 831

different scale of LCB-P levels between WT and Atdpl1-1 plants 832

Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834

Pst infection 835

Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836

with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837

infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838

AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839

bars indicate significant differences between the pathogen-treated WT sample 840

and the control sample and asterisks on Atdpl1-1 bars indicate significant 841

differences in total species between the pathogen-treated WT sample and the 842

pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843

Plt001 Plt0005) Asterisks have only been considered for the total species 844

displaying the same fatty acid or hydroxylationunsaturation degree Results are 845

the mean of four to five independent biological experiments plusmn SD 846

Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848

infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852

(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853

significant differences between the pathogen-treated WT sample and the 854

control sample and asterisks on Atdpl1-1 bars indicate significant differences 855

between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856

sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857

Asterisks have only been considered for the total species displaying the same 858

fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859

five independent biological experiments plusmn SD 860

Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862

plants upon pathogen infection 863

significant differences between the pathogen-treated sample and the control 868

sample and asterisks on Atdpl1-1 bars indicate significant differences between 869

the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870

according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871

only been considered for the total species displaying the same fatty acid or 872

hydroxylationunsaturation degree Results are the mean of four to five 873

independent biological experiments plusmn SD 874

Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876

inoculation 877

Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878

Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879

DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880

plusmn SD of three replicates per experiment The experiment was repeated three 881

times with similar results 882

Figure 7 Time-course of programmed cell death marker gene expression 884

after B cinerea or Pst infection 885

Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886

(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887

AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888

representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889

and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890

mutant plants with five biological replicates with comparable results 891

Figure 8 Transient ROS production in response to pathogen infection in 893

WT and Atdpl1-1 mutant plants 894

Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895

to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896

were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897

108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898

repetitions Three independent experiments were performed with similar results 899

Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901

dihydrosphingosine on electrolyte leakage in response to pathogen 902

infection in WT plants 903

A and B B cinerea conidia suspension was deposited on leaves of WT and 904

Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905

solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906

Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907

infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908

containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909

spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910

DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911

mean plusmn SD of three replicates per experiment The experiment was repeated 912

three times with similar results 913

dihydrosphingosine on ROS production in response to pathogen infection 916

in WT plants 917

Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918

response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919

and F) infection Leaf disks were immersed in a solution containing 100 microM of 920

t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921

Pst Error bars represent SE of the mean from 12 biological repetitions Three 922

independent experiments were performed with similar results 923

Figure 11 Expression levels of JA and SA pathway-associated genes in 925

WT and Atdpl1-1 mutant plants during B cinerea infection 926

Results are expressed as the fold increase in the transcript level compared with 927

the untreated control (time 0 h) referred to as the times1 expression level Values 928

shown are means plusmn SD of duplicate data from one representative experiment 929

among five independent repetitions 930

WT and Atdpl1-1 mutant plants during Pst infection 933

Figure 13 Analysis of phytohormone accumulation in stressed WT and 939

Atdpl1-1 mutant plants 940

JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941

following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942

indicate significant differences between WT and Atdpl1-1 samples according to 943

Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944

SD from one representative experiment among five independent repetitions 945

Figure 14 Schematic overview of interconnections between sphingolipid 947

metabolism cell death and defense signaling pathways in Atdpl1 mutant 948

plants upon pathogen attack 949

Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950

and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951

metabolism may also indirectly modulate cell death through its tightly 952

connection (double-headed dashed arrow) as positive andor negative regulator 953

to jasmonate andor SA signaling pathways respectively Reduced cell death 954

and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955

more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956

single-headed arrows indicate activation double-headed arrows indicate 957

unknown regulatory mechanism Ald aldehyde Ethan-P 958

phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959

Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960

kinase 1 961

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Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041

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El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049

El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052

Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055

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Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060

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Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065

Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068

Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071

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Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077

Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079

Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081

LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085

Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088

Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092

Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096

Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100

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Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110

Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112

Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116

Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118

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Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124

Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126

Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129

Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133

Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135

Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139

Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142

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Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149

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Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

3 Control DC3000 AvrRPM1

A B C D

E F G H

WT WT Atdpl1-1 Atdpl1-1

Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by

Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results

0 12 24 36 48

(microS

) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1

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Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1

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Hpi Hpi

Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results

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Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1

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Time (min)

Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1

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Time (min)

Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1

Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results

0 100 200 300 400

Time (min)

Control t180-P Bc Bc + t180-P

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Control t180-P DC3000 DC3000+t180-P

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Control t180-P AvrRPM1 AvrRPM1+t180-P

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Control d180 Bc Bc + d180

0 30 60 90 Time (min)

Control d180 DC3000 DC3000+d180

0 30 60 90 Time (min)

Control d180 AvrRPM1 AvrRPM1+d180

Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results

0 12 24 36 48

Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions

n PDF12 CHIT

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Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions

SA JA JA-Ile

WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi

SA JA JA-Ile

WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi

Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions

Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1

LCB-Ps

Cell death

Necrotrophic pathogen B cinerea

Hemibiotrophic pathogen Pst

JA JA-Ile SA

plant cell Sphingolipid metabolism

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

tolerance susceptibility

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Parsed Citations

Modifications of sphingolipid content affect tolerance to 18

hemibiotrophic and necrotrophic pathogens by modulating 19

plant defense responses in Arabidopsis 20

Magnin-Robert Maryline1 Le Bourse Doriane2 Markham Jonathan2 Steacutephan 22

Dorey1 Cleacutement Christophe1 Baillieul Fabienne1 and Dhondt-Cordelier 23

Sandrine1 24

25 1 Uniteacute de Recherche Vigne et Vin de Champagne (URVVC-EA 4707) 26

Laboratoire Stress Deacutefenses et Reproduction des Plantes SFR Condorcet FR 27

CNRS 3417 Universiteacute de Reims Champagne-Ardenne BP 1039 F-51687 28

Reims cedex 2 France 29 2 Center for Plant Science Innovation and Department of Biochemistry 30

University of Nebraska-Lincoln Beadle Center 1901 Vine Street Lincoln NE 31

68588 USA 32

One-sentence summary 35

Sphingolipids play a key role in plant defense towards different lifestyle 36

pathogens by modulating cell death ROS accumulation and jasmonate 37

signaling pathway 38 39

ABSTRACT 52

INTRODUCTION 79

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

ABSTRACT 52

INTRODUCTION 79

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

ABSTRACT 52

INTRODUCTION 79

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

INTRODUCTION 79

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

RESULTS 178

plant response 180

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

towards Pst 355

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

pathogens 392

infection 467

DISCUSSION 470

mechanism 542

processes 673

Chemicals 679

L-77 002 731

ROS production 767

al (2014) 782

infection 788

FIGURE LEGENDS 801

pathogen 822

Pst infection 835

inoculation 877

in WT plants 917

kinase 1 961

ownloaded on M

ll rights reserved

Control Bc

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

0 1 2 3 4 5 6 Control

DC3000 AvrRPM1

A B C D

E F G H

8 Control Bc

25 Control Bc

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

WT Atdpl1-1

Control

DC3000

AvrRPM1

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Fatty acid

d180 d181 t180 t181 0

Control

DC3000

AvrRPM1

WT Atdpl1-1

0 12 24 36 48

(microS

0 6 12 18 24

(microS

0 12 24 36 48 0

0 12 24 36 48 Rel

0 12 24 36 48 0

0 12 24 36 48

Hpi Hpi

0 100 200 300 400

Time (min)

0 30 60 90

Time (min)

0 20 40 60 80 100

Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 100 200 300 400

Time (min)

0 30 60 90 Time (min)

0 12 24 36 48

n PDF12 CHIT

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8

0 12 24 36 48

0 2 4 6 8 10 0 12 24 36 48

0 12 24 36 48

0 12 24 36 48 0

0 12 24 36 48

ERF1 CHIT

0 12 24 36 48

0 500 1000 1500

SA JA JA-Ile

LCB-Ps

Cell death

JA JA-Ile SA

Ethan-P + Ald

LOH123 FAH12

LCBK SPHK1

LCB-P Pase

AtDPL1

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Parsed Citations

Documents

1 Running head: Sphingolipids in plant defense responses 2 ...€¦ · 16/9/2015 · 164 in response to B. cinerea infection and in comparison to Pst infection. For this 165 purpose,