1

RESEARCH ARTICLE 1

Secretion of Phospholipase Dδ Functions as a Regulatory Mechanism in 2

Plant Innate Immunity 3

4

Jingjing Xinga,b,1

, Xiaojuan Lia,c,1

, Xiaohua Wanga, Xueqin Lv

c, Li Wang

a, Liang 5

Zhanga, Yingfang Zhu

b, Qianhua Shen

d, František Baluška

e, Jozef Šamaj

f, and 6

Jinxing Lina,c,2

7

8 aInstitute of Botany, Chinese Academy of Sciences, Beijing 100093, China 9

bKey Laboratory of Plant Stress Biology, School of Life Sciences, Henan University, 10

Kaifeng 457001, China 11 cBeijing Advanced Innovation Center for Tree Breeding by Molecular Design and 12

College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 13

100083, China 14 dState Key Laboratory of Plant Cell and Chromosome Engineering, Centre for Molecular 15

Agrobiology, Institute of Genetics and Developmental Biology, Chinese Academy of 16

Sciences, Beijing100101, China 17 eInstitute of Cellular and Molecular Botany, Rheinische Friedrich-Wilhelms- University 18

Bonn, Department of Plant Cell Biology, Bonn D-53115, Germany 19 fCentre of the Region Hana for Biotechnological and Agricultural Research, Faculty of 20

Science, Palacky University, Olomouc 78301, Czech Republic 21 1These authors contributed equally to this work. 22

2To whom correspondence should be addressed. Email: linjx@ibcas.ac.cn 23

Short title: Secretion of phospholipase Dδ in innate immunity 24

One-sentence summary: Membrane microdomains and PLDδ exocytosis regulate PLDδ 25

accumulation during penetration resistance in plant innate immunity. 26

The author(s) responsible for distribution of materials integral to the findings presented in 27

this article in accordance with the policy described in the Instructions for Authors 28

(www.plantcell.org) is Jinxing Lin (linjx@ibcas.ac.cn). 29

ABSTRACT 30

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function 31

in multiple signal transduction cascades; however, the mechanisms regulating PLDs in 32

response to pathogens remain unclear. Here, we found that Arabidopsis thaliana PLDδ 33

Plant Cell Advance Publication. Published on October 9, 2019, doi:10.1105/tpc.19.00534

©2019 American Society of Plant Biologists. All Rights Reserved

2

accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria 34

graminis f. sp. hordei (Bgh). Using fluorescence recovery after photobleaching (FRAP) 35

and single-molecule analysis, we observed higher PLDδ density in the plasma membrane 36

after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance 37

energy transfer with fluorescence lifetime imaging microscopy (FRET-FLIM) showed 38

that the interaction between PLDδ and the microdomain marker AtREMORIN 1.3 39

(AtREM 1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, 40

efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further 41

unveiled a tradeoff between Brefeldin A (BFA)-resistant and sensitive pathways in 42

secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion 43

involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. 44

Analysis of phosphatidic acid (PA), H2O2 and jasmonic acid (JA) levels, and expression 45

of related genes indicated that the relocalization of PLDδ is crucial for its activation to 46

produce PA and initiate ROS and JA signaling pathways. Together, our findings revealed 47

that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering 48

PA-mediated signaling in plant innate immunity. 49

INTRODUCTION 50

Phospholipids, the bilayer-forming structural components of membranes, have novel and 51

unexpected roles in cell signaling (Wang and Chapman, 2013). The phospholipase D 52

(PLD) family of enzymes, which directly generate phosphatidic acid (PA) through the 53

hydrolysis of structural phospholipids, plays a vital role in lipid-based signaling cascades 54

in plants. Arabidopsis thaliana has 12 genes that encode PLDs of six types (three genes 55

for PLDα, two for PLDβ, three for PLDγ, one for PLDδ and PLDε, and two for PLDζ) 56

and these types have distinguishably biochemical and regulatory properties (Zhang et al., 57

2003; Hong et al., 2016; Takáč et al., 2019). Considerable work has focused on the 58

regulatory functions of PLD signaling pathways, which alter cellular and physiological 59

3

processes in response to different stimuli, including abscisic acid (ABA), auxin, reactive 60

oxygen species, and freezing (Bargmann and Munnik, 2006; Guo et al., 2012; Li et al., 61

2004; Mishra et al., 2006; Zhang et al., 2003). Distinct from other PLDs, PLDδ is 62

activated by oleic acid and serves as a direct link between the plasma membrane (PM) 63

and the microtubule cytoskeleton (Gardiner et al., 2001; Pleskot et al., 2013; Wang and 64

Wang, 2001). Previous studies of the specific properties of PLDδ have focused on its role 65

in the responses to abiotic stresses, such as tolerance of freezing, dehydration, and 66

high-salt stress, as well as in ABA-induced leaf senescence (Jia et al., 2013; Katagiri et 67

al., 2001; Li et al., 2004). 68

Increasing evidence suggests that phospholipid-derived molecules and PLD enzymes 69

play roles in plant–pathogen interactions (Kachroo and Kachroo, 2009; Laxalt and 70

Munnik, 2002; Pinosa et al., 2013). For example, in rice (Oryza sativa) challenged with 71

the bacterial pathogen Xanthomonas oryzae, PLDα1-like PLDs cluster in the plasma 72

membrane adjacent to the bacterial cells in leaves undergoing an immune response. The 73

translocation of PLDs in response to pathogen attack suggests that these PLDs are 74

activated in response to pathogens and function in the resistance interaction (Wang et al., 75

2006; Young et al., 1996). PA, a vital signaling lipid produced by PLD, has also been 76

reported to activate multiple cellular responses in plant defense, including the oxidative 77

burst and mitogen-activated protein kinase cascades (Rentel et al., 2004; Rizzo et al., 78

2000). Furthermore, PA accumulates in response to pathogen-associated molecular 79

patterns (PAMPs), inducing pathogen-related gene expression and cell death (Testerink 80

and Munnik, 2011). As PLD is a pivotal regulator of phospholipid signaling in plant cells, 81

its location is closely associated with its function. 82

Plant innate immunity endows entire species of plants with resistance to potentially 83

infectious pathogens, such as viruses, bacteria, and fungi. Epidermal penetration 84

resistance, a key aspect of innate immunity, hinders fungi from entering the plant and 85

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involves the localized formation of cell wall appositions known as papillae (Nielsen et al., 86

2012). In Arabidopsis, two separate pathways enhance penetration resistance; one 87

pathway involves syntaxin (PEN1) and the other involves a combination of peroxisomal 88

β-glycosyl hydrolase (PEN2) and the PM-localized ABC transporter PEN3 (Clay et al., 89

2009; Lipka et al., 2008; Lipka et al., 2005; Stein et al., 2006). During these 90

well-described plant defense processes, PLDδ is also reported to be involved in the basal 91

defense and non-host resistance response (Pinosa et al., 2013). Nevertheless, the 92

functions of phospholipid signaling pathways during these plant defense processes have 93

remained largely unknown, and the translocation of PLDδ and downstream signaling in 94

the Arabidopsis–Blumeria graminis f. sp. hordei (Bgh) plant–fungus interactions remain 95

to be further elucidated. 96

Earlier studies have focused on the functions of PA in plant defense signaling (Rentel 97

et al., 2004; Rizzo et al., 2000); however, the relocalization and accumulation of PLDδ in 98

response to pathogens remained to be examined and may be a key step for signal 99

transmission in plant innate immunity. In this study, we examined PLDδ exocytosis in 100

plant innate immunity during plant–pathogen interactions, with particular emphasis on 101

the spatio-temporal dynamics of PLDδ translocation. Furthermore, we explored whether 102

the partitioning of PLDδ in plant defense is associated with membrane microdomains. 103

Our comprehensive analyses offer insight into the diverse regulation of PLDδ secretion in 104

penetration resistance and may also serve as a model of how membrane phospholipases 105

control phospholipid signaling in response to pathogens. 106

RESULTS 107

PLDδ Aggregates in Plasma Membrane Microdomains during Bgh Infection 108

To characterize the subcellular localization of Arabidopsis PLDδ in penetration resistance 109

against Bgh, we expressed a PLDδ-green fluorescent protein (GFP) fusion under the 110

5

control of the 35S promoter and the native PLDδ promoter in wild type and pldδ mutants. 111

To test whether PLDδ-GFP retained function, we then examined the resistance phenotype 112

of wild-type (Col-0), p35S:PLDδ-GFP/Col-0, pPLDδ:PLDδ-GFP/pldδ, and pldδ plants. 113

Measurement of penetration rates and hypersensitive response (HR)-associated cell death 114

in Bgh-infected leaves showed that PLDδ-GFP retained function and PLDδ was required 115

for penetration resistance (Supplemental Figure 1). We further analyzed the subcellular 116

localization of PLDδ-GFP using confocal laser scanning microscopy (CLSM). Upon Bgh 117

infection, a striking accumulation of PLDδ-GFP was observed at the Bgh penetration sites 118

and GFP signal remained localized at the papillae in the extracellular space after 119

plasmolysis (Figure 1A–1G). When the membrane in the papillae was stained with the 120

fluorescent lipophilic dye FM4-64, the red signal of this dye overlapped with the GFP 121

signal at penetration sites, revealing colocalization of the preformed papillae with 122

PLDδ-GFP (Figure 1H–1K). These results showed that PLDδ was targeted mainly to the 123

membranes of papillae in the extracellular space during infection by Bgh. 124

To study the role of PLDδ in defense responses, we treated the plants with the fungal 125

elicitor chitin, as described previously (Underwood and Somerville, 2013) and used 126

fluorescence correlation spectroscopy (FCS) to measure the density of PLDδ-GFP in the 127

PM in vivo (Li et al., 2011). We found that the average density of PLDδ-GFP in the PM 128

was 7.9 ± 0.5 molecules μm2

in the resting condition (no chitin), but that of the 129

chitin-treated cells was 44% higher, at 14.1 ± 1.5 molecules μm2

. Cycloheximide 130

(CHX), an inhibitor of de novo protein synthesis, suppressed this increase, resulting in a 131

PLDδ-GFP protein density of only 5.7 ± 0.1 molecules μm2

in plants treated with CHX 132

plus chitin (Figure 2A and B). Moreover, immunoblot analysis showed that the 133

abundance of PLDδ-GFP increased in response to chitin in the PM and in the entire cell 134

and that CHX pretreatment inhibited this rise (Figure 2C, Supplemental Figure 2). Using 135

total internal reflection fluorescence microscopy (TIRFM), we found that the 136

fluorescence intensities of PLDδ-GFP at the PM were not homogeneous and more 137

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PLDδ-GFP aggregation foci occurred in the chitin-treated plants (Figure 2D and E). 138

To determine how the cell recruited PLDδ-GFP to the PM, we examined the dynamics 139

of PLDδ-GFP within the PM using fluorescence recovery after photobleaching (FRAP). 140

The FRAP analysis showed that in the chitin-treated cells, PLDδ-GFP had a shorter 141

average half-life (t1/2, chitin = 5.93 ± 0.29 min, t1/2, control = 7.36 ± 0.35 min) in the PM and a 142

higher percentage of recovery (chitin-treated: 38.82 ± 1.51%, control: 33.64 ± 0.97%) 143

compared to the resting condition (Figure 2F and G). The fluorescence recovery curve of 144

membrane-resident proteins describes the sum of the lateral mobility of the protein in the 145

membrane and the exchange of proteins between cytoplasmic vesicles and the membrane 146

via exocytosis and endocytosis. 147

To separate these effects, we divided the initial bleaching regions into top, middle, and 148

bottom sectors; lateral mobility from adjacent membrane regions will preferentially 149

increase recovery in the top and bottom sectors, but exocytosis-mediated exchange will 150

contribute evenly to all three sectors. We found that the three regions recovered at equal 151

rates (Supplemental Figure 3A–D and Table 1). When we photobleached GFP signals on 152

the cell surface and measured FRAP signals from the center, the periphery, and the 153

entirety of the bleached areas over time, the results showed that these three regions also 154

recovered at equal rates (Supplemental Figure 4). These results indicated that PLDδ-GFP 155

is primarily recruited from the cytoplasm to the PM via exocytosis; therefore, the rate of 156

recovery represents the rate of exocytosis. Moreover, we examined the regions next to the 157

bleached regions (magenta circles in Supplemental Figure 4) and found that the 158

fluorescence signal was increased by chitin treatments compared to that under control 159

conditions (Supplemental Figure 4). Based on this, we found that the exocytosis rate of 160

PLDδ-GFP showed a 15% increase in chitin-treated cells compared to the control cells 161

(Figure 2H). 162

Protein Sorting into Microdomains Is Related to the Focal Accumulation of PLDδ 163

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PMs are highly organized structures that contain diverse coexisting microdomains (Jarsch 164

et al., 2014), and previous proteomics studies have detected PLDδ in the 165

detergent-resistant membrane fraction from plants (Demir et al., 2013; Shahollari et al., 166

2005). To investigate whether the localization of PLDδ is associated with microdomains 167

during penetration resistance, we used AtREM1.3 as a marker of sterol-rich membrane 168

microdomains and created transgenic plants coexpressing AtREM1.3-mCherry and 169

PLDδ-GFP. At 48 h postinfection (hpi) with Bgh, AtREM1.3-mCherry accumulated in the 170

papillae and colocalized with the accumulation of PLDδ-GFP in foci (Figure 3A–D), 171

implying that the papillary extracellular membrane also included microdomains and that 172

PLDδ was contained within these microdomains during the penetration resistance 173

response. 174

We quantified the colocalization of AtREM1.3-mCherry and PLDδ-GFP using the 175

protein proximity index (PPI), revealing that the mean PPI value for PLDδ-GFP and 176

AtREM1.3-mCherry was 0.90 ± 0.05 after chitin exposure, but only 0.66 ± 0.06 in the 177

resting condition. This indicated that the degree of colocalization between PLDδ-GFP and 178

AtREM1.3-mCherry increased from “more than moderate” to “very strong” during the 179

PAMP response (Figure 3E–I). 180

We further used fluorescence resonance energy transfer with fluorescence lifetime 181

imaging microscopy (FRET-FLIM) to confirm the interaction between PLDδ-GFP and 182

AtREM1.3-mCherry. The fluorescence lifetime of PLDδ-GFP alone in the PM of 183

epidermal cells was ~2.38 ± 0.04 ns. In the transgenic line coexpressing free-GFP and 184

AtREM1.3-mCherry, the mean GFP fluorescence lifetime of Free-GFP (2.35 ± 0.05 ns) 185

showed no meaningful difference from that of PLDδ-GFP alone. However, the average 186

GFP fluorescence lifetime was strongly reduced in the plants coexpressing PLDδ-GFP 187

and AtREM1.3-mCherry (2.23 ± 0.04 ns). After treatment with chitin, the fluorescence 188

lifetime of PLDδ-GFP in these plants showed a strong reduction (to 1.98 ± 0.06 ns) in 189

8

comparison to the coexpressing plants in the control condition, with a FRET efficiency of 190

16.7% (Figure 3J–N). 191

To better understand the molecular mechanisms underlying the partitioning of PLDδ 192

into PM microdomains, we then investigated the dynamic behavior of individual 193

PLDδ-GFP fluorescent spots inside living cells using single-particle tracking (SPT) in 194

continuous images (Supplemental Figure 5A and C, Movies S1 and S2). Next, we 195

obtained histograms of the diffusion coefficients, which we measured by fitting the 196

particle trajectories to a Gaussian function to characterize the global mobility of 197

fluorescent spots in each treatment group, in which the Gaussian peaks (Ĝ) were defined 198

as the characteristic values for the diffusion coefficients (Cui et al., 2018; Wu et al., 199

2019). Under control conditions (no chitin), the diffusion coefficients of PLDδ-GFP 200

reflected two populations, with Ĝ values of 9.55 × 103

μm2/s (47.13%, SE 8.51 × 10

3 to 201

1.07 × 102

μm2/s) and 4.67 × 10

3 μm

2/s (52.86%, SE 3.98 to 5.50 × 10

3 μm

2/s) 202

(Supplemental Figure 5E). Under chitin treatment, the pattern was the same, with Ĝ 203

values of 9.77 × 103

μm2/s (58.69%, 9.33 × 10

3 to 1.02 × 10

2 μm

2/s) and 4.57 × 10

3 204

μm2/s (41.30%, SE 4.27 to 4.90 × 10

3 μm

2/s) (Supplemental Figure 5E). 205

By contrast, in seedlings treated with methyl-β-cyclodextrin (MβCD), a 206

sterol-disrupting reagent, TIRFM analysis of the PLDδ-GFP fluorescent spots revealed 207

that the histogram of diffusion coefficients yielded a one-population distribution with a Ĝ 208

value of 8.91 × 103

μm2/s (SE 8.79 to 9.03 × 10

3 μm

2/s) in MβCD-treated cells and a Ĝ 209

value of 9.12 × 103

μm2/s (SE 8.95 to 9.29 × 10

3 μm

2/s) in cells pretreated with MβCD 210

and then treated with chitin (Supplemental Figure 5E). Thus, MβCD treatment caused the 211

percentage of PLDδ-GFP fluorescent spots with a fast diffusion coefficient to increase 212

and caused the subpopulation with the slow diffusion coefficient to disappear. 213

Evaluating the diffusion mode by analyzing the mean square displacement (MSD) of 214

the trajectories of individual PLDδ-GFP molecules over time (Supplemental Figure 5G), 215

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we found that most PLDδ-GFP molecules were restricted to specific zones and had 216

restricted diffusion with or without chitin treatment. After pretreatment with MβCD, the 217

movement type of PLDδ-GFP molecules changed from restricted diffusion to Brownian 218

diffusion (Supplemental Figure 5G). Moreover, we performed experiments to examine 219

whether the PLDδ-generated PA was affected by the sterol-enriched microdomain 220

location. When the membrane sterol was predepleted by MβCD treatment, PLD activity 221

was decreased and the chitin-induced PA production was reduced compared to the chitin 222

treatment without MβCD (Supplemental Figure 6). 223

To test the effect of membrane microdomain disruption on the recruitment of 224

PLDδ-GFP to sites of attempted penetration by Bgh, we syringe-infiltrated leaves with 225

different concentrations of MβCD 1 h before inoculation with Bgh and then observed the 226

localization of PLDδ-GFP at Bgh penetration sites at 48 hpi (Supplemental Figure 7A and 227

B). Increasing MβCD concentrations caused progressively lower PLDδ-GFP 228

accumulation rates, ranging from 39.8 ± 0.6% for leaves treated with 0.5 μM MβCD to 229

16.8 ± 1.9% at 2 μM MβCD (Supplemental Figure 7C). We also determined the impact 230

of MβCD on Bgh penetration and found that MβCD treatment increased the penetration 231

rate to 19.81 ± 1.04%, approximately twice that measured after control treatment with 232

ddH2O (10.07 ± 1.19%) (Supplemental Figure 8A). 233

Pathogen-Inducible PLDδ Recruited in a BFA-Sensitive Manner 234

Since exocytosis of PLDδ increased in response to pathogen, we next tried to identify the 235

secretory pathway that trafficks PLDδ to the papillae. We employed the 236

vesicle-trafficking inhibitor brefeldin A (BFA), which inhibits certain ADP ribosylation 237

factor/guanine nucleotide exchange factors (ARF-GEFs), to address this question. In 238

plants treated with 50 μM BFA plus FM4-64, PLDδ-GFP showed weak intracellular 239

agglomeration that colocalized with positive BFA compartments labeled with FM4-64. In 240

addition, most punctate PLDδ-GFP structures were intact and did not accumulate in BFA 241

10

bodies (Figure 4A–C). Exposure of seedlings to chitin after BFA pretreatment led to an 242

obvious accumulation of PLDδ-GFP in BFA bodies, but many PLDδ-GFP signals 243

remained unaffected by BFA (Figure 4D–F). Quantitative analysis of PLDδ exocytosis 244

showed that the numbers of BFA bodies labeled with PLDδ-GFP increased to 481.20% of 245

control in response to chitin exposure, but the numbers of BFA-resistant vesicles marked 246

by PLDδ-GFP did not significantly change under either control or chitin treatment 247

conditions (Figure 4G and H). Cells pretreated with CHX had fewer BFA-resistant 248

vesicles and BFA bodies labeled by PLDδ-GFP (Supplemental Figure 9), indicating that 249

the secretion of PLDδ involved BFA-resistant and -sensitive pathways that were both 250

derived from de novo protein synthesis. FRAP analysis also showed that the average 251

half-life increased from 5.93 ± 0.29 min to 7.27 ± 0.26 min, and the percentage of 252

recovery decreased from 38.82 ± 1.51% to 29.21 ± 1.73% in plants treated with chitin 253

following BFA pretreatment (Figure 4I–L). In addition, we detected a 24.7% decrease of 254

the exocytosis rate in the presence of BFA (Figure 4M). 255

To further investigate whether PLDδ-GFP was associated with BFA-sensitive secretion 256

during Bgh attack, we syringe-infiltrated leaves with 300 μM BFA 1 h before inoculation 257

with Bgh and observed the localization of PLDδ-GFP at Bgh penetration sites at 48 hpi. 258

In the BFA-treated leaves, PLDδ-GFP often did not accumulate at the sites of attempted 259

Bgh penetration (Figure 4N and O), and the frequency of normal papillary PLDδ-GFP 260

accumulation decreased from 90.3 ± 5.1% to 25.8 ± 6.2% (Figure 4P). Moreover, BFA 261

treatment led an increase in the penetration rate of Bgh (Supplemental Figure 8B). 262

Bgh-Triggered PLDδ Associates with PEN1 in Vivo at the PM and Papillary 263

Extracellular Membrane 264

Previous studies revealed that treatment with BFA blocks the accumulation of PEN1 265

(Nielsen et al., 2012); therefore, we next asked whether the trafficking pathway of PLDδ 266

is associated with PEN1. In the transgenic plants coexpressing PLDδ-GFP and 267

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mCherry-PEN1, PLDδ and PEN1 clearly colocalized at the preformed papillae after Bgh 268

inoculation (Figure 5A–D). PPI was used to quantify the fraction of colocalized 269

molecules in these plants, revealing a mean value of 0.23 ± 0.05 for the proximity of 270

PLDδ-GFP to mCherry-PEN1 in the resting condition, which increased to 0.63 ± 0.07 271

after treatment with chitin (Supplemental Figure 10A–E), representing a change in the 272

degree of colocalization between PLDδ and PEN1 from “weak” to “more than moderate.” 273

We also used FLIM to detect FRET between PLDδ-GFP and mCherry-PEN1 in the PM 274

of epidermal cells. FRET-FLIM analysis revealed a reduction in the fluorescence lifetime 275

of PLDδ-GFP from 2.39 ± 0.05 ns in the PLDδ-GFP-expressing plants to 2.22 ± 0.02 ns 276

in the PLDδ-GFP/mCherry-PEN1-coexpressing plants, whereas the GFP fluorescence 277

lifetime did not decrease in the transgenic lines coexpressing Free-GFP/mCherry-PEN1 278

(2.36 ± 0.04 ns). 279

When the PLDδ-GFP/mCherry-PEN1-coexpressing plants were treated with chitin for 280

1 h, the fluorescence lifetime of PLDδ-GFP further decreased to 1.99 ± 0.05 ns and 281

yielded a FRET efficiency of 17.0%, indicating that chitin enhanced the interaction of 282

PLDδ and PEN1 (Figure 5E–I). Using coimmunoprecipitation to analyze the possible 283

interaction between PLDδ and PEN1, we found that mCherry-PEN1 immunoprecipitated 284

with PLDδ-GFP in transgenic plants coexpressing the two proteins, indicating that PEN1 285

interacts with PLDδ in vivo (Figure 5J). 286

PLDδ Is Trafficked to the Papillae via VAMP721/722-Mediated Secretion 287

PEN1 interacts with VAMP721/722 to mediate a pathway involved in secretion for the 288

execution of penetration resistance (Kwon et al., 2008b); therefore, we hypothesized that 289

PLDδ may share the same exocytosis pathway as PEN1 or play a role in the function of 290

the PEN1–SNAP33–VAMP721 complex. We generated transgenic plants coexpressing 291

PLDδ-GFP and mCherry-VAMP721 to further confirm the pathway is involved in 292

trafficking of PLDδ. At 48 hpi with Bgh, PLDδ-GFP and mCherry-VAMP721 colocalized 293

12

in the papillae (Figure 6A–D). More importantly, of the 173 PLDδ-GFP-labeled 294

compartments observed in the cytoplasm, 153 (88.4%) colocalized with cytoplasmic 295

bodies labeled with mCherry-VAMP721 after treatment with chitin for 1 h (Figure 6E–J). 296

Similar phenomena were observed in the transgenic plants coexpressing PLDδ-GFP and 297

mCherry-VAMP722 (Supplemental Figure 11A–J). 298

To investigate whether trafficking of PLDδ protein to the papillae is affected by 299

VAMP721/722-mediated secretion, we obtained PLDδ-GFP vamp721/

, PLDδ-GFP 300

vamp722/

, PLDδ-GFP vamp721+/

vamp722/

, and PLDδ-GFP vamp721/

vamp722+/

301

plants. In the vamp721/

, vamp722/

, vamp721+/

vamp722/

, and vamp721/

302

vamp722+/

mutant backgrounds, PLDδ protein was properly targeted to papillae after 303

incubation with Bgh for 48 h (Figure 6K–N). To quantify the effect of VAMP721/722 in 304

PLDδ trafficking, we counted the PLDδ-GFP foci in papillae in the PLDδ-GFP 305

vamp721/

and PLDδ-GFP vamp722/

plants and found that deficiency of 306

VAMP721/722 led to decreases in the frequency of PLDδ-GFP accumulation on papillae 307

from 78.55 ± 4.20% to 41.26 ± 3.78% in vamp721/

and 40.94 ± 3.05% in vamp722/

at 308

48 hpi (Figure 6O). Compared to the PLDδ-GFP vamp721+/+

vamp722+/+

control plants, 309

the targeting efficiency of PLDδ-GFP in PLDδ-GFP vamp721+/

vamp722/

and 310

PLDδ-GFP vamp721/

vamp722+/

plants further decreased, to 12.94 ± 3.02% and 16.02 311

± 6.32%, respectively, at 48 hpi (Figure 6O). 312

PLDδ Function in Penetration Resistance Is Diminished in the Absence of the 313

SNARE Complex 314

PLD-derived PA is reported to play an essential role in basal defense and non-host 315

resistance (Pinosa et al., 2013). Therefore, we tested whether the mistargeting of PLDδ to 316

papillae in SNARE mutants could influence the resistance function of PLDδ. Using a 317

biosensor based on the PA-binding protein Spo20p fused to a fluorescent reporter, we 318

found that PA accumulated in the pathogen entry site in a pattern similar to the 319

13

distribution of PLDδ-GFP (Figure 7A–D). The pldδ mutants showed decreased 320

chitin-induced PA production compared to the wild type following chitin treatment. 321

Consistent with those observations, chitin-induced PA production was reduced in the 322

pen1 and vamp721/722 mutants, as confirmed by the relative levels of 323

phosphatidylcholine (PC) and phosphatidylethanolamin (PE), two main substrates of 324

PLDδ, measured in pldδ, pen1, and vamp721/722 mutants (Figure 7E, Supplemental 325

Figure 12). 326

Furthermore, we analyzed the levels of H2O2 and jasmonic acid (JA) in pldδ, pen1, 327

and vamp721/722 mutants and wild-type plants under chitin treatment. The pldδ mutants 328

exhibited much lower H2O2 and JA accumulation than the wild-type plants, indicating 329

that the loss of PLDδ disrupted the plants’ ability to produce H2O2 and JA in response to 330

chitin (Figure 7F, Supplemental Figure 13). More importantly, the production of H2O2 331

and JA after chitin treatment decreased in the absence of the SNARE complex (Figure 7F, 332

Supplemental Figure 13). 333

To explore the molecular basis for the susceptible phenotype of pldδ and snare 334

mutants, we tested the expression levels of the important plant defense genes PDF1.2 335

(PLANT DEFENSIN GENE 1.2), PDK1.1 (3-PHOSPHOINOSITIDE DEPENDENT 336

PROTEIN KINASE-1), PHI1 (PHOSPHATE-INDUCED 1.1), and RBOHF 337

(RESPIRATORY BURST OXIDASE HOMOLOG F). RT-qPCR analysis showed that the 338

transcript levels of PDF1.2, PDK1.1, PTI1, and RBOHF were dramatically lower in pldδ 339

and snare mutants than in wild-type plants (Figure 7G and H, Supplemental Figure 13). 340

These results suggest that loss of function of PEN1, VAMP721, and VAMP722 attenuates 341

the function of PLDδ in response to pathogens. 342

DISCUSSION 343

Innate immunity helps plants defend themselves against potentially infectious pathogens, 344

14

such as viruses, bacteria, and fungi. For powdery mildew fungi, resistance to penetration 345

at the attack site of the fungus involves a well-described innate immunity process that 346

depends on the formation of papillae, which are rich in antimicrobial compounds, at the 347

site of attempted penetration. The strong focal accumulation of proteins involved in 348

penetration resistance at papillae is considered to be important for their function (Assaad 349

et al., 2004; Stein et al., 2006). In this study, we found that the loss of PLDδ caused 350

defects in penetration resistance, in agreement with the conclusion reported recently 351

(Zhang et al., 2018). Furthermore, we demonstrated that PLDδ was targeted to the 352

papillary extracellular membrane, which is the extracellular site of the host membrane 353

The PM provides a physical barrier for the cell and a transfer station for vesicle 354

trafficking in many processes, including penetration resistance. Previous studies used 355

transmission electron microscopy to reveal large numbers of exosomes, termed 356

paramural bodies (PMBs), within the papillary matrix and clusters of multivesicular 357

bodies (MVBs) in close proximity to the site of pathogen attack, indicating that the 358

trafficking destination of the MVB cargos was the PM and that PMBs were also derived 359

from the PM (An et al., 2006; Nielsen et al., 2012). Here, our immunoblotting assay 360

showed a higher abundance of PLDδ-GFP protein following chitin treatment and a lower 361

PLDδ-GFP abundance in the PM following treatment with CHX. The highly dynamic 362

nature of PM proteins means that analyzing the density and dynamics of PLDδ molecules 363

within living cells during the pathogen response poses a challenge; therefore, we applied 364

single-molecule approaches with high spatial and temporal accuracy to characterize the 365

dynamics of PLDδ in the PM. Using FCS and TIRFM analysis, we found PLDδ-GFP 366

accumulated in the PM under pathogen stimulation. By measuring the spatial and 367

temporal dynamics of individual fluorescent spots of PLDδ directly at the plasma 368

membrane in living cells, our in vivo analysis offers another powerful way to understand 369

the dynamics of PLDδ and its regulatory mechanisms. 370

15

The PM contains highly compartmentalized structures that are partitioned into 371

different types of microdomains, which function in the transduction of various signals in 372

plant cells, especially during the early stages of fungal infection (Bhat et al., 2005) and 373

during other PAMP-induced signaling processes (Fujiwara et al., 2009; Keinath et al., 374

2010; Stanislas et al., 2009). AtREM1.3 resides in sterol-rich microdomains in the PM 375

(Demir et al., 2013) and is associated with fungal infection (Bozkurt et al., 2014). Our 376

results showed that the interaction between PLDδ and AtREM1.3 was enhanced in the 377

presence of chitin, suggesting that the pathogen-triggered, newly translated PLDδ 378

proteins are sorted into AtREM1.3-labeled sterol-rich microdomains. Parallel 379

experiments using MβCD plus chitin treatment showed that sterol depletion led to 380

decreased PLDδ-GFP accumulation and lower PA production. Since distinct subcellular 381

localizations of PLDs led to the production of PA at specific regions of cell membranes 382

(Liu et al., 2013), and PLDs might be recruited to subdomains at the PM (Novák et al., 383

2018), we proposed that the microdomain localization of PLDδ contributes to the 384

activation of PLDδ and might be related to the local production of the lipid secondary 385

messenger PA. 386

In exocytosis, membrane-bound vesicles fuse with the PM and release their contents to 387

the outside, a process that is closely involved with plant immunity (Kwon et al., 2008a). 388

Accumulating evidence from studying the interaction of Arabidopsis with the Bgh fungus 389

points to a key role of exocytosis in plant innate immunity (Yun et al., 2008). Although 390

substantial genetic analysis has focused on the pathogen-trigged immune response, the 391

regulatory mechanism of exocytosis in plant–pathogen interactions has largely been 392

neglected. In the present study, we detected a rapid recovery and an increased exocytosis 393

rate of PLDδ after stimulation by chitin. From the different recovery rates after 394

photobleaching, we conclude that the PLDδ was rapidly recruited from the cytoplasm to 395

the PM by secretion following chitin treatment and that exocytosis provides a fast and 396

efficient way to control PLDδ activity in response to pathogen attack. 397

16

In plants, exocytosis via the endomembrane system and secreted proteins are crucial 398

for cell wall deposition and modification in response to biotic stimuli (Surpin et al., 399

2004). The ARF-GEF inhibitor BFA, a well-known inhibitor of secretory and endocytic 400

pathways, is a useful diagnostic tool for recognizing the mode of exocytosis (Beck et al., 401

2012; Ding et al., 2014; Kleine-Vehn et al., 2006). In our study, no conventional signal 402

peptide sequence was found in PLDδ predicted by the SignalP 3.0 server 403

(http://www.cbs.dtu.dk/services/SignalP/) or the TargetP 1.1 server 404

(http://www.cbs.dtu.dk/services/TargetP/), indicating that PLDδ was exported by a signal 405

peptide-independent secretory process. We further found that the secretion of PLDδ can 406

occur in both BFA-resistant and BFA-sensitive manners. The BFA-resistant pathway of 407

PLDδ appears to be unaffected in response to pathogen stimuli, while the exocytosis of 408

PLDδ by the BFA-sensitive pathway significantly increased. These findings suggested 409

that PLDδ may constitutively be recycled in a BFA-resistant manner in resting 410

conditions, but upon perception of pathogen, the BFA-sensitive pathway is dramatically 411

enhanced. These observations provide strong evidence that plants have evolved a 412

mechanism to respond to pathogen attack by a trade-off that alters the dynamic 413

equilibrium between BFA-resistant and -sensitive exocytic pathways. 414

Vesicle trafficking functions as a vital part of the extracellular immune response to 415

ascomycete pathogens in Arabidopsis (Collins et al., 2003; Kwon et al., 2008b). 416

Vesicle-associated secretion mediated by PEN1 and its SNARE partners SNAP33 and 417

endomembrane-resident VAMP721/722 can enable the execution of penetration 418

resistance, and this secretion pathway can be blocked by treatment with BFA (Nielsen et 419

al., 2012; Underwood and Somerville, 2013; Zhang et al., 2019). In our study, BFA 420

effectively blocked the secretion and accumulation of PLDδ-GFP, which was similar to 421

its effect on PEN1. Moreover, we found that chitin significantly enhanced the 422

colocalization and interaction of PLDδ-GFP and mCherry-PEN1, and the deficiency of 423

VAMP721/722 led to a decrease in papillary PLDδ-GFP accumulation. Taken together, 424

17

these findings demonstrated that PEN1-associated VAMP721/722-mediated secretion 425

enables the translocation of PLDδ during attack by powdery mildew fungi. 426

PA generated by PLDs is considered as a secondary messenger and interacts with 427

effector proteins such as protein kinases, phosphatases, NADPH oxidases, modulating 428

their activity, thus amplifying the signal to initiate plant defense signaling (Rentel et al., 429

2004; Rizzo et al., 2000; Wang et al., 2006). Zhang et al (2018) reported that PLDδ 430

knockout plants showed enhanced susceptibility to lettuce powdery mildew 431

(Golovinomyces cichoracearum, Gc UCSC1), but they did not detect the changes of JA 432

levels after 5 days postinfection (dpi) under Gc UCSC1 attack (Zhang et al., 2018). In our 433

study, we observed that pldδ mutants exhibited markedly lower levels of PA, H2O2, and 434

JA than wild-type plants, partly due to the differences in the timing of examination and 435

different effects on JA levels induced in various pathogen species. Likewise, the genes 436

related to PA, ROS and JA signaling were down-regulated in pldδ mutants as compared 437

with wild-type plants, indicating that the absence of PLDδ disturbed the plants’ ability to 438

produce H2O2 and JA in response to the chitin stimulus. Moreover, snare mutants and 439

pldδ mutants showed similar phenotypes in response to pathogen stimuli, suggesting that 440

the delivery of PLDδ mediated by the SNARE complex contributed to the resistance 441

function of PLDδ. In combination with the accumulation of PA at papillae, these findings 442

support the hypothesis that the translocation of PLDδ to papillae depends on a 443

PEN1/VAMP721/722-mediated secretion pathway, which is accompanied by PLDδ 444

activation and production of PA, thus triggering PA-mediated signaling in the plant 445

defense response. 446

In summary, our investigation of the dynamic behaviors of PLDδ has provided a more 447

global view of the mechanism of translocation of this phospholipase in the immune 448

response to pathogens. The focal concentration of PLDδ at pathogen entry sites is 449

important for its function and is regulated by diverse secretory pathways. Consequently, 450

PM microdomains likely harbor unique sets of signaling proteins or serve as signaling 451

18

platforms in penetration resistance, acting as transfer stations to deliver 452

pathogen-inducible PLDδ to the papillae. This study produced three novel findings: (1) 453

secretion of PLDδ functions as a fast and efficient way to control PLDδ accumulation in 454

response to pathogen attack; (2) plants have evolved a dynamic equilibrium between 455

BFA-resistant and -sensitive exocytic pathways in the secretion of PLDδ under diverse 456

conditions, and the exocytosis of pathogen-inducible PLDδ in BFA-sensitive manner 457

requires PEN1-associated VAMP721/722-mediated secretion; and (3) AtREM1.3-labeled 458

sterol-rich microdomains act as essential platforms for the transportation and 459

accumulation of PLDδ. Taken together, these findings reveal a novel regulatory 460

mechanism involving membrane microdomains and exocytosis in the relocalization of 461

PLDδ, aiding in the execution of penetration resistance against a fungal pathogen. 462

METHODS 463

Plant and Fungal Growth 464

The Arabidopsis thaliana pldδ mutant expressing the transgene PLDδ-GFP driven by the 465

endogenous PLDδ promoter and the pldδ mutant (SALK_023247) was used in all 466

experiments (Pinosa et al., 2013). The p35S:AtREM1.3-mCherry vectors were described 467

previously by Xue et al. (2018). The vamp721/

, vamp722/

, vamp721+/

vamp722/

, 468

and vamp721/

vamp722+/

mutants and Arabidopsis expressing the transgene 469

pVAMP721/722:mCherry-VAMP721/722 were obtained from Zhang et al. (2011). The 470

p35S:AtREM1.3-mCherry vectors were transformed into Col-0, and seedlings were 471

selected with 70 μg/mL hygromycin. For Arabidopsis coexpressing PLDδ-GFP and 472

mCherry-PEN1, PLDδ-GFP and mCherry-VAMP721/722, PLDδ-GFP and 473

AtREM1.3-mCherry, F1 lines derived from crosses between PLDδ-GFP and 474

mCherry-PEN1, mCherry-VAMP721/722, or AtREM1.3-mCherry, respectively. The 475

plants were grown at 21°C, with 12 h of white light (light intensity of 120 μmol photons 476

m−2

s−1

) per day. The barley powdery mildew fungus (Blumeria graminis f. sp. hordei, 477

19

isolate K1) was propagated on barley (Hordeum vulgare) at 18°C in an illuminated 478

incubator. 479

Drug Treatments 480

The inhibitors BFA, CHX, and MβCD were obtained from Sigma-Aldrich. CHX and BFA 481

were made into stock solutions with DMSO (50 mM for CHX and 50 mM for BFA), and 482

MβCD was dissolved in deionized water to yield a 200 mM stock solution. The PAMP 483

chitin solutions were generated by dissolving hydrolyzed chitin (purified from crab shells, 484

Sigma-Aldrich) in deionized water to a concentration of 100 μg·mL1

. FM4-64 (obtained 485

from Invitrogen) was kept as a 5 mM stock solution and diluted to a 5 μM working 486

solution with half-strength liquid Murashige and Skoog medium (1/2× liquid MS). The 487

BFA treatment combined with fungal inoculation was performed as previously described 488

(Nielsen et al., 2012). For other inhibitor treatments, seedlings were incubated in 1/2× 489

liquid MS medium containing appropriate drugs for the following times: 30 min for BFA, 490

30 min of pretreatment with BFA and 30 min for BFA plus 100 μg·mL1

chitin, 30 min 491

for MβCD, and 30 min pretreatment with MβCD plus 100 μg·mL1

chitin. Seedlings were 492

then transferred onto a slide with inhibitor solution and covered with a coverslip for 493

CLSM or variable angle (VA)-TIRFM imaging. 494

FM4-64 Detection, Plasmolysis, and FRAP Analysis Using Confocal Microscopy 495

The leaves from adult Arabidopsis plants were dipped in a solution of 0.01% (v/v) 496

Silwet/0.2% (w/v) propidium iodide to stain the Bgh spores (Nielsen et al., 2012). 497

Membrane staining was performed by incubating the adult leaves with 3 μM FM4-64 for 498

30 min before observation, and plasmolysis experiments were conducted by incubating 499

the adult leaves with 1 M mannitol for 5 min before observation. An FV1000ME 500

multiphoton laser scanning microscope (Olympus) was used to observe the FRAP of the 501

plants, and 488- and 458-nm laser lines operating at 100% were used to bleach the 502

20

PLDδ-GFP from a region of the same size in every treatment. Images were collected 1 503

min before bleaching, immediately after bleaching, and once a minute after bleaching for 504

a total of 20 min. The fluorescence recovery data obtained were corrected for bleaching 505

during imaging as previously described (Konopka et al., 2008; Underwood and 506

Somerville, 2013), and fitted curves were made using Origin 8.0 software (Origin Lab 507

Corporation). The exocytosis rate was calculated according to the methods previously 508

described (Luo et al., 2016). 509

VA-TIRFM and Single-Particle Fluorescence Image Analysis 510

The PLDδ-GFP dynamics were recorded using a VA-TIRFM technique on an inverted 511

microscope (IX-71; Olympus) with a total internal reflective fluorescence illuminator and 512

a 100× oil-immersion objective (Olympus; numerical aperture = 1.45) (Fan et al., 2013; 513

Li et al., 2012; Li et al., 2011; Wang et al., 2015a; Wang et al., 2013; Wang et al., 2015b; 514

Wang et al., 2018). The method described by Wang et al. (2015b) was employed for 515

single-particle tracking, which was the basis of the kinetic parameter analysis of 516

PLDδ-GFP. The diffusion coefficient was determined according to the methods 517

previously described (Li et al., 2011; Wang et al., 2015b; Xiao et al., 2008). Trajectories 518

with a length of >10 frames were kept for MSD and diffusion coefficient analysis. The 519

MSD calculation of PLDδ-GFP spots was performed using the following equation: 520

521

MSD(t) =1

L−n∑ (r(s + n) − r(s))2L−n−1

s=0 [S1] 522

where n = t/t, L is the length of the trajectory, and r(s) is the two-dimensional position of 523

the particle in frames (Goulian and Simon, 2000). The diffusion coefficient for a spot was 524

determined by fitting a line to MSD with n running from 1 to the largest integer ≤L/4 525

(Saxton, 1997). In the text, a single (or multiple) Gaussian fit(s) of the diffusion 526

coefficient (D) histograms represents the diffusion coefficients of PLDδ-GFP spots. 527

21

Origin software (OriginLab) was used to determine the SE values of the position of the 528

peak (denoted Ĝ) in the form of an upper and a lower diffusion coefficient. Moreover, 529

parameters were performed according to Espenel et al. (2008) and used to determine the 530

motional modes (Brownian, restricted diffusion). When linearly fitted to the MSD, the 531

MSD-t plot was a straight line with a slope of 4D, representing a typical trajectory for 532

spots undergoing Brownian diffusion. If the MSD-t plot showed a negative deviation 533

from a straight line with a slope of 4D, the motion was defined as restricted diffusion. 534

Single fluorescent spots of PLDδ-GFP were detected in a time-lapse series of up to 100 535

images per sequence, which were acquired with a 200-ms exposure time. 536

FCS and PPI Analysis 537

The FCS analysis was performed using the point-scanning mode on a Leica TCS SP5 538

FCS microscope equipped with a 488-nm argon laser, in-house coupled correlator, and 539

Avalanche photodiode. Spontaneous fluctuations in fluorescence intensity were generated 540

by the diffusion of PLDδ-GFP molecules in and out of the focal plane, which can alter the 541

local concentration of the fluorophores. The density of PLDδ-GFP was detected by a laser 542

focused at the PM and calculated according to the protocol previously described by Li et 543

al. (2011). PPI was performed on confocal images using PPI software (Wu et al. 2010; 544

Zinchuk et al. 2013; Cui et al., 2019). The quantification of the degree of colocalization 545

between PLDδ-GFP and mCherry-PEN1 or AtREM1.3-mCherry was based on the review 546

by Zinchuk et al. (2013). 547

FRET-FLIM Analysis 548

FRET-FLIM analysis was performed using an inverted Olympus FV1200 microscope 549

equipped with a PicoQuant picoHarp300 controller. The excitation at 488 nm was 550

performed by a picosecond pulsed diode laser at a repetition rate of 40 MHz, via a water 551

immersion objective (60×, numerical aperture = 1.2). The emitted light was filtered with 552

22

a 520/35-nm bandpass filter and detected by an MPD SPAD detector. Data were collected 553

and analyzed using the SymphoTime 64 software (PicoQuant). 554

Coimmunoprecipitation and Immunoblot Analysis 555

To monitor the abundance of PLDδ-GFP in the different treatment groups, total proteins 556

were extracted from the PLDδ-GFP transgenic plants grown on half-strength MS medium 557

for 3 weeks using the EZ extraction protocol (Martinez-Garcia et al., 1999). For 558

membrane protein extraction, the PLDδ-GFP plants were ground into a fine powder in 559

liquid nitrogen and mixed with extraction solution (25 mM Tris, pH 7.5, 2 mM EDTA, 2 560

mM DTT, 15 mM mercaptoethanol, 0.5% BSA, 0.25 M sucrose, 10% glycerol, and one 561

complete protease inhibitor tablet [Roche Applied Science] per 50 mL extraction buffer). 562

After being mixed in a 1:1 (v:v) ratio with SDS-PAGE buffer, the protein extraction 563

solution was heated at 95°C for 15 min and then centrifuged at 12,000 rpm (10,625 g) for 564

5 min to remove cellular debris. The supernatants containing total proteins and membrane 565

proteins were subjected to 8% SDS-PAGE. An immunoblot analysis was performed 566

following the protocol described by Fan et al. (2013). For coimmunoprecipitation, plants 567

coexpressing PLDδ-GFP and mCherry-PEN1 were harvested, and the assay was 568

performed as previously described (Fan et al., 2013; Serino and Deng, 2007). 569

PA Analysis by Mass Spectrometry and PLD Activity Assay 570

For PA extraction, the seedlings of every genotypes were harvested under chitin treatment 571

for 0 min, 15 min, 30 min. The harvested seedlings were transferred immediately into 2 572

mL of isopropanol with 0.01% butylated hydroxytoluene at 75°C. Chloroform/methanol 573

(2:1) was used to extract the tissue with three additional times with 2 h of agitation each 574

time. Then the tissues were heated overnight at 105°C and weighed. A triple quadrupole 575

tandem mass spectrometry (MS/MS) equipped for electrospray ionisation (ESI) was 576

employed to analyze lipid samples. The PAs in each class were quantified by comparison 577

23

with two internal standards for the class. Data processing was performed as previously 578

described (Welti et al., 2002). 579

PLD activity was assayed spectrophotometrically by measuring the free choline 580

released upon PC hydrolysis. Plant tissues were harvested under resting condition and the 581

treatments of chitin, MβCD and MβCD plus chitin. The harvested tissues were then 582

incubated with PC substrate and the reaction mixture containing 50 mM Tris-HCl, pH 8.0, 583

20 mM CaCl2, 1.7 mM 4-aminoantipyrine, 9 mM sodium 2-hydroxy-3, 584

5-dichlorobenzenesulfonate, 0.5 U choline oxidase and 0.5 U peroxidase for 30 min at585

30°C. The absorbance measurements at 500 nm were carried out to quantify the amounts 586

of free choline released. One unit of PLD activity was calculated as described by previous 587

study (Abdelkafi and Abousalham, 2011). 588

Statistics 589

All experimental analyses were conducted in consultation with a statistician. 590

Quantification and statistical parameters are reported in legends of each figure, including 591

error bars (SD or SE), n values, repeats of experiments, and test types. Student’s t-test 592

was used to describe differences between two groups. For those involving multiple group 593

comparisons, ANOVA followed by a post hoc test was applied. In the figures, statistically 594

significant differences are shown with asterisks as follows: *P < 0.05, **P < 0.01 and 595

***P < 0.001. 596

Accession Numbers 597

Sequence data from this article can be found in The Arabidopsis Information Resource 598

(TAIR) database under the following accession numbers: PLDδ (AT4G35790), AtRem1.3 599

(At2g45820), VAMP721 (AT1G04750), VAMP722 (AT2G33120), and PEN1 600

(AT3G11820). 601

24

Supplemental Data 602

Supplemental Figure 1. PLDδ is involved in non-host resistance to Bgh (supports Figure 603

1). 604

Supplemental Figure 2. The abundance of PLDδ-GFP is influenced by CHX (supports 605

Figure 2). 606

Supplemental Figure 3. FRAP analysis revealing the dynamics of PLDδ-GFP with or 607

without stimulation by chitin (supports Figure 2). 608

Supplemental Figure 4. Dynamics of PLDδ-GFP at the plasma membrane estimated by 609

FRAP (supports Figure 2). 610

Supplemental Figure 5. The effect of MβCD on PLDδ-GFP dynamics (supports Figure 611

3). 612

Supplemental Figure 6. The levels of phosphatidic acid and PLD activity under 613

stimulation of pathogen (supports Figure 3). 614

Supplemental Figure 7. MβCD blocked the accumulation of PLDδ-GFP at the Bgh entry 615

sites (supports Figure 3). 616

Supplemental Figure 8. Penetration rate of Bgh affected by MβCD or BFA (supports 617

Figure 3 and Figure 4). 618

Supplemental Figure 9. Subcellular localization of PLDδ in the presence of CHX 619

(Supports Figure 4). 620

Supplemental Figure 10. The colocalization of PLDδ-GFP and mCherry-PEN1 analyzed 621

by protein proximity index (supports Figure 5). 622

Supplemental Figure 11. The colocalization of PLDδ-GFP and mCherry-VAMP722 in 623

the cytoplasm and papillae (supports Figure 6). 624

Supplemental Figure 12. Relative levels of PC and PE levels in the absence of PLDδ 625

and the SNARE complex in response to pathogen stimuli (supports Figure 7). 626

Supplemental Figure 13. JA level and expression of JA-related genes in wild-type, pldδ, 627

pen1, vamp721/

, and vamp722/

mutant plants in response to chitin (supports Figure 7). 628

25

Supplemental Table 1. Primers used for qPCR. 629

Supplemental Movie 1. VA-TIRFM imaging of PLDδ-GFP spots in the control condition 630

at the PM of Arabidopsis leaf epidermal cells. 631

Supplemental Movie 2. VA-TIRFM imaging of PLDδ-GFP spots at the PM of 632

Arabidopsis leaf epidermal cells under chitin treatment. 633

Supplemental Movie 3. VA-TIRFM imaging of PLDδ-GFP spots at the PM of 634

Arabidopsis leaf epidermal cells treated with 10 mM MβCD. 635

Supplemental Movie 4. VA-TIRFM imaging of PLDδ-GFP spots at the PM of 636

Arabidopsis leaf epidermal cells under MβCD + chitin treatment. 637

Author contributions 638

J.J.X., X.J.L., X.H.W, and J.X.L. designed the research; J.J.X. and X.Q.L. performed the 639

research; J.J.X., X.J.L., L.W., X.Q.L., and L.Z. analyzed data; J.J.X., X.J.L., Q. H. S., F. 640

B., M. B., J. S., Y. F, Z and J.X.L. wrote the article. 641

ACKNOWLEDGMENTS 642

We thank Prof. Mats X. Andersson (University of Gothenburg, Sweden) for kindly 643

providing the transgenic seeds expressing PLDδ-GFP driven by the endogenous PLDδ 644

promoter and the pldδ mutant. We also thank Prof. Ying Fu (China Agricultural 645

University, Beijing) for kindly providing the seeds expressing the transgene 646

pPEN1::mCherry-PEN1. We thank the members of the Imaging Core 647

Facility, Technology Center for Protein Sciences, Tsinghua University, for assistance with 648

using the FV1200 LSCM with the Picoquant FLIM/FCS system. This work is supported 649

by the National Natural Science Foundation of China (31530084, 31622005, 31900162), 650

the Program of Introducing Talents of Discipline to Universities (111 project, B13007) 651

and the ERDF project "Plants as a tool for sustainable global development" (No. 652

CZ.02.1.01/0.0/0.0/16_019/0000827). 653

26

FIGURE LEGENDS 654

Figure 1. The Subcellular Localization of PLDδ-GFP Following Bgh Infection. (A–B) 655

Fluorescence (A) and fluorescence merged with bright-field (B) images showing that 656

PLDδ-GFP is localized at the epidermal cell membrane of the control Arabidopsis leaves. 657

(C–D) Fluorescence (C) and fluorescence merged with bright-field (D) images 658

demonstrating the localization of PLDδ-GFP at the Bgh penetration sites. The white 659

arrowheads indicate the accumulation of PLDδ-GFP. (E–G) Plasmolysis upon Bgh 660

infection showed the focal accumulation (arrowhead) of PLDδ-GFP in the papillae of the 661

periplasmic space. The fluorescence image (E) is merged with the bright-field image (F) 662

in (G). (H–K) The focal accumulation of PLDδ-GFP (H) overlaps completely (J) with the 663

FM4-64-stained papillae (I) at 48 hpi with Bgh. (K) Bright-field image of the papillae in 664

(H–J). Scale bars: 10 μm in each panel. 665

666

Figure 2. Density and Dynamics of PLDδ-GFP at the Plasma Membrane. (A) 667

Live-cell imaging of leaf epidermal cells expressing PLDδ-GFP, with the numbers 668

indicating the detection areas for FCS analysis. (B) FCS analysis determined the 669

differences in PLDδ-GFP density at the plasma membrane following treatment with 670

chitin or CHX plus chitin (n = 50, 96, and 74 measurements for the control, chitin, and 671

CHX plus chitin, respectively, from 12 seedlings for each condition. ***P < 0.001, 672

ANOVA and post-hoc Tukey’s test). (C) Immunoblot analysis showed the abundance of 673

PLDδ-GFP in total proteins and membrane proteins in response to chitin or CHX + chitin 674

treatment. Actin and CBB (Coomassie Brilliant Blue) were used as the internal controls. 675

(D–E) Distribution of PLDδ-GFP particle fluorescence on the plasma membrane in the 676

control (D) or chitin-treated cells (E) shown by pseudocolor images (blue-yellow-red 677

palette). (F) FRAP time course of PLDδ-GFP with or without chitin treatment. White 678

squares indicate bleached regions. (G) Fluorescence recovery curves of the 679

photobleached region of interest. (H) Exocytosis rates of PLDδ-GFP with or without 680

chitin treatment. *P < 0.05, Student’s t-test. Bars represent means, error bars represent 681

standard error (SE). Scale bars: 10 μm in A and F, 3 μm in D–E. 682

683

Figure 3. Localization of PLDδ-GFP in Plasma Membrane Microdomains. (A–D) 684

Colocalization of PLDδ-GFP (A) and AtREM1.3-mCherry (B) in the papillae, with 685

merged (C) and bright-field (D) images. (E–G) Live-cell imaging of leaf epidermal cells 686

expressing PLDδ-GFP (E), AtREM1.3-mCherry (F) and the merged image (G) in the 687

control conditions. (H–I) Protein proximity index between PLDδ-GFP and 688

AtREM1.3-mCherry with or without chitin treatment (n=10, with 15 regions of interest in 689

the control condition and 30 in the chitin treatment). (J–M) Live-cell imaging (using 690

confocal microscopy) of fluorescence lifetime distribution of PLDδ-GFP in the plants 691

27

expressing PLDδ-GFP alone (J), coexpressing Free-GFP/ AtREM1.3-mCherry (K) and 692

co-expressing PLDδ-GFP/AtREM1.3-mCherry. L and M indicate the fluorescence 693

lifetime of PLDδ-GFP with (L) or without chitin treatment (M). AtREM1.3 is a marker of 694

membrane microdomains. (N) FRET-FLIM analysis revealed the fluorescence lifetime of 695

PLDδ-GFP (n = 12, with 15 regions of interest in the plants expressing PLDδ-GFP alone 696

and 15 regions in the plants co-expressing PLDδ-GFP/AtREM1.3-mCherry under control 697

conditions and 19 under chitin treatment). Bars represent means, error bars in all panels 698

represent SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test in H; ANOVA and 699

post-hoc Tukey’s test in K). Scale bars: 10 μm in A–G, 2 μm in J–M. 700

701

Figure 4. Effect of BFA on the Exocytosis and Accumulation of PLDδ. (A–H) 702

Subcellular localization of PLDδ in the presence of the vesicle-trafficking inhibitor BFA 703

with (D–F) or without chitin treatment (A–C). Costaining with FM4-64 (B, E) highlights 704

the plasma membrane and BFA bodies. White arrowheads indicate BFA-resistant vesicles, 705

and the magenta arrowheads indicate BFA bodies. G and H show the quantitation of BFA 706

bodies (G) and BFA-resistant vesicles of PLDδ-GFP (H). For each experiment, at least 30 707

epidermal cells from 4 seedlings were scored (n = 3). (I–J) FRAP time course of 708

PLDδ-GFP with BFA pretreatment and chitin treatment. The white rectangle indicates the 709

bleached region (I). The initial bleached region was further subdivided into three sectors, 710

indicated by the green, red, and blue rectangles (J). (K) Fluorescence recovery curves of 711

the photobleached region of interest. (L) Fluorescence recovery curves of divided regions 712

of interest. Curves represent the best fits of mean values of six independent FRAP 713

experiments on PLDδ-GFP. (M) Exocytosis rates of PLDδ-GFP with or without BFA 714

treatment. (N–P) Accumulation of PLDδ-GFP at sites of Bgh attempted penetration with 715

(O) or without pretreatment with BFA (N). Arrowheads indicate the Bgh attempted 716

penetration sites. For each leaf, at least 49 germinated spores were scored (n = 3). Bars 717

represent means, error bars in all panels represent SD. **P < 0.01, ***P < 0.001, 718

Student’s t-test. Scale bars: 20 μm in A–F and N–O; 10 μm in I; 5 μm in J. 719

720

Figure 5. Colocalization and Interaction Analysis of PLDδ with PEN1 in Living 721

Plant Cells. (A–D) Colocalization of PLDδ-GFP (A) and mCherry-PEN1 (B) in the 722

papillae, with the merged (C) and bright-field (D) images. (E–H) Live-cell imaging 723

(using confocal microscopy) of fluorescence lifetime distribution of PLDδ-GFP in the 724

plants expressing PLDδ-GFP alone (E), coexpressing Free-GFP/mCherry-PEN1 (F) and 725

co-expressing PLDδ-GFP/mCherry-PEN1 (G–H). G and H indicate the fluorescence 726

lifetime of PLDδ-GFP with (H) or without chitin treatment (G). (I) FRET-FLIM analysis 727

revealed the change in the fluorescence lifetime of PLDδ-GFP (n = 10, with 15 regions of 728

interest in the plants expressing PLDδ-GFP alone and in the plants co-expressing 729

PLDδ-GFP/mCherry-PEN1 under both control condition and chitin treatment). (J) 730

Co-immunoprecipitation of PEN1 with PLDδ in transgenic Arabidopsis. Total protein 731

28

extracts from PLDδ-GFP and PLDδ-GFP/mCherry-PEN1 transgenic plants were 732

immunoprecipitated (IP) with anti-GFP. IP proteins were detected by immunoblotting 733

with anti-mCherry antibody. Bars represent means, error bars in I represent SD. ANOVA 734

and post-hoc Tukey’s test for I. *P < 0.05. Scale bar: 10 μm in A–D; 2 μm in E–H. 735

736

Figure 6. PLDδ is Trafficked to the Papillae Via VAMP721/722-mediated Secretion 737

in Response to Chitin. (A–D) Colocalization of PLDδ-GFP (A) and mCherry-VAMP721 738

(B) in the papillae at the Bgh infection sites, with the merged (C) and bright-field (D) 739

images. (E–J) Colocalization (G, J) of PLDδ-GFP (E, H) and mCherry-VAMP721 (F, I) in 740

the cytoplasm under control conditions (E–G) or chitin treatment (H–J). Arrowheads 741

indicate the cytoplasmic bodies labeled by GFP or mCherry. (K–N) Fluorescence images 742

demonstrating the accumulation of PLDδ-GFP at the penetration sites of Bgh in 743

PLDδ-GFP vamp721-/-

plants (K), PLDδ-GFP vamp722-/-

plants (L), in PLDδ-GFP 744

vamp721+/-

vamp722-/-

(M) and PLDδ-GFP vamp721-/-

vamp722+/-

plants (N). The white 745

arrowheads in K–N indicate the accumulation of PLDδ-GFP. (O) The number of foci 746

were enumerated for at least 49 germinated spores per leaf for six leaves per genotype (n 747

= 6). Bars represent means, error bars in all panels represent SD. ANOVA and post-hoc 748

Tukey’s test for O, **P < 0.01, ***P < 0.001. Scale bar: 10 μm in A–N. 749

750

Figure 7. PA levels, ROS accumulation, and Defense-related Gene Expression in the 751

Absence of PLDδ and the SNARE Complex in Response to Pathogen Stimuli. (A–D) 752

Focal accumulation of PLDδ-GFP (A) and a fluorescent phosphatidic acid (PA) biosensor 753

(B) in the papillae at the Bgh entry site, with bright-field (C) and merged (D) images. The 754

PA sensor is the PA-binding domain of the yeast SNARE Spo20p fused to the fluorescent 755

protein mCherry. (E) Total PA in Col-0, pldδ, pen1, vamp721-/-

, and vamp722-/-

plants 756

measured by mass spectrometry under chitin treatment for 0, 15, and 30 min. Relative PA 757

levels compared to WT at time zero (control) are presented. (F) Total H2O2 in Col-0, pldδ, 758

pen1, vamp721-/-

, and vamp722-/-

plants measured by a micro hydrogen peroxide (H2O2) 759

assay kit under chitin treatment for 0, 15, and 30 min. Relative H2O2 levels compared to 760

WT at time zero (control) are presented. (G–H) Expression of defense-related genes in 761

Col-0, pldδ, pen1, vamp721-/-

, and vamp722-/-

mutant plants in response to chitin. 762

Transcript levels were normalized to ACTIN2. Relative expression levels compared to 763

WT at time zero (control) are presented. Data bars represent the mean (± SD) of three 764

repeats. The asterisk (*) in E and F indicates the statistically significant difference in the 765

level of PC and PE compared to respective control. Student’s t-test, *P < 0.05, **P < 0.01. 766

Scale bars: 10 μm in A–D. 767

768

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Figure 1. The Subcellular Localization of PLDδ-GFP Following Bgh Infection. (A–B)

Fluorescence (A) and fluorescence merged with bright-field (B) images showing that PLDδ-

GFP is localized at the epidermal cell membrane of the control Arabidopsis leaves. (C–D)

Fluorescence (C) and fluorescence merged with bright-field (D) images demonstrating the

localization of PLDδ-GFP at the Bgh penetration sites. The white arrowheads indicate the

accumulation of PLDδ-GFP. (E–G) Plasmolysis upon Bgh infection showed the focal

accumulation (arrowhead) of PLDδ-GFP in the papillae of the periplasmic space. The

fluorescence image (E) is merged with the bright-field image (F) in (G). (H–K) The focal

accumulation of PLDδ-GFP (H) overlaps completely (J) with the FM4-64-stained papillae (I) at

48 hpi with Bgh. (K) Bright-field image of the papillae in (H–J). Scale bars: 10 μm in each panel.

Figure 2. Density and Dynamics of PLDδ-GFP at the Plasma Membrane. (A) Live-cell

imaging of leaf epidermal cells expressing PLDδ-GFP, with the numbers indicating the detection

areas for FCS analysis. (B) FCS analysis determined the differences in PLDδ-GFP density at

the plasma membrane following treatment with chitin or CHX plus chitin (n = 50, 96, and 74

measurements for the control, chitin, and CHX plus chitin, respectively, from 12 seedlings for

each condition. ***P < 0.001, ANOVA and post-hoc Tukey’s test). (C) Immunoblot analysis

showed the abundance of PLDδ-GFP in total proteins and membrane proteins in response to

chitin or CHX + chitin treatment. Actin and CBB (Coomassie Brilliant Blue) were used as the

internal controls. (D–E) Distribution of PLDδ-GFP particle fluorescence on the plasma

membrane in the control (D) or chitin-treated cells (E) shown by pseudocolor images (blue-

yellow-red palette). (F) FRAP time course of PLDδ-GFP with or without chitin treatment. White

squares indicate bleached regions. (G) Fluorescence recovery curves of the photobleached

region of interest. (H) Exocytosis rates of PLDδ-GFP with or without chitin treatment. *P < 0.05,

Student’s t-test. Bars represent means, error bars represent standard error (SE). Scale bars:

10 μm in A and F, 3 μm in D–E.

Figure 3. Localization of PLDδ-GFP in Plasma Membrane Microdomains. (A–D)

Colocalization of PLDδ-GFP (A) and AtREM1.3-mCherry (B) in the papillae, with merged (C)

and bright-field (D) images. (E–G) Live-cell imaging of leaf epidermal cells expressing PLDδ-

GFP (E), AtREM1.3-mCherry (F) and the merged image (G) in the control conditions. (H–I)

Protein proximity index between PLDδ-GFP and AtREM1.3-mCherry with or without chitin

treatment (n=10, with 15 regions of interest in the control condition and 30 in the chitin

treatment). (J–M) Live-cell imaging (using confocal microscopy) of fluorescence lifetime

distribution of PLDδ-GFP in the plants expressing PLDδ-GFP alone (J), coexpressing Free-

GFP/ AtREM1.3-mCherry (K) and co-expressing PLDδ-GFP/AtREM1.3-mCherry. L and M

indicate the fluorescence lifetime of PLDδ-GFP with (L) or without chitin treatment (M).

AtREM1.3 is a marker of membrane microdomains. (N) FRET-FLIM analysis revealed the

fluorescence lifetime of PLDδ-GFP (n = 12, with 15 regions of interest in the plants expressing

PLDδ-GFP alone and 15 regions in the plants co-expressing PLDδ-GFP/AtREM1.3-mCherry

under control conditions and 19 under chitin treatment). Bars represent means, error bars in all

panels represent SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test in H; ANOVA and

post-hoc Tukey’s test in K). Scale bars: 10 μm in A–G, 2 μm in J–M.

Figure 4. Effect of BFA on the Exocytosis and Accumulation of PLDδ. (A–H) Subcellular

localization of PLDδ in the presence of the vesicle-trafficking inhibitor BFA with (D–F) or without

chitin treatment (A–C). Costaining with FM4-64 (B, E) highlights the plasma membrane and

BFA bodies. White arrowheads indicate BFA-resistant vesicles, and the magenta arrowheads

indicate BFA bodies. G and H show the quantitation of BFA bodies (G) and BFA-resistant

vesicles of PLDδ-GFP (H). For each experiment, at least 30 epidermal cells from 4 seedlings

were scored (n = 3). (I–J) FRAP time course of PLDδ-GFP with BFA pretreatment and chitin

treatment. The white rectangle indicates the bleached region (I). The initial bleached region

was further subdivided into three sectors, indicated by the green, red, and blue rectangles (J).

(K) Fluorescence recovery curves of the photobleached region of interest. (L) Fluorescence

recovery curves of divided regions of interest. Curves represent the best fits of mean values of

six independent FRAP experiments on PLDδ-GFP. (M) Exocytosis rates of PLDδ-GFP with or

without BFA treatment. (N–P) Accumulation of PLDδ-GFP at sites of Bgh attempted penetration

with (O) or without pretreatment with BFA (N). Arrowheads indicate the Bgh attempted

penetration sites. For each leaf, at least 49 germinated spores were scored (n = 3). Bars

represent means, error bars in all panels represent SD. **P < 0.01, ***P < 0.001, Student’s t-

test. Scale bars: 20 μm in A–F and N–O; 10 μm in I; 5 μm in J.

Figure 5. Colocalization and Interaction Analysis of PLDδ with PEN1 in Living Plant Cells.

(A–D) Colocalization of PLDδ-GFP (A) and mCherry-PEN1 (B) in the papillae, with the merged

(C) and bright-field (D) images. (E–H) Live-cell imaging (using confocal microscopy) of

fluorescence lifetime distribution of PLDδ-GFP in the plants expressing PLDδ-GFP alone (E),

coexpressing Free-GFP/mCherry-PEN1 (F) and co-expressing PLDδ-GFP/mCherry-PEN1 (G–

H). G and H indicate the fluorescence lifetime of PLDδ-GFP with (H) or without chitin treatment

(G). (I) FRET-FLIM analysis revealed the change in the fluorescence lifetime of PLDδ-GFP (n

= 10, with 15 regions of interest in the plants expressing PLDδ-GFP alone and in the plants co-

expressing PLDδ-GFP/mCherry-PEN1 under both control condition and chitin treatment). (J)

Co-immunoprecipitation of PEN1 with PLDδ in transgenic Arabidopsis. Total protein extracts

from PLDδ-GFP and PLDδ-GFP/mCherry-PEN1 transgenic plants were immunoprecipitated

(IP) with anti-GFP. IP proteins were detected by immunoblotting with anti-mCherry antibody.

Bars represent means, error bars in I represent SD. ANOVA and post-hoc Tukey’s test for I. *P

< 0.05. Scale bar: 10 μm in A–D; 2 μm in E–H.

Figure 6. PLDδ is Trafficked to the Papillae Via VAMP721/722-mediated Secretion in

Response to Chitin. (A–D) Colocalization of PLDδ-GFP (A) and mCherry-VAMP721 (B) in the

papillae at the Bgh infection sites, with the merged (C) and bright-field (D) images. (E–J)

Colocalization (G, J) of PLDδ-GFP (E, H) and mCherry-VAMP721 (F, I) in the cytoplasm under

control conditions (E–G) or chitin treatment (H–J). Arrowheads indicate the cytoplasmic bodies

labeled by GFP or mCherry. (K–N) Fluorescence images demonstrating the accumulation of

PLDδ-GFP at the penetration sites of Bgh in PLDδ-GFP vamp721-/- plants (K), PLDδ-GFP

vamp722-/- plants (L), in PLDδ-GFP vamp721+/- vamp722-/- (M) and PLDδ-GFP vamp721-/-

vamp722+/- plants (N). The white arrowheads in K–N indicate the accumulation of PLDδ-GFP.

(O) The number of foci were enumerated for at least 49 germinated spores per leaf for six

leaves per genotype (n = 6). Bars represent means, error bars in all panels represent SD.

ANOVA and post-hoc Tukey’s test for O, **P < 0.01, ***P < 0.001. Scale bar: 10 μm in A–N.

Figure 7. PA levels, ROS accumulation, and Defense-related Gene Expression in the

Absence of PLDδ and the SNARE Complex in Response to Pathogen Stimuli. (A–D) Focal

accumulation of PLDδ-GFP (A) and a fluorescent phosphatidic acid (PA) biosensor (B) in the

papillae at the Bgh entry site, with bright-field (C) and merged (D) images. The PA sensor is the

PA-binding domain of the yeast SNARE Spo20p fused to the fluorescent protein mCherry. (E)

Total PA in Col-0, pldδ, pen1, vamp721-/-, and vamp722-/- plants measured by mass

spectrometry under chitin treatment for 0, 15, and 30 min. Relative PA levels compared to WT

at time zero (control) are presented. (F) Total H2O2 in Col-0, pldδ, pen1, vamp721-/-, and

vamp722-/- plants measured by a micro hydrogen peroxide (H2O2) assay kit under chitin

treatment for 0, 15, and 30 min. Relative H2O2 levels compared to WT at time zero (control) are

presented. (G–H) Expression of defense-related genes in Col-0, pldδ, pen1, vamp721-/-, and

vamp722-/- mutant plants in response to chitin. Transcript levels were normalized to ACTIN2.

Relative expression levels compared to WT at time zero (control) are presented. Data bars

represent the mean (± SD) of three repeats. The asterisk (*) in E and F indicates the statistically

significant difference in the level of PC and PE compared to respective control. Student’s t-test,

*P < 0.05, **P < 0.01. Scale bars: 10 μm in A–D.

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DOI 10.1105/tpc.19.00534; originally published online October 9, 2019;Plant Cell

Shen, Frantisek Baluska, Jozef Samaj and Jinxing LinJingjing Xing, Xiaojuan Li, Xiaohua Wang, Xueqin Lv, Li Wang, Liang Zhang, Yingfang Zhu, Qian-Hua

Functions as a Regulatory Mechanism in Plant Innate ImmunityδSecretion of Phospholipase D

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