1

RESEARCH ARTICLE 1 2

Pectinmethyesterases Modulate Plant Homogalacturonan Status 3

in Defenses Against the Aphid Myzus persicae 4 5

Christian Silva-Sanzanaa, Jonathan Celiz-Balboaa, Elisa Garzoc, Susan E. Marcusd, 6 Juan Pablo Parra-Rojasa, Barbara Rojasa, Patricio Olmedoa, Miguel A. Rubilara, 7 Ignacio Riosa, Rodrigo A. Chorbadjiane, Alberto Fereresc, Paul Knoxd, Susana Saez-8 Aguayoa,*, Francisca Blanco-Herreraa,b,* 9

a Centro de Biotecnología Vegetal, Facultad de Ciencias Biológicas, Universidad Andrés 10 Bello, Santiago, Chile. 11 b Millennium Institute for Integrative Biology (IBio), Santiago, Chile. 12 c Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Científicas, Madrid, 13 Spain. 14 d Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, 15 United Kingdom. 16 e Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, 17 Santiago, Chile. 18

19 * Corresponding authors:20 Francisca Blanco-Herrera (mblanco@unab.cl)21 Susana Saez-Aguayo (susana.saez@unab.cl)22

23 Short title: Role of HG status during plant-aphid interaction 24

25 One-sentence summary: The role of homogalacturonan status modulated by 26 PME/PMEI13 is a central component of the early defense responses of Arabidopsis 27 against infestation by the green peach aphid, M. persicae. 28

29 30

The author responsible for distribution of materials integral to the findings presented in this 31 article in accordance with the policy described in the Instructions for Authors 32 (www.plantcell.org) is: Francisca Blanco-Herrera (mblanco@unab.cl) 33

34 35

ABSTRACT 36

Because they suck phloem sap and act as vectors for phytopathogenic viruses, 37

aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has 38

been described as a defensive element for plants during infections with 39

phytopathogens. However, its role during aphid infestation remains unexplored. 40

Using immunofluorescence assays and biochemical approaches, the HG 41

methylesterification status and associated modifying enzymes during the early 42

stage of Arabidopsis thaliana infestation with the green peach aphid Myzus 43

persicae were analyzed. Additionally, the influence of PME activity on aphid 44

Plant Cell Advance Publication. Published on May 24, 2019, doi:10.1105/tpc.19.00136

©2019 American Society of Plant Biologists. All Rights Reserved

2

settling and feeding behavior was evaluated by free choice assays and the 45

Electrical Penetration Graph technique, respectively. Our results revealed that HG 46

status and HG-modifying enzymes are significantly altered during the early stage of 47

the plant-aphid interaction. Aphid infestation induced a significant increase in total 48

PME activity and methanol emissions, concomitant with a decrease in the degree 49

of HG methylesterification. Conversely, inhibition of PME activity led to a significant 50

decrease in the settling and feeding preference of aphids. Furthermore, we 51

demonstrate that the PME inhibitor AtPMEI13 has a defensive role during aphid 52

infestation, since pmei13 mutants are significantly more susceptible to M. persicae 53

in terms of settling preference, phloem access, and phloem sap drainage. 54

55

INTRODUCTION 56

Phytophagous insects have developed different strategies to extract nutrients from 57

plants to complete their life cycle, resulting in a direct impairment of host health 58

and performance. Of the phytophagous insects that affect commercial crops, 59

aphids have a greater impact due to the nutrient losses caused by colonies 60

draining plants and promoting saprophytic fungal growth, thus significantly 61

decreasing crop yields (Östman et al., 2003; Dedryver et al., 2010). Moreover, 62

viruses transmitted by aphids are the most relevant risk factor for the target crop. 63

Indeed, aphids function as vectors for approximately 50% of the 700-known insect-64

borne viruses (Hooks and Fereres, 2006; Dedryver et al., 2010). Consequently, 65

aphids are one of the most costly pests in terms of pesticide treatments (Murray et 66

al., 2013). 67

The aphid feeding process starts when the stylet penetrates the host and then 68

moves towards the phloem through intercellular pathways, such as cell wall 69

matrices, middle lamellae and gas spaces, until sieve elements are reached 70

(Kimmins, 1986; Tjallingii and Esch, 1993). Most cells along the stylet pathway are 71

briefly punctured (typically for 5–10 s), but the stylets are always withdrawn from 72

the cells and then continue along the intercellular route until sieve elements are 73

found (Tjallingii and Esch, 1993). 74

3

Intercellular cell wall polysaccharides are a main component of the intercellular 75

stylet pathway. These macromolecules share common features among vascular 76

plants, and consist of cellulose microfibrils anchored to the cell membrane, cross-77

linked by and embedded in matrices of hemicellulose and pectic polymers (Ridley 78

et al., 2001; Wolf et al., 2012). In this context, homogalacturonan (HG) has been 79

found to participate in different plant developmental and defensive processes 80

(Ridley et al., 2001; Gramegna et al., 2016). HG is a homopolymer of galacturonic 81

acid (GalA) residues, which can be methyl-esterified at C-6 and may carry acetyl 82

groups on O-2 and O-3 (Ridley et al., 2001). According to the current model of HG 83

synthesis, it has been established that HGs are synthesized in the Golgi apparatus 84

in a highly methylesterified state, and then secreted into the cell wall (Ibar and 85

Orellana, 2007). In the cell wall, the methylesterification status may be modified by 86

the action of pectin methylesterases (PME), which remove the methylester groups 87

(E.C. 3.1.1.11). In turn, these reactions of HG de-methylesterification, are 88

regulated by the proteinaceous PME inhibitors (PMEI) (Hothorn et al., 2004; Caffall 89

and Mohnen 2009; Levesque-Tremblay, 2015; Saez-Aguayo et al., 2013). 90

The degree and pattern of HG methylesterification are key factors influencing the 91

mechanical properties of cell walls, and hence in controlling plant development 92

(Peaucelle et al., 2008, Levesque-Tremblay, 2015). In fact, depending on the 93

methylesterification degree, HG domains can be directed into different fates, i) 94

polymer breakdown by polygalacturonases (PGs, EC 3.2.1.15) and pectate lyase 95

(EC 4.2.2.2) causing cell wall loosening and ii) ionic cross-linking with other de-96

methylesterified HG chains through calcium ion bridges creating the so-called “egg 97

box” structures leading to cell wall stiffening and reduced matrix porosity (Braccini 98

et al., 1999; Willats et al., 2001; Levesque-Tremblay, 2015). 99

HG modification and degradation are important factors during the attack of 100

pathogens or phytophagous insects possessing cell wall-degrading enzymes 101

(CWDE) such as PMEs, PGs and PLs as virulence factors (Cantu et al., 2008; 102

Malinovsky et al., 2014). The evidence linking HG to the defensive responses of 103

plants includes the broad spectrum of pathogen resistance or susceptibility 104

4

phenotypes that are created by altering HG modifying enzymes in different plant 105

species (Cantu et al., 2008). Although the evidence relating to HG metabolism 106

during aphid feeding is limited, it is thought that the presence of HG modifying 107

enzymes such as PMEs and PGs, in the saliva of aphids, could facilitate stylet 108

penetration through the intercellular matrix (McAllan and Adams, 1961; Dreyer and 109

Campbell, 1987; Ma et al., 1990). Additionally, by exploring the transcriptional 110

profiles of Arabidopsis thaliana plants attacked by different pathogens and 111

phytophagous insects, De Vos et al. (2005) found that the PECTIN 112

METHYLESTERASE 13 (AtPMEI13) gene was specifically up-regulated during 113

Myzus persicae feeding, yet was unchanged during the interaction with other 114

attackers studied. Despite this valuable information, there still exists a lack of 115

detailed mechanistic understanding about the role of HG during plant-116

aphid interactions. 117

The focus of the present work was to characterize the dynamics of HG and its 118

modifying enzymes during the early stage of aphid infestation and how these 119

changes could influence the aphid settling and feeding behavior. To this end, the 120

globally distributed aphid M. persicae and A. thaliana were used as the plant-aphid 121

interaction model. Here we show that during early aphid infestation, total PME 122

activity and methanol emission increases with a concomitant decrease in the 123

degree of HG methylesterification. Exogenous inhibition of total PME activity leads 124

to a significant decrease in aphid settling preference in WT Col-0 plants. 125

Furthermore, by exploiting the results obtained by De Vos et al. (2005), the 126

inhibitory activity of AtPMEI13 and its defensive role during aphid infestation were 127

isolated and characterized. Due to the marked preference of M. persicae to settle 128

on pmei13 plants, concomitant with longer phloem sap ingestions on these 129

mutants compared to the WT genotypes, it has been demonstrated that AtPMEI13 130

is a resistance factor during aphid colonization in A. thaliana. 131

132

RESULTS 133

134

5

Determination of early infestation stage during M. persicae-A. thaliana 135

interaction 136

Prior to the experiments and analysis, in order to determine the time scales of the 137

early infestation stages, the proper sampling time for the experiments was 138

established. We decided to establish as the early aphid infestation stage the time 139

that aphids took to perform the first sustained phloem ingestion from the first 140

contact with the Arabidopsis leaf (aphid landing). To this, we used the Electrical 141

Penetration Graph (EPG) technique which creates distinct fluctuating voltage 142

patterns referred to as EPG waveforms, which in turn, has been experimentally 143

related to different feeding processes or activities performed by the insect, in this 144

case the aphid M. persicae (Figure 1A). 145

The EPG results showed that wingless adult M. persicae aphids settled on wild-146

type leaves (WT), achieved the first sustained phloem ingestion after 271.2 + 34.8 147

min (4.5 ± 0.5 hours) from landing (Figure 1B). Thus, considering that aphids took 148

approximately 5 hours to perform the first sustained phloem ingestion (and adding 149

1 hour to cover possible variations) we established as an early infestation stage 150

the first 6 hours of plant aphid interaction and based on this timing further 151

experiments were done. Additionally, EPG analysis revealed that after the first 152

host penetration, performed 4.2 minutes after landing, host tissues were probed by 153

aphids approximately 35 times (35 probes), and within these probing events, M. 154

persicae performed an average of 145 membrane punctures, visualized as 155

potential drops (Figure 1A and 1B). Moreover, during the first 360 minutes (6 h) M. 156

persicae spent just 85.1 minutes in non-probing activities i.e. with their stylets out 157

of host plant (Figure 1B). Therefore, this confirmed that aphids perform an 158

exhaustive examination by constantly probing the host tissues during the early 159

infestation stage (6 hours of plant-aphid interaction). 160

161

Early stage of aphid infestation increases the total PME activity, methanol 162

emissions and abundance of de-methylesterified HG 163

Considering that pectin methylesterase (PME) activity and the HG 164

methylesterification status has been described as defense-related elements during 165

6

pathogen attack (Cantu et al., 2008; Osorio et al., 2008; Raiola et al., 2011; 166

Bethke et al., 2014), it was decided to measure the total PME activity and its 167

consequent effects on the HG methylesterification degree and methanol emissions 168

(Figure 2A) during the early stage of plant-aphid interaction. Our results showed 169

that total PME activity increased approximately 20% in aphid-infested leaves of 170

Arabidopsis with respect to the control plants i.e. non-infested (Figure 2B). 171

Consequently, the degree of methylesterification of HG decreased significantly by 172

19% (Figure 2C), concomitant with a 3-fold increase in the methanol emissions in 173

the aphid-infested plants compared to the control condition (Figure 2D). 174

To visualize the cell wall modifications that occurred as a result of the increase in 175

PME activity, immunofluorescence assays on infected and control Arabidopsis 176

leaves were performed. The immunofluorescence assays that were done to 177

visualize the HG methylesterification status, in situ, support the results obtained in 178

Figures 2B and C. The LM19 monoclonal antibody, which targets the de-179

methylesterified domains of HG, showed a doubling of the signal in the 180

parenchyma tissue and lower epidermis of infested leaves, with respect to the 181

control condition (Figure 3; Supplemental Figures 1 and 2). Additionally, some 182

replicates with LM19 antibody, revealed HG de-methylesterification zones 183

localized close to aphid bodies and stylets (Supplemental Figure 1), suggesting 184

that HG modifications could be occurring as a consequence of stylet penetration 185

through the pectic matrix. On the other hand, a significant 30% reduction in the 186

signal of the LM20 antibody, which recognizes highly methylesterified HG, was 187

observed in the aphid-infested leaves compared to the non-infested plants (Figure 188

3; Supplemental Figure 3). Besides, HG epitopes were measured by ELISA; 189

however, no differences were detected during aphid infestation by this method 190

(discussed below, Supplemental Figure 4). Therefore, these results showed that 191

early aphid infestation induced an increase in the total PME activity with the 192

consequent de-methylesterification of HG and methanol release. 193

194

Early stage of aphid infestation increases the calcium cross-linked HG and 195

alters the total PL activity 196

7

Once HG chains are de-methylesterified in cell walls, they may be directed to two 197

different fates: i) polymer breakdown by polygalacturonases (PGs) and/or pectate 198

lyases (PLs) or ii) interact ionically with other de-methylesterified HG chains 199

through calcium bridges creating the so-called “egg box” structures (Braccini et al., 200

1999; Willats et al., 2001) (Figure 4A). Then, as the early M. persicae infestation 201

process induces HG de-esterification, which of these two subsequent steps 202

(PG/PL breakdown or “egg box” arrangement) could be occurring in early aphid-203

infested plants were investigated. To achieve this, total PG and PL activities were 204

measured. The results revealed that the total PG activity remain unchanged 205

(Figure 4B) concomitant with a significant increase in total PL activity (Figure 4C) 206

in the aphid-infested plants with respect to the control condition. 207

Since the other possible fate of de-methylesterified HG is the ion cross-linking, we 208

visualized these epitopes by using the monoclonal antibody 2F4. Interestingly, it 209

was found that the “egg box” arrangement of HG is significantly more abundant in 210

infested plants, since a significant tripling in the signal of 2F4 antibody was 211

measured mainly in the lower epidermis and parenchyma tissue of the aphid-212

infested leaves compared to the control condition (Figure 4D and E; Supplemental 213

Figure 5). Thus, suggesting that during early M. persicae, infestation changes in 214

HG structure leads to an increase in the abundance of both de-methylesterified 215

and ion cross-linked HG. 216

217

Exogenous modulation of PME activity and methanol emissions influence 218

the aphid settling preference 219

The above results revealed that HG methylesterification status is significantly 220

altered during the early plant-aphid interaction (Figures 2 and 3; Supplemental 221

Figures 1 to 3). However, we cannot distinguish whether these HG alterations 222

correspond to a defensive mechanism of the host plant or to the consequences of 223

the aphid infestation/feeding process. In order to gain an insight into this question 224

it was decided to investigate how different levels of PME activity of the host plant 225

could influence the aphid behavior in terms of settling preference. 226

8

The first approach was to exogenously modulate the total PME activity of WT Col-227

0 plants and then subject these plants to a free choice assay, which reveals the 228

preference of the aphids to settle on the most suitable host to establish a new 229

colony (Poch et al., 1998). This was achieved by infiltrating one group of plants 230

with 1 mg/mL of Epigallocatechin gallate (EGCG, Sigma Aldrich), which has been 231

described as a specific chemical inhibitor of global PME activity (Lewis et al., 232

2008). Then, a second group of plants was infiltrated with 15 U/mL of orange peel 233

PME (Sigma Aldrich) (Figure 5A). After 1 hour of the infiltration procedure, treated 234

plants plus a water infiltrated control group (mock), were subjected to the free 235

choice assay. The result shows that treatment with the chemical PME inhibitor 236

EGCG resulted in approximately 10% reduction in total PME activity (Figure 5B) 237

concomitant with a 2.7-fold reduction of methanol emissions (Figure 5D) compared 238

to the infiltration control (mock). On the other hand, infiltration with the commercial 239

orange peel PMEs cocktail increased the total PME activity by 15% (Figure 5B) 240

compared to the mock infiltrated plants, while methanol emissions showed no 241

differences between both conditions (Figure 5D). Free choice assays on these 242

treated plants showed no significant differences in aphid preference when 243

compared with the increased PME activity group of plants (PME infiltrated) with 244

the control condition (mock) (Figure 5C). Interestingly, a significant reduction in 245

aphid settling was observed for the reduced PME activity plants (EGCG infiltrated), 246

since only 20% of the total aphid population preferred those plants as host 247

compared to the 38% and 42% of the aphid population which preferred to settle on 248

mock and orange peel PME treated plants, respectively (Figure 5C). 249

Moreover, it is known that methanol is a critical volatile defense signal emitted 250

during phytophagous insect feeding (Baldwin et al., 2006; Von Dahl et al., 2006) 251

and considering that our results show increased methanol emissions in aphid-252

infested plants (Figure 2D), it was decided to investigate how methanol emissions 253

could influence the host settling preference of M. persicae. To accomplish this and 254

based on methanol emissions from infested plants which averaged 0.09% v/v (900 255

ppm; Figure 2D), a methanol solution of 0.1% v/v was prepared to infiltrate WT 256

Col-0 leaves and then these plants were subjected to an aphid free choice assay 257

9

using water infiltrated plants as controls (mock). As shown in Figure 5E, the results 258

revealed that aphids significantly prefer to settle on methanol infiltrated plants, 259

since 60% of the total aphid population chose those plants as host compared to 260

the 40% of insects that chose the mock plants. These results suggest that both 261

exogenous modulation of PME activity and methanol emission in Arabidopsis 262

leaves could influence the M. persicae settling preference. However, considering 263

that the infiltration procedure could lead to unknown changes in the plant 264

physiology and consequently alter the aphid behavior, a second approach was 265

designed in order to determine the influence of the PME activity over the settling 266

behavior of aphids. 267

268

PMEI13 possess in vitro and in vivo inhibitory activity of PMEs and pmei13 269

mutant lines are more susceptible to M. persicae settling 270

Expression analysis using microarray published by De Vos et al. (2005) showed 271

that a PME inhibitor (PMEI13) is specifically up-regulated during M. persicae 272

infestation of A. thaliana. Considering this data, the potential role of PMEI13 during 273

the plant-aphid interaction was evaluated. Two T-DNA insertional mutant lines 274

were identified in the locus At5g62360/PMEI13 and were designated as pmei13-1 275

(background Col-0) and pmei13-2 (background WS4) (Supplemental Figure 6A). 276

Expression analysis using RT-PCR and RT-qPCR were done on pmei13-1 and 277

pmei13-2 mutant plants. Amplification of the full-length coding sequence of 278

PMEI13 in both pmei13-1 and pmei13-2 mutant lines confirmed that both mutants 279

are knock-down lines, with a decrease of 65.5% and 57.1% in PMEI13 transcript 280

accumulation in comparison to their corresponding WT genotypes, respectively 281

(Supplemental Figures 6B and 6C). Then in order to characterize the PME-282

inhibiting capacity of PMEI13, the inhibitory effect of recombinant PMEI13 on 283

global PME activity of WT plants by using a gel diffusion assay was determined, 284

as described by Saez-Aguayo et al. (2013 and 2017). The results presented in 285

Supplemental Figure 6D show that the induced bacterial culture containing the 286

recombinant PMEI13 (PMEI13x6his + IPTG) has 30% and 23% less global PME 287

activity than cultures containing the empty vector (EV + IPTG) and the non-288

10

induced PMEI13 construct (PMEI13x6his – IPTG), respectively. Thus, these 289

results confirm that PMEI13 is an inhibitor of pectin methylesterase activity. 290

To confirm the in vivo inhibitor activity of PMEI13 in Arabidopsis, total PME activity 291

was measured in four-week-old pmei13-1 and pmei13-2 plants. Results show that 292

both pmei13 mutant lines possess higher total PME activity compared to the WT 293

genotypes. pmei13-1, showed 14% more PME activity compared to WT Col-0 294

while pmei13-2 exhibited 11% more PME activity compared to WT WS4 295

(Supplemental Figure 7A). These significant increases in total PME activity 296

observed in pmei13 mutants were consistent with the increased abundance of de-297

methylesterified HG epitopes (Figure 6A and 6B; Supplemental Figures 8 and 9) 298

and concomitant with a significant decrease of 7% and 11% in the degree of 299

methylesterification of pmei13-1 and pmei13-2, respectively compared with their 300

WT genotypes (Supplemental Figure 7B). In addition, both mutant lines possess 301

higher methanol emissions compared to WT genotypes. pmei13-1 showed a 302

significant increase of approximately 72 ppm related to WT Col-0. While pmei13-2 303

showed an increase of approximately 164 ppm when compared to WT WS4 304

(Supplemental Figure 7C). 305

Given that PMEI13 specifically responds to M. persicae attack (De Vos et al., 306

2005) and that pmei13 lines basally possess higher levels of total PME activity 307

with respect to the wild type genotypes (Supplemental Figure 7A), it was decided 308

to further investigate these mutants. This was done to evaluate the influence of 309

PME activity over aphid settling and feeding behavior. Thus, this represents an 310

alternative approach to the exogenously manipulated PME levels, and it is not 311

subject to the possible side effects of infiltration procedures since PME activity is 312

basally and endogenously modulated in pmei13 mutant lines compared to WT 313

genotypes. Hence, once characterized, the role of PMEI13 in plant defense in 314

terms of aphid settling preference was investigated. Both mutant lines (pmei13-1 315

and pmei13-2) were subjected to a dual choice assay against their respective WT 316

backgrounds. Figure 6C shows that both pmei13 mutant plants are significantly 317

more preferred by M. persicae. Approximately 71% of the total aphid population 318

chose the pmei13-1 mutant line to settle, compared to the 29% of the population 319

11

that chose the WT Col-0 genotype. This aphid behavior was observed 2 hours 320

after the start of the assay and showed no significant fluctuation during the 24 321

hours of the experiment (Figure 6C). In the case of pmei13-2, 62% of the total 322

aphid population preferred to settle on this mutant line and 38% of insects chose 323

the WT WS4 as host (Figure 6C), showing no further variations during the 24 324

hours of the assay. Thus, loss of function of AtPMEI13 resulted in a significant 325

susceptibility to aphids settling on Arabidopsis therefore confirming the influence of 326

the plant PME activity over the settling behavior of M. persicae. 327

328

The pmei13 mutant plants are more susceptible to phloem nutrient drainage 329

by M. persicae 330

HG and its modifying enzymes have been described as an element controlling cell 331

wall rheology (Braccini et al., 1999; Willats et al., 2001) and defense responses 332

sensing and triggering (Cantu et al., 2008; Lionetti et al., 2017). Thus, taking into 333

account that pmei13 lines possess increased PME activity and a lower degree of 334

HG methylesterification (Supplemental Figure 7A and 7B) it was decided to 335

investigate how these alterations could influence the aphid feeding behavior. By 336

using EPG technology, the aphid feeding variables associated to mechanical or 337

chemical traits of host plants was analyzed. This could determine the global host 338

resistance or susceptibility to aphid colonization. The results showed that 339

significant differences between genotypes were found in feeding activities related 340

to the phloem ingestion phase and phloem accessibility (Tables 1 and 2). In the 341

case of pmei13-1, aphids spent more than twice as long (155.2 min) sucking 342

nutrients from the phloem (waveform E2) compared to WT Col-0 (66.8 min) (Table 343

1). Sustained phloem sap ingestions (E2 waveform > 10 min) were 2.6 times 344

longer when M. persicae fed on pmei13-1 (145.5 min) instead of WT Col-0 (54.6 345

min), and the mean duration of the longest phloem ingestions was 3.4 times longer 346

on the mutant genotype (119.5 min) compared to its wild type (35.9 min). 347

Whereas, the non-phloem phase was significantly shorter in pmei13-1 mutants 348

(304.3 min) compared to WT Col-0 (405.6 min) (Table1), which is consistent since 349

a longer non-phloem phase directly implies that aphids spend more time on other 350

12

activities, different to phloem salivation or ingestion. No significant differences 351

were detected for other probing/feeding activities such as intercellular probing 352

(waveform C), xylem ingestion (waveform G) and cell puncturing (waveform pd). 353

Moreover, M. persicae was able to perform the first host probe significantly faster 354

on pmei13-1 (1.9 min) than on WT Col-0 (4.5 min). Similarly, it took significantly 355

less time for aphids to perform the first phloem ingestion when fed on pmei13-1 356

(157.0 min) compared to WT Col-0 (226.4 min) (Table 1). 357

In the case of pmei13-2, the longest phloem ingestions were significantly longer in 358

the mutant genotype (154.7 min) compared to WT WS4 (89.0 min), while other 359

parameters related to phloem ingestion (waveform E2) showed no significant 360

differences (Table 2). However, the duration of sustained phloem ingestion (E2 361

waveform > 10 min) showed a clear tendency (p=0.080) to be longer when aphids 362

were fed on pmei13-2 (199.7 min) compared to WT WS4 (147.0 min). The same 363

phenomenon was observed for the total duration of the phloem ingestion phase 364

(waveform E2), since this variable tended to be longer for pmei13-2 (211.6 min) 365

than for WT WS4 (161.1 min, p=0.093). Moreover, as with pmei13-1, significantly 366

less time was needed by aphids to perform the first phloem ingestion when fed on 367

pmei13-2 (106.1 min) compared to WT WS4 (159.9 min) (Table 2). Likewise, 368

aphids required significantly less time to perform the first sustained phloem 369

ingestion (E2 > 10 min) on pmei13-2 (163.9 min) compared to WT WS4 (234.1 370

min) (Table 2). No significant differences were found for other probing/feeding 371

activities (waveforms np, C, pd, G, F and E1). 372

373

DISCUSSION 374

Early aphid infestation induces an increase in PME activity, methanol 375

emissions and de-methylesterified HG 376

HG and its modifying enzymes have been associated with plant defense 377

mechanisms from bacterial and fungal pathogen attacks, however its defensive 378

role during aphid infestation remains uncertain. Here, the dynamics of HG and its 379

modifying enzymes were described during the early aphid infestation stage (6 h) 380

13

and the defensive role of the Arabidopsis PME inhibitor (PMEI13) in terms of aphid 381

settling and feeding behavior was characterized. 382

The results revealed that during the first 6 hours of aphid infestation the total PME 383

activity increased significantly in infested Arabidopsis leaves with the consequent 384

increase in the abundance of de-methylesterified HG and methanol release 385

(Figure 2). It has been proposed that de-methylesterification of HG is a key step to 386

elicit an efficient defense response (Osorio et al., 2008). Conversely, the defense 387

eliciting activity of HG oligomers (oligogalacturonides) is significantly reduced 388

when their degree of methylesterification increases (Jin and West, 1984; Navazio 389

et al., 2002). Therefore, taking into account the results and the previous evidence, 390

the HG de-methylesterification process that is taking place during the first 6 hours 391

of M. persicae-Arabidopsis interaction could represent part of the elicitation of an 392

efficient defense response against the insect. However, a detailed study of the 393

defense pathways, its dynamics, and related phytohormones are needed to 394

support this hypothesis. With respect to the increased methanol emissions on 395

aphid-infested plants (Figure 2D), it is described that this molecule acts as a 396

volatile signal recognized by plants, but also by phytophagous insects (Komarova 397

et al., 2014). For example; methanol emitted by mechanically wounded tobacco 398

leaves (methanol emitter plants) acts as a signaling molecule that is involved in 399

plant-to-plant communication, promoting antibacterial resistance on non-wounded 400

neighboring plants (methanol receivers) (Dorokhov et al., 2012). Additionally, PME 401

overexpressing Nicotiana tabacum plants possessing higher levels of methanol 402

emissions compared to the wild type genotype showed a dramatic increase in 403

resistance to different phytophagous insects such as caterpillars, aphids and 404

whiteflies (Dixit et al., 2013). However, the study carried out by von Dahl et al. 405

(2006) showed that by applying methanol to plants (in quantities that mimic the 406

caterpillar feeding) the performance of the attacking larvae increases significantly. 407

This antecedent is in part similar to the results shown in Figure 5E, where 408

methanol-treated plants (at a concentration that mimics the aphid-infested plants) 409

were significantly more attractive to aphids which is consistent with the aphid 410

settling preference observed for PME or EGCG treated plants, since plants with 411

14

more methanol emissions are significantly more preferred by M. persicae (Figure 412

5C, D and E). These results provide a potential insight for future studies aiming to 413

understand methanol signaling during aphid infestation. 414

415

Early aphid infestation promotes the increase in de-methylesterified and ion 416

cross-linked HG forms 417

After de-methylesterification, HG can be cleaved by PG/PL or cross-linked through 418

calcium ions (Braccini et al., 1999; Willats et al., 2001). Considering that early 419

aphid infestation promotes the de-methylesterification of HG in Arabidopsis (Figure 420

2; Supplemental Figures 1 to 3), which of the two fates is taking place for HG in 421

aphid-infested leaves were investigated. The results show that both de-422

methylesterified and ion cross-linked HG epitopes increased in aphid infested 423

leaves while highly methylesterified HG decreased (Figures 2 and 3; Figures 4D 424

and 4E; Supplemental Figures 1 to 5). 425

Although, quantification of epitope abundance in cell wall extracts by ELISA 426

showed no difference between infested and non-infested leaves (Supplemental 427

Figure 4), immunofluorescence experiments revealed that the HG de-428

methylesterification zones in aphid-infested leaves could be notably 429

heterogeneous in pattern, distribution and size. This probably depends on the time 430

that an aphid spent in the same probing site (Supplemental Figure 1). Thus, 431

altered HG from zones where aphids remained attached could be diluted with the 432

rest of the non-probed cell walls of the same infested leaves. Another possible 433

explanation could be that the critical HG component that shows 434

methylesterification differences between the treatments corresponds to the 435

fractions solubilized by KOH and/or cellulose. In these cases, all methylesters 436

were removed by the alkali treatment and thus HG methylesterification cannot be 437

assessed. 438

Concerning the influence that the different HG forms possess over the mechanical 439

properties of the extracellular matrix, studies carried out by atomic force 440

microscopy (AFM) have shown that de-methylesterification of pectin contributes to 441

an increase in elasticity of meristematic cells in Arabidopsis (Peaucelle et al., 442

15

2011). On the other hand, calcium cross-linked HG has been associated to a 443

decreased elasticity and increased stiffness through in vitro assays (Fraeye et al., 444

2010; Ngouémazong et al., 2012). Conversely, these results indicate that both HG 445

forms (de-methylesterified and ion cross-linked) increase in aphid-infested plants 446

(Figures 3 and 4). It is not possible to argue with the influence of the mechanical 447

properties of this HG form over the stylet penetration through the extracellular 448

matrix, and further mechanical studies must be done to support this hypothesis. 449

However, an interesting clue regarding this idea could be extracted from the EPG 450

analysis, since it revealed that aphids reached the phloem significantly faster on 451

pmei13 mutants (Table 1 and Table 2), which in turn possess a significant lower 452

basal degree of HG methylesterification compared to the WT genotypes 453

(Supplemental Figure 7D). 454

455

Exogenous and endogenous modulation of total PME activity in Arabidopsis 456

influence the M. persicae settling preference 457

In order to understand the influence of PME activity over M. persicae settling 458

behavior, the first approach was to exogenously modulate the total PME activity by 459

infiltrating a commercial PME cocktail and a chemical inhibitor of PME (EGCG) 460

into WT Col-0 plants and to then expose these plants to a free choice assay with 461

mock infiltrated plants. The results of this experiment showed that aphids 462

significantly preferred to settle on plants with higher levels of PME activity (PME 463

infiltrated and Mock) compared with plants with lower PME activity levels (EGCG 464

infiltrated) (Figure 5). However, it is important to mention that the EGCG inhibitor, 465

exogenous PMEs (non-self PMEs) and methanol infiltration procedures could lead 466

to unknown side effects on physiological status of the infiltrated plants, which in 467

turn could alter aphid behavior. 468

For this reason, the second approach was to obtain PME or PMEI mutant plants of 469

Arabidopsis possessing different basal levels of PME activity compared to WT 470

genotypes. It is known that PMEs and PMEIs correspond to large protein families 471

in A. thaliana, possessing 66 and 69 putative genes encoding for PMEs and 472

PMEIs, respectively (Wolf et al., 2009). Additionally, the potential redundancy and 473

16

promiscuity of the PMEI isogenes and the lack of information about their specific 474

interacting PME partners (Wolf et al., 2009; Jolie et al., 2010; Sénechal et al., 475

2015) hinder the investigation of their individual physiological roles. Fortunately, by 476

exploring the transcriptional profiles of A. thaliana attacked by different pathogens 477

and phytophagous insects, De Vos et al. (2005) found that the PECTIN 478

METHYLESTERASE 13 (AtPMEI13) gene was specifically up regulated during M. 479

persicae feeding. Once the genotype and phenotype characterization of pmei13 480

lines was done (Supplemental Figures 6 to 9), both mutant lines (pmei13-1 and 481

pmei13-2) were subjected to a dual free choice assay against their respective WT 482

genotypes (WT Col-0 and WT WS4), revealing that M. persicae significantly 483

preferred to settle on pmei13 plants (Figure 6C). Thus, aphids are significantly 484

more attracted by pmei13 plants which possess higher PME activity levels, 485

methanol emissions and de-methylesterified HG abundance. 486

487

The pmei13 plants are more susceptible to phloem access and drainage by 488

M. persicae 489

EPG assays have been used to dissect and identify the elements that compose 490

the global susceptibility or resistance phenotypes of several plant species to 491

different phloem feeder insects (Poch et al., 1998; Garzo et al., 2002; Klingler et 492

al., 2005; Le Roux et al., 2008). Thus, in order to gain insights into the specific 493

factors influenced by the lack of PMEI13 over aphid infestation performance, the 494

complete probing and feeding behavior profile of M. persicae on pmei13 mutant 495

lines using the EPG technique (Table 1 and Table 2) was evaluated. The results of 496

this analysis showed that both pmei13 mutant genotypes are significantly more 497

susceptible than their respective WT, in terms of phloem accessibility and phloem 498

sap drainage by M. persicae (Table 1 and 2). 499

The fact that the duration of the longest phloem ingestions was significantly 500

greater in both pmei13 mutants compared to their WT genotypes (Table 1 and 2), 501

allow us to suggest that a specific trait of the phloem cells or nutrient composition 502

could be influenced by the loss of the inhibitory activity of AtPMEI13. It has been 503

described that differences in the duration of phloem ingestions (waveform E2) can 504

17

be caused by the presence of deterrent compounds or a clogging mechanism in 505

the phloem (Harrewijn and Kayser 1997; Klingler et al., 1998, Tjallingii, 2006; 506

Zhang et al., 2011). In these cases, the duration of phloem salivation (waveform 507

E1), that always precedes phloem ingestion (waveform E2), is significantly longer 508

since this feeding activity corresponds to an aphid mechanism to overcome the 509

defensive elements of the host (Tjallingii, 2006, Peng and Walker, 2018). 510

However, the EPG analysis showed no differences on phloem salivation 511

(waveform E1) between pmei13 and WT plants, and hence the existence of a 512

phloem trait different to clogging systems or deterrent compounds presence may 513

be involved. In addition, pmei13 plants exhibited a clear susceptibility in terms of 514

phloem accessibility, since aphids took significantly less time to reach the phloem 515

phase (waveform E) in both mutant lines (Table 1 and 2). This result suggests the 516

participation of a pre-phloem mechanism related to changes in the intercellular 517

matrix rheology or anatomical variations that could hinder phloem access to stylets 518

in WT genotypes. This hypothesis can be supported in part by the lower HG 519

methylesterification degree of pmei13 plants which has been previously correlated 520

with increased flexibility of cell walls in Arabidopsis (Peaucelle et al., 2011), hence 521

stylet movement towards sieve elements could be less hindered in pmei13 lines 522

compared to WT genotypes. 523

Until now, due to the limited knowledge available it has not possible to understand 524

the role of each member of the PMEI family during aphid infestation in 525

Arabidopsis. However, by studying the feeding behavior profile of M. persicae in 526

PMEI6 over-expresser plants (characterized in Saez-Aguayo et al., 2013) it was 527

found that none of the feeding activities studied were altered compared to WT Col-528

0 (Supplemental Table 1). The first insights suggest that not all members of the 529

PMEI family in Arabidopsis possess the same level of influence over the plant-530

aphid interaction as that observed with PMEI13. This is probably due to the 531

interaction with specific PME partners expressed during aphid infestation. 532

533

METHODS 534

Plant Material and Growth Conditions 535

18

The Arabidopsis thaliana WT Col-0 accession and the pmei13-1 (Salk_035506) T-536

DNA mutant line were obtained from the ABRC (http://abrc.osu.edu/) using the 537

SIGnAL Salk collection (Alonso et al., 2003). The WT WS4 and pmei13-2 538

(FLAG_58B07) mutant were obtained from the Versailles Arabidopsis Stock Center 539

(http://publiclines.versailles.inra.fr/tdna). Homozygous lines were identified by PCR 540

using the primers indicated in Supplemental Table 2 using genomic DNA. Plants 541

were grown in peat-vermiculite soil in a controlled environment chamber (21-26 ºC, 542

photoperiod of 16 h light and 8 h dark, 65% relative humidity, and cold white LED 543

lamps providing 120 μmol m-2s-1 of light intensity) 544

545

Insect growth conditions and infestation treatment 546

Parthenogenetic colonies of the green peach aphid Myzus persicae were 547

maintained on pesticide-free Brassica oleracea var. capitata plants grown in Peat 548

Moss substrate. Growth chambers were set to 25 ºC + 2 ºC and a 55 – 65% 549

relative humidity. Fully expanded leaves of four-week-old A. thaliana (WT Col-0) 550

plants were each infested with 20 wingless adult aphids. Aphids were confined on 551

each fully expanded leaf using clip cages built with 300 µm sheer mesh. After 6 h 552

of plant-aphid interaction, insects were removed, and plant tissue was immediately 553

frozen in liquid nitrogen and stored at -80ºC containers. Non-infested plants were 554

used as the control condition. 555

556

Expression Analysis 557

RNA extractions were performed using an RNeasy Plus Micro Kit (Qiagen). One 558

microgram of total RNA was used as a template for first-strand cDNA synthesis 559

with an oligo(dT) primer and SuperScript II (Thermo Fisher Scientific), according to 560

the manufacturer’s instructions. For RT-PCR analysis, the primers described in 561

Supplemental Table 2 were used to amplify the entire coding sequence of PMEI13 562

from single-stranded cDNA in the wild-type Col-0, WS4, pmei13-1, and pmei13-2 563

19

plants. The primers used to amplify EF1aA4 were those described by North et al. 564

(2007). RT-qPCR was performed using the Fast EvaGreen qPCR Master Mix kit 565

(Mx3000P; Stratagene). Reactions contained 1 µL of 1:2 diluted cDNA in a total 566

volume of 10 µL. Reactions were performed using primers that had been 567

previously tested for their efficiency rates and sensitivity in a cDNA dilution series. 568

The quantification and normalization procedures were performed using the 569

equation described in Saez-Aguayo et al. (2017). EF1aA4 and UBC9 were used as 570

reference genes, and all primers used in this study are described in Supplemental 571

Table 2 572

573

Determination of total pectin methylesterase (PME), polygalacturonase (PG) 574

and pectate lyase (PL) activities 575

For determination of global PME, PG and PL total activities, total protein extracts 576

were obtained by homogenizing 100 µg of tissue in 150 µL of extraction buffer (1 M 577

NaCl, 0.2 M Na2HPO4, 0.1 M citric acid, pH 6.5). The homogenates were incubated 578

at 4°C for 1.5 h, cleared twice by centrifugation at 15.000 g for 10 min at 4°C, and 579

the supernatant recovered. Protein concentration was determined based on a BSA 580

standard curve using a Pierce BCA Protein Assay Kit (Thermo Scientific). 581

Global PME activity was quantified by the radial gel diffusion assay. Equal 582

quantities of protein (8 μg) were loaded into 5 mm diameter wells in gels prepared 583

with 0.1% (w/v) of 85% esterified citrus fruit pectin (Sigma-Aldrich), 1% (w/v) 584

agarose, 12.5 mM citric acid, and 50 mM Na2HPO4, pH 6.5. After incubation 585

overnight at 28°C, plates were stained with 0.01% ruthenium red for 45 min and 586

destained with distilled water. The total area of stained halos was measured using 587

ImageJ software. Results were expressed as relative percentages respect to the 588

halo area measured in non-infested controls. 589

To determine global PG activity, an enzymatic reaction was performed by mixing 590

0.3 mL of reaction buffer (200 mM NaCl, 200 mM Na-Acetate, pH 4.5), 400 µg total 591

protein extract in 0.1 mL extraction buffer and 0.3 mL of 1% polygalaturonic acid 592

20

(PGA). Reactions were started by incubation at 37 °C for 60 min and finished by 593

heating at 100°C for 5 minutes. To quantify reducing sugars produced by PG 594

activity, 100 µL DNS reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to 595

100 µL of the reaction mix and heated at 100°C for 30 min. Tubes were ice chilled 596

for 5 min and 1 mL distilled water was added. The formation of reducing groups 597

was quantified against a standard curve of galacturonic acid (US Biological) and 598

measured at 540 nm. PG activity was defined as the amount of enzyme required to 599

produce 1 µg GalA h-1mg-1 of proteins. 600

Pectate lyase enzyme activity was determined as described in Uluisik et al., 2016 601

with minor modifications. Briefly, 50 µg of total protein extracts were incubated in a 602

solution containing 0.12% w/v of polygalacturonic acid, 30 mM Tris-HCl pH 8.5 and 603

0.15 mM CaCl2. The absorbance of products with double bonds released was 604

measured at 232 nm in an initial time (T0) and after incubation at 30 °C for 15 605

hours (T15). Results are expressed as the delta of absorbance between T15 and 606

T0 at 232 nm. 607

608

Determination of methanol emissions by Full evaporation Headspace Gas 609

Chromatography 610

Stomata vapor was collected by individually enclosing the Arabidopsis rosettes 611

inside 50 mL tube during 5 min at 60 °C. The tubes were placed immediately on ice 612

for 5 min. Rosettes were removed, and the resulting condensed vapor on the tube 613

walls was collected by centrifugation at 5,000 rpm for 3 min. Finally, 1µL of the 614

collected vapor was placed in a 10 mL headspace sample vial to carry out the full 615

evaporation of the sample at 80 °C for 20 min before HS-GC measurement. 616

HS-GC measurements were done with a TRACETM 1300 (Thermo Scientific, 617

Waltham, United States) gas chromatograph coupled to a flame ionization detector 618

(FID). The column used was a Rtx®-5 w/Integra-Guard® (Restek, Bellefonte, 619

United States) 30 m long, 0.32 mm internal diameter and a film thickness of 0.25 620

µm. Chromatographic method was modified from Tiscione et al. (2011), using 621

21

Helium as a gas carrier. Run conditions were as follows: Inlet 90 °C with an split 622

ratio of 5:1, gas carrier flow of 3 mL/min; Oven 35 °C for 2 mins with a ramp of 25 623

°C/min until 90 °C, hold for 0.8 min; FID detector at 300 °C, hydrogen flow rate of 624

40 ml/min and air flow rate of 450 mL/min. To test the detection limit of the GC-FID, 625

a 5-point calibration curve was done with 1.0, 0.5, 0.1, 0.05 and 0.01 PPM of 626

methanol. Every point of the calibration curve was measured in triplicate with 5 min 627

total time per run. 628

629

Preparation of alcohol-insoluble residues (AIR) 630

Plant tissue (200 mg) was ground in liquid nitrogen and rinsed three times with 10 631

mL 80% (v/v) aqueous ethanol at room temperature for 1 h. Then, lipids were 632

removed by rinsing three times with 1:1 (v/v) methanol: chloroform followed by 633

three rinses with 100% acetone. The final AIR materials were dried at room 634

temperature by evaporation overnight. 635

636

Acid hydrolysis and sugar quantification 637

Two milligrams of AIR were hydrolyzed for 1 h with 400 µL of 2 M Trifluoroacetic 638

acid (TFA) at 121 °C. TFA was evaporated at 60 °C with nitrogen gas and samples 639

were washed twice with 400 µL isopropanol, and then dried with nitrogen gas. 640

Hydrolyzed samples were suspended in 1 mL deionized water, sonicated during 15 641

min and filtered through a syringe filter (pore size: 0.45 µm) and used for HPAEC-642

PAD analysis as described below. Inositol and allose were used as the internal 643

controls for TFA hydrolysis. 644

A Dionex ICS3000 ion chromatography system, equipped with a pulsed 645

amperometric detector, a CarboPac PA1 (4x 250 mm) analytical column, and a 646

CarboPac PA1 (4 x 50 mm) guard column was used to quantify sugars. The 647

separation of neutral sugars was performed at 40 °C with a flow rate of 1 mL/min 648

using an isocratic gradient of 20 mM NaOH for 20 min followed by a wash with 200 649

mM NaOH for 10 min. After every run, the column was equilibrated in 20 mM 650

22

NaOH for 10 min. Separation of acidic sugars was performed using 150 mM 651

NaOAc and 100 mM NaOH for 10 min at a flow rate of 1 mL/min at 40 °C. 652

Standard curves of neutral sugars (D-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, and 653

D-Man) or acidic sugars (D-GalA and D-GlcA) were used for quantification. 654

655

Determination of homogalacturonan epitopes abundance by ELISA 656

Samples (5 mg) of leaf AIR materials (see above) were sequentially extracted with 657

2 ml of water, CDTA, KOH and cellulose as described elsewhere (Wang et al. 658

2019) to generate soluble fractions containing HG. Extracts were sequentially 659

diluted on to microtiter plates and incubated overnight at 4 °C. Plates were then 660

washed vigorously with tap water six times and shaken dry. Microtiter plate wells 661

were blocked using 200 µl per well of milk-PBS (5%, 1X) for 2 h at room 662

temperature. The plates were washed nine times in tap water and shaken dry. 663

Primary HG-directed antibodies (LM19 and LM20) were added at 100 µl per well as 664

tenfold dilutions of hybridoma cell culture supernatants in milk-PBS (5%, 1X), and 665

incubated for 1h. In the case of 2F4, a Tris-based saline buffer was used and the 666

hybridoma supernatant diluted 200-fold. Plates were washed nine times in tap 667

water, and shaken dry before the secondary antibody incubation. Secondary 668

antibodies (anti-rat or anti-mouse horseradish peroxidase-conjugated IgG; Sigma-669

Aldrich) were added at 100 µl per well at 1000-fold dilution in milk-PBS (5%, 1X) for 670

1 h at room temperature. After extensive washing in tap water, microtiter plates 671

were developed using 100 µl substrate per well (0.1 M sodium acetate buffer, pH 6, 672

1% 3,3,5’5’-TetramethylBenzidine, 0.006% H2O2). The enzyme reaction was 673

stopped by addition of 50 µl of 2.5 M H2SO4 to each well, and the absorbance of 674

each well at 450 nm was determined. 675

676

Determination of HG degree of methylesterification (DM) 677

One mg of AIR was mixed with 100 µL deionized water in 1.5 mL tubes, and HG 678

methyl groups were released by alkali treatment by adding 100 µL of 1 M NaOH 679

23

during 1 hour at 4ºC. Reactions were neutralized by adding 100 µL of 1 M HCl. In a 680

separate 2 mL tube, methanol oxidation by alcohol oxidase was carried out by 681

mixing 100 µL of 200 mM Tris-HCl buffer pH 7.5, 40 µL of 3 mg/mL N-682

methylbenzothiazolinone-2-hydrazone (MBTH), 25 µL of the previously demethyl-683

esterified sample and 20 µL of 0.02 U/µL alcohol oxidase. After a brief vortex, the 684

reaction mix was incubated for 20 min at 30 ºC. Colorimetric reactions were started 685

by incorporation of 200 µL sulfamic acid/ ferric ammonium sulfate (0.5% w/v with 686

deionized water), a brief vortex and incubation for 20 min at room temperature. 687

Finally, 600 µL deionized water was added, and absorbance was measured at 620 688

nm. Results were expressed as µmol methanol released per gram of AIR 689

(methanol µmol/g of AIR). 690

691

Tissue processing, embedding, and sectioning 692

After infestation treatments, rosette leaves were stored in FAA fixative solution 693

(3.7% formaldehyde; 5% glacial acetic acid; 50% ethanol) for at least 2 weeks at 4 694

ºC. Fixed tissue was dehydrated by serial incubations at 4 ºC in solutions with an 695

increasing concentration of ethanol from 10% to 100%. LR White resin (Sigma 696

Aldrich) was infiltrated into the tissue by incubations at 4 ºC in serial solutions with 697

increasing concentrations of resin (Ethanol 100%: LR White, from 4:1 to 1:4) plus 698

two overnight infiltrations with pure resin at room temperature. Once infiltrated, 699

tissues were placed into hard gelatin capsules, submerged in resin and 700

polymerized at 50 ºC for 24 h. One µm sections were obtained from resin blocks 701

with a rotatory micrometer and placed on glass slides pretreated with Vectabond 702

(Vector Laboratories) for immunofluorescence assays. 703

704

Immunofluorescence and confocal microscopy 705

LM19 and LM20 rat monoclonal antibodies were used to target un-esterified and 706

highly methylesterified HGs, respectively (Verhertbruggen et al., 2009). 707

Additionally, the 2F4 mouse antibody was used to target “egg box” epitopes formed 708

24

due to the dimeric association of un-esterified HGs chains through calcium ions 709

(Liners et al., 1989; Liners and Van Cutsem, 1992). Slides with sections were 710

incubated for 30 min at room temperature with 5% fat-free milk (Svelty) dissolved 711

in 1x PBS to block non-specific binding sites and washed once with 1x PBS. 712

Primary antibody diluted 1 in 5 in milk-PBS (5%, 1X) was added and incubated at 713

room temperature for 90 min. Three washes with 1x PBS were done before 714

incubation with the secondary antibody. Alexa Fluor 488 goat anti-rat or anti-mouse 715

(Life Technologies) was diluted 1 in 100 in milk-PBS (5%, 1X) and incubated with 716

sections at room temperature for 60 min. Samples were then washed three times 717

with 1x PBS. A solution of 0.25 mg/ml Calcofluor White (Sigma Aldrich) in 1x PBS 718

was added for 5 min to stain all cell walls. Two washes with 1x PBS were carried 719

out, and the anti-fade agent Citifluor (Agar Scientific) was added before coverslip 720

mounting. Images were taken with Leica TCS LSI confocal microscope with a 721

PlanApo 5X/0.5 LWD objective lens. Immunolabeling of different conditions was 722

done at least in triplicate, and the most representative image of the corresponding 723

batch was chosen. 724

725

Quantification of immunofluorescence signal 726

Antibody and Calcofluor White signal areas were measured with ImageJ software 727

Schneider et al., (2012). Each image was transformed to a 8-bit image, and the 728

threshold was adjusted for each channel (Calcofluor white and Alexa Fluor 488) 729

using the same threshold parameters for all biological replicates. Then, signal 730

areas were selected with the option “create selection” and measured with the menu 731

tool “measure”. For each image, the area of antibody signal was measured and 732

represented as percentage respect to the total cell walls area (Calcofluor white 733

signal). Then, measures obtained for the infested condition were normalized to the 734

control condition (non-infested) assigned as 100%. Compared images (i.e., 735

Infested versus non-infested slices) correspond to samples obtained from the 736

same plant culture batch and within the same immunofluorescence experiment. 737

738

25

Cloning of PMEI13 739

The PMEI13 CDS was cloned using cDNA synthesized from A. thaliana leaf RNA 740

as template. Sequences without the signal peptide were PCR-amplified using 741

PfuUltra II fusion HS DNA polymerase (Agilent) and the following primers pair: 742

FWPMEI13NP: 5’-CACCACAACAACAACAACTACAA-3’ and 5’-743

TTAGCCATGAATAGAAGCAAAGTG-3’. Resulting PCR products were inserted 744

into the pENTRTM/D-TOPO® cloning vector according to the standard protocol 745

(Thermo Fisher Scientific) to generate the entry clone pENTR-PMEI13NP. The C-746

terminal His6 fusion was obtained by recombining the entry clones pENTR-747

PMEI13NP with the destination vector Champion™ pET300/NT-DEST using LR 748

clonase (Thermo Fisher Scientific). Escherichia coli strain BL21 (DE3) was 749

subsequently transformed. 750

751

Heterologous expression of PMEI13 in Escherichia coli 752

E. coli carrying the recombinant PMEI13 (PMEI13-His6) construct were grown in753

LB medium with 100 µg/mL ampicillin at 37°C with shaking until OD600 of ≈0.6. 754

Expression of the fusion protein was induced by the addition of 0.2 mM IPTG and 755

incubation at 37°C for 3 h. The bacterial pellet was harvested by centrifugation. 756

Extraction of proteins was performed by resuspension of this in 50 mM phosphate 757

buffer pH 7.5, and 100 mM NaCl, followed by sonication. The supernatant was 758

collected by centrifugation, and the protein concentration was determined as 759

described above. Proteins were analyzed by SDS-PAGE using a 12% acrylamide/ 760

bis-acrylamide gel and stained with Coomassie Brilliant Blue G-250. 761

762

Characterization of PMEi13 inhibiting activity 763

Total protein extract (30 µg) from the different E. coli cultures were put in contact 764

with 30 µg of total extract protein of WT plants. Then using a gel diffusion assay, as 765

described in Saez-Aguayo et al. (2013 and 2017), the inhibitory effect of 766

recombinant PMEI13 on global PME activity of WT plants was determined. PME 767

26

activity was normalized to the average EV activity (EV, the expression vector 768

without the PMEI13 insert). 769

770

Probing and feeding behavior of M. persicae on A. thaliana plants using the 771

Electrical Penetration Graph (EPG) technique 772

Once an explorer aphid has landed on a potential host, a complex and integrative 773

evaluation of gustatory, olfactory and mechano-sensory parameters assesses the 774

suitability of the host to establish a new colony (Schoonhoven et al., 2005; Van 775

Emden and Harrington, 2017). This early host examination step is critical to accept 776

or reject a potential host, and in this way, we defined as the early plant-aphid 777

interaction as the time elapsed between aphid landing and the first sustained 778

phloem sap ingestion (>10 min) (Tjallingii and Mayoral, 1992; Schoonhoven et al., 779

2005). To establish the optimal sampling time related to early stage plant-aphid 780

interaction we performed a detailed feeding behavior study of M. persicae on A. 781

thaliana WT Col-0, as reference host, using Electrical Penetration Graphs (EPG). 782

Additionally, probing and feeding behavior profiles of wingless adult M. persicae 783

aphids was evaluated on 4-week-old WT Col-0, WT WS4, pmei13-1, and pmei13-2 784

plants. 785

EPG consists of an electrical circuit composed of the aphid and the host plant. 786

Once the insect stylets penetrate the host plant, the circuit is closed, and a 787

fluctuating voltage is created depending on the stylets tip position and the activity 788

inside the plant tissues (Fig. 1A). This fluctuating voltage creates distinct patterns 789

referred to as EPG waveforms, which in turn, are experimentally related to different 790

feeding processes or activities performed by the insect, in our case the aphid M. 791

persicae (Fig. 1A). Electrical penetration graphs were recorded using a Giga-4 792

channel DC-EPG and a Giga-8 DC-EPG device with 1 GΩ of input resistance 793

(EPG Systems, Wageningen, The Netherlands). The EPG devices were connected 794

to PC computers via a USB analog/digital converter card (DI- 710; DATAQ 795

Instruments, Akron, OH). M. persicae individuals were immobilized on a pipette tip 796

coupled to a vacuum pump, and an 18.5 µm diameter gold wire was attached to 797

27

the aphid dorsum with water-based conductive silver glue paint (EPG Systems, 798

Wageningen, The Netherlands). The other end of the gold wire was glued with a 799

droplet of paint to a copper extension wire (2 cm in length), which was inserted into 800

the input of the EPG probe, which in turn was connected to the Giga-4 or Giga-8 801

devices. The EPG circuit was completed by inserting a copper electrode (10 cm 802

length, 2 mm diameter) into the soil of the pot. Aphids were starved for 803

approximately 1 h to acclimatize between the time of wiring and the beginning of 804

EPG recording. Wired aphids were placed on fully expanded rosette leaves. 805

Probing and feeding behavior was monitored for 8 h for each aphid/plant 806

combination. Plants and aphids were used only once for each EPG recording. EPG 807

signals were acquired and analyzed using Stylet+ software for Windows (EPG 808

Systems, Wageningen, The Netherlands). 809

810

Analysis of electrical penetration graph (EPG) waveforms 811

EPG variables were processed using the EPG-Excel Data Workbook version 5.0 812

developed by the laboratory of Dr. Fereres (Sarria et al., 2009). Recordings in 813

which aphids exhibited aberrant behavior (no feeding during the first hour) were 814

discarded. 815

The EPG waveforms associated with specific stylet tip positions and activities 816

when aphids probe and feed on plants are well characterized (Tjallingii, 1978). 817

Waveform “Np” represents non-probing behavior (no stylet contact with the leaf 818

tissue), waveform “C” represents the intercellular stylet pathway where the insects 819

show a cyclic activity of mechanical stylet penetration and secretion of saliva, and 820

waveform pd (Potential drops) represents brief (4–12 s) intracellular stylet 821

punctures during the pathway phase (C). Two waveforms related to phloem 822

activity were recorded: waveform “E1”, which represents salivation into phloem 823

sieve elements at the beginning of the phloem phase, and waveform “E2”, which is 824

correlated with passive phloem sap uptake from the sieve element. Furthermore, 825

waveform “G” represents an active intake of xylem sap, and waveform “F” 826

represents derailed stylet mechanics. The number of waveform events per insect 827

28

was calculated using the sum of the number of events of a particular waveform 828

divided by the total number of insects under each treatment. The total duration of 829

the waveform event per insect was calculated using the sum of durations of each 830

event of a particular waveform made by each insect that produced that waveform 831

divided by the total number of insects under each treatment. 832

833

Free choice assays 834

The choice assay was performed in order to evaluate the aphid settling preference 835

for a specific genotype or treatment. 836

The influence of PME activity on host choice by M. persicae was evaluated by 837

exogenously modulating global PME activity of four-week-old WT Col-0 and then 838

subjecting these plants to a free choice assay. One group of plants was infiltrated 839

with 1 mg/mL of Epigallocatechin gallate (EGCG, Sigma Aldrich), which has been 840

described as a specific chemical inhibitor of total PME activity (Lewis et al., 2008). 841

Then, the second group of plants was infiltrated with 15 U/mL of orange peel PME 842

(Sigma Aldrich). Treated plants, plus water infiltrated control group (mock), were 843

then subjected to a free choice assay. One plant of each treatment was placed 844

within the choice arena, consisting of a 10 mm diameter transparent acrylic 845

platform connecting the three individual plant pots. Thirty wingless adult aphids 846

were released in the middle of the platform, equidistantly from test plants. After 24 847

h, the total number of aphids per plant was registered. Results were expressed as 848

a percentage of aphid preference, considering the settled aphid as the 100%. 849

The influence of methanol, released during PME reaction, was evaluated on aphid 850

settling behavior by infiltrating a solution of methanol (0.1% v/v) into three rosette 851

leaves of four-week-old WT Col-0 plants. Methanol treated plants along with mock 852

plants were then used in a free choice assay. One plant of each condition was 853

placed within the choice arena. Thirty wingless adult aphids were released in the 854

middle of the platform, equidistantly from both treated plants. After 24 h, the total 855

number of aphids per plant was registered. Results were expressed as a 856

percentage of aphid preference, considering the settled aphid as the 100% 857

29

The role of PMEI13 during aphid infestation was evaluated in terms of aphid 858

settling behavior by subjecting both pmei13 mutant lines along with their 859

respective WT genotypes to a free choice assay according to Poch et al. (1998). 860

Two mutants and two WT plants were placed equidistantly from the center of a 19 861

cm diameter plates containing Murashige and Skoog medium supplemented with 862

2% (w/v) sucrose and set with 0.8% (w/v) agar. Free choice tests were done by 863

challenging three-week-old A. thaliana plants with 22 wingless adult aphids 864

released right in the center of the choice plates. The number of aphids per plants 865

was registered at 2, 6, 12 and 24 hours after release. At least five choice plates 866

were assayed in parallel. 867

868

Statistical Analysis 869

Technical replicates were averaged to give a single value for each biological 870

replicate and then these values were used to perform the Student t-test and to 871

calculate the standard error (SE). Therefore, Student t-test was done with at least 872

3 replicates (n= 3) which correspond to the average of at least three technical 873

replicates for each biological replicate. Student t-test was performed using 874

GraphPad Prism version 5.00, GraphPad Software, La Jolla California USA. 875

SE was calculated by dividing the standard deviation (of the biological replicates) 876

by the square root of the number of biological replicates. In the case of EPG 877

results, Mann-Whitney U test was performed using IBM SPSS Statistics, Version 878

20.0. Armonk, New York, USA. 879

880

Accession numbers 881

Accession number of Arabidopsis pmei13 mutants used in the present work 882

correspond to Salk_035506 (pmei13-1) and FLAG_58B07 (pmei13-2) 883

884

885

Supplemental Data 886

30

Supplemental Figure 1 (Supports Figures 2 and 3). HG de-887

methylesterification zones in aphid-infested leave has different patterns, 888

distribution and sizes. 889

890

Supplemental Figure 2 (Supports Figures 2 and 3). Early aphid infestation 891

increases the abundance of de-methylesterified HG epitopes. 892

893

Supplemental Figure 3 (Supports Figure 3). Early aphid infestation 894

decreases the abundance of highly methylesterified HG epitopes. 895

896

Supplemental Figure 4 (Supports Figures 2 to 4). Influence of early aphid 897

infestation on HG epitopes and sugar composition in Arabidopsis leaves. 898

899

Supplemental Figure 5 (Supports Figure 4). Early aphid infestation increases 900

ion cross-linked HG epitopes. 901

902

Supplemental Figure 6 (Supports Figure 6; Tables 1 and 2). Identification of 903

pmei13 mutant lines and characterization of PMEI13 inhibitory activity. 904

905

Supplemental Figure 7 (Supports Figure 6; Tables 1 and 2). Characterization 906

of pmei13 mutant phenotypes. 907

908

Supplemental Figure 8 (Supports Figure 6). pmei13-1 mutant possesses 909

increased abundance of de-methylesterified HG respect to WT Col-0. 910

911

Supplemental Figure 9 (Supports Figure 6). pmei13-2 mutant possesses 912

increased abundance of de-methylesterified HG respect to WT WS4. 913

914

Supplemental Table 1 (Supports Tables 1 and 2). M. persicae feeding 915

behavior is not altered on PMEI6-OE plants. 916

917

31

Supplemental Table 2. Sequences of primers used in this study. 918

919

Acknowledgments: This work was supported by the Fondo Nacional de 920

Desarrollo Científico y Tecnológico [1170259; 11160787]; PAI 79170136, and 921

Instituto Milenio iBio - Iniciativa Científica Milenio MINECON. 922

Thanks to Helen North, from de the IJPB-INRA (Versailles, France) for the gift of 923

the PMEI6 overexpressor, WT WS4 and pmei13-2 genotypes. 924

The authors would like to thank Michael Handford (Universidad de Chile) for 925

language support. 926

927

Author contributions: FB-H, CS-S and SS-A conceived the study and designed 928

the experimental strategy; PK and SEM designed and supervised immunolabelling 929

experiments; RAC, AF and EG designed and supervised aphid settling and 930

feeding behavior experiments; JC-B and SS-A cloned and assayed the inhibiting 931

capacity in vitro of PMEI13; JPP-R and SS-A quantified monosaccharaides by 932

HPAEC-PAD; CS-S and MAR quantified methanol; BR and IR carried out 933

expression analysis by RT-qPCR; CS-S and PO carried out the measurements of 934

enzymatic activities, CS-S and EG performed statistical analysis; CS-S, SS-A and 935

FB-H wrote the manuscript with input from PK, AF, EG, and RAC. 936

Tables 937

Non-sequential variables Genotype mean SE p

Total duration of np WT Col-0 114.4 14.4 0.237

pmei13-1 92.3 11.3

Total probing time WT Col-0 365.6 14.4 0.257

pmei13-1 387.2 11.3

Total duration of C WT Col-0 219.2 11.9 0.158

pmei13-1 191.5 17.2

Total duration of pd WT Col-0 16.9 1.3 0.137

pmei13-1 13.8 1.3

Total duration of G WT Col-0 15.4 4.4 0.834

pmei13-1 13.9 4.3

Total duration of F WT Col-0 61.3 19.2 0.072

pmei13-1 22.5 10.7

Total duration of E1 WT Col-0 3.9 0.6 0.435

32

pmei13-1 4.0 1.0

Total duration of E2 WT Col-0 66.8 10.5 *0.034

pmei13-1 155.2 26.5

Duration of the longest E2 WT Col-0 35.9 5.7 *0.033

pmei13-1 119.5 26.3

Duration of sustained E2 WT Col-0 54.6 10.3 *0.040

pmei13-1 145.5 27.2

Total duration of E WT Col-0 70.6 10.6 *0.030

pmei13-1 159.2 26.5

duration of non-phloematic phase WT Col-0 405.6 10.4 **0.007

pmei13-1 304.3 26.5

Sequential variables Genotype mean SE p

Time to 1st probe from start of EPG WT Col-0 4.5 1.2 *0.050

pmei13-1 1.9 0.4

Time from 1st probe to 1st E2 WT Col-0 226.4 27.7 *0.044

pmei13-1 157.0 27.6

Time from 1st probe to 1st E2 sust WT Col-0 287.2 32.8 0.268

pmei13-1 232.8 33.1

Table 1. pmei13-1 plants are more susceptible to phloem accessibility and 938

drainage by aphid feeding. Electrical Penetration Graph (EPG) recordings during 939

8 hours were performed in order to analyze the feeding behavior profile of adult 940

Myzus persicae aphids on pmei13-1 and WT Col-0. Mean and SE were calculated 941

from n=20 (20 independent EPG recordings). Asterisks represent significant 942

differences determined by the Mann-Whitney U test: *(p<0.05). np: Non-probing; C: 943

Intercellular probing; pd: Cell punctures (potential drop); G: Xylem ingestion; F: 944

Stylet derail; E1: Phloem salivation; E2: Phloem ingestion. 945

946

Non-sequential variables Genotype mean SE p

Total duration of np WT WS4 83.6 12.0 0.364

pmei13-2 66.6 8.7

Total probing time WT WS4 396.4 12.0 0.364

pmei13-2 413.4 8.7

Total duration of C WT WS4 194.4 13.4 0.496

pmei13-2 179.3 13.8

Total duration of pd WT WS4 13.8 1.1 0.982

pmei13-2 13.9 1.3

Total duration of G WT WS4 18.5 6.1 0.193

pmei13-2 7.1 2.8

33

Total duration of F WT WS4 21.0 7.7 0.648

pmei13-2 15.7 6.7

Total duration of E1 WT WS4 3.4 0.3 0.803

pmei13-2 3.4 0.4

Total duration of E2 WT WS4 161.1 22.9 0.093

pmei13-2 211.6 19.4

Duration of the longest E2 WT WS4 89.0 16.4 *0.019

pmei13-2 154.7 19.9

Duration of sustained E2 WT WS4 147.0 23.9 0.080

pmei13-2 199.7 20.3

Total duration of E WT WS4 164.5 22.9 0.097

pmei13-2 215.0 19.1

Duration of non-phloematic phase WT WS4 315.5 22.9 0.097

pmei13-2 265.0 19.1

Sequential variables Genotype mean SE p

Time to 1st probe from start of EPG WT WS4 2.3 0.6 0.51

pmei13-2 2.9 0.8

Time from 1st probe to 1st E2 WT WS4 159.9 20.4 *0.028

pmei13-2 106.1 12.5

Time from 1st probe to 1st E2 sust WT WS4 234.1 26.2 *0.028

pmei13-2 163.9 22.4

Table 2. pmei13-2 plants are more susceptible to phloem accessibility and 947

drainage by aphid feeding. Electrical Penetration Graph (EPG) recordings during 948

8 hours were performed in order to analyze the feeding behavior profile of adult 949

Myzus persicae aphids on pmei13-2 and WT WS4. Mean and SE were calculated 950

from n=20 (20 independent EPG recordings). Asterisks represent significant 951

differences determined by the Mann-Whitney U test: *(p<0.05). np: Non-probing; C: 952

Intercellular probing; pd: Cell punctures (potential drop); G: Xylem ingestion; F: 953

Stylet derail; E1: Phloem salivation; E2: Phloem ingestion. 954

955

956

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of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the 1139

green peach aphid Myzus persicae. BMC plant biology, 11(1), 11. 1140

1141

41

FIGURE LEGENDS 1142

Figure 1. Determination of sampling time for early aphid infestation stage. 1143

Electrical Penetration Graphs (EPG) were performed to evaluate feeding behavior 1144

of M. persicae using Arabidopsis (WT Col-0) as a host with the aim of determining 1145

the proper sampling time related to the early infestation stage. 1146

(A) Shows a schematic representation of the biological activities of the aphid stylet1147

inside the host plant and its corresponding EPG waveform. The arrow points to the 1148

potential drop related to the stylet entry into the sieve elements. C: Intercellular 1149

probing; pd: Cell puncture (potential drop); E1: Phloem salivation; E2: Phloem 1150

ingestion; Ue: Upper epidermis; Vb: Vascular bundle; St: Stylet. 1151

(B) Illustrates the EPG variables analyzed to define the timing of the early aphid1152

infestation stage. Mean and SE were calculated from n=20 (20 independent EPG 1153

recordings). 1154

1155

Figure 2. Early plant-aphid interaction increases global PME activity and 1156

decreases the degree of HG methylesterification. 1157

(A) Is a schematic representation of the HG demethylesterification process. Pectin1158

methylesterases (PME) catalysed by HG demethylesterification and their activity is 1159

regulated by their proteinaceous inhibitor (PMEIs). 1160

(B) Shows total PME activity that was measured after 6 hours of M. persicae-1161

Arabidopsis interaction with 4-week-old WT Col-0 plants. Total protein extracts 1162

from rosette leaves of WT Col-0 plants were used to measure global PME activity. 1163

Values are expressed as relative PME activity and normalized to the average WT 1164

Col-0 activity (non-infested). Error bars represent SE from n=3 (Three individual 1165

plants). 1166

(C) Is the degree of methylesterification after 6 hours of M. persicae- Arabidopsis1167

interaction. Error bars represent SE from n=3 (Three individual plants). 1168

(D) Illustrates methanol emissions that was measured after 6 hours of M. persicae-1169

Arabidopsis interaction with 4-week-old WT Col-0 plants by full evaporation 1170

headspace gas chromatography (FE HS-GC). Values correspond to part per 1171

million (ppm) of methanol in 1µl of collected transpiration vapour. Error bars 1172

42

represent SE from n=4 (Four individual plants). Asterisks represent any significant 1173

differences determined by the Student t-test: **(p < 0.005). 1174

1175

Figure 3. Early aphid infestation increases the abundance of de-1176

methylesterified HG epitopes. Representative transversal sections of 4-week-old 1177

WT Col-0 A. thaliana leaves immunolabeled with LM19 and LM20 monoclonal 1178

antibodies to target de-methylesterified HG (green) and highly methylesterified HG 1179

(yellow) respectively. Calcofluor White was applied to stain all cell walls (magenta). 1180

The images show a close up of the lower epidermis and parenchyma surrounding 1181

the main vascular bundle of leaves of non-infested and infested plants. Le: Lower 1182

epidermis; Vb: Vascular bundle; Pa: Parenchyma. The scale bar is equal to 50 µm. 1183

The graphs show the relative fluorescence signal of each antibody. Values were 1184

normalized respect to the non-infested condition. Error bars represent the SE 1185

obtained from 4 biological replicates (4 leaves from different plants, from different 1186

culture batches). The asterisks represent significant differences determined by the 1187

Student t-test: *(p < 0.05); **(p < 0.005). 1188

1189

Figure 4. Early stage of aphid infestation increases the calcium cross-linked 1190

HG and alters the total PL activity. 1191

(A) Is the schematic representation of the two possible fates of de-methylesterified 1192

HGs: (1) To form a stable ion cross-linked structure with other de-methylesterified 1193

HG blocks via calcium, or (2), to be degraded by the hydrolytic action of 1194

polygalacturonases (PGs) or pectate lyases (PLs). 1195

(B) Shows the total PG activity that was measured after 6 hours of M. persicae- 1196

Arabidopsis interaction. Error bars represent SE from n=3 (Three individual plants). 1197

(C) Is the total PL activity that was measured after 6 hours of M. persicae- 1198

Arabidopsis interaction. Error bars represent SE from n=4 (Four individual plants). 1199

(D) Indicates the representative transversal sections of 4-week-old WT Col-0 A. 1200

thaliana leaves immunolabeled with 2F4 monoclonal antibody to target ion-cross 1201

linked HG (cyan). Calcofluor White was applied to stain all the cell walls (magenta). 1202

Images show a close up of the lower epidermis and parenchyma surrounding the 1203

43

main vascular bundle of leaves of non-infested and infested plants. Le: Lower 1204

epidermis; Vb: Vascular bundle; Pa: Parenchyma. Scale bar is equal to 50 µm. 1205

(E) Is the relative fluorescence signal of 2F4 antibody. Values are normalized with1206

respect to the non-infested condition. Error bars represent the SE obtained from 3 1207

biological replicates (3 leaves from different plants, from different culture batches). 1208

Asterisks represent any significant differences determined by the Student t-test: *(p 1209

< 0.05). 1210

1211

Figure 5. Exogenous modulation of total PME activity alters aphid host 1212

choice behavior. 1213

(A) Is a schematic representation of PME activity and methanol release. A1214

commercial PME cocktail and EGCG were used to increase and to inhibit global 1215

PME activity, respectively. Infiltration of methanol was performed to mimic the 1216

induction of HG demethylation during early aphid feeding. 1217

(B) Shows the total PME activity that was exogenously modified in WT Col-0 plants1218

by infiltrating leaves with commercial orange peel PME, or the PME activity 1219

inhibitor EGCG. Total protein extracts from infiltrated leaves were used to measure 1220

PME activity. Values are expressed as relative PME activity and normalized to the 1221

average value in non-infiltrated WT Col-0 leaves (Mock). Error bars represent SE 1222

from n=3 (Three individual plants). 1223

(C) Illustrates a choice assay that was performed to evaluate the aphid settling1224

preference on plants with exogenously altered PME activity. Thirty aphids were 1225

released equidistantly from orange peel PME- and EGCG-infiltrated plants and 1226

allowed to freely choose their host. After 24 hours, the total number of aphids per 1227

plant was counted. Error bars represent SE from 5 independent choice tests. 1228

(D) Shows the methanol emissions measured on plants with exogenously modified1229

PME activity by full evaporation headspace gas chromatography (FE HS-GC). 1230

Values correspond to part per million (ppm) of methanol in 1µl of collected 1231

transpiration vapour. Error bars represent SE from n=4 (Four individual plants). 1232

(E) Is a choice assay which was performed to evaluate the aphid settling1233

preference on plants infiltrated with methanol (0.1% v/v). Thirty aphids were 1234

44

released equidistantly from mock and methanol treated plants and allowed to freely 1235

choose their host. After 24 hours the total number of aphids per plant was counted. 1236

Error bars represent SE from 5 independent choice tests. Asterisks represent 1237

significant differences determined by the Student t-test: *(p<0.05); **(p < 0.005); 1238

**(p < 0.001). 1239

1240

Figure 6. pmei13 mutants possess increased abundance of de-1241

methylesterified HG and are more susceptible to M. persicae settling 1242

compared to WT genotypes. 1243

(A) Are representative transversal sections of 4-week-old WT Col-0, WT WS4,1244

pmei13-1 and pmei13-2 leaves immunolabeled with LM19 monoclonal antibody to 1245

target de-methylesterified HG (green). Calcofluor White was applied to stain all the 1246

cell walls (magenta). Le: Lower epidermis; Vb: Vascular bundle; Pa: Parenchyma. 1247

Scale bar is equal to 50 µm. 1248

(B) Illustrate the relative fluorescence signal of LM19 antibody. Values of pmei131249

mutant lines were normalized respect to its respective WT genotypes. Error bars 1250

represent the SE obtained from 3 biological replicates (3 leaves from different 1251

plants, from different culture batches). 1252

(C) Shows a choice assay performed to evaluate the aphid settling preference on1253

pmei13 mutant lines. Thirty aphids were released equidistantly from WT Col-0 and 1254

pmei13-1, or WT WS4 and pmei13-2. Aphids were allowed to freely choose their 1255

host and the total number of aphids per plant was counted after 24 hours. Error 1256

bars represent SE from 5 independent choice tests. Asterisks represent significant 1257

differences as determined by the Student t-test: *(p<0.05); *** (p<0.001). 1258

1259

1260

1261

Figure 1. Determination of sampling time for early aphid infestation stage.

Electrical Penetration Graphs (EPG) were performed to evaluate feeding behavior of M. persicae using

Arabidopsis (WT Col-0) as a host with the aim of determining the proper sampling time related to the early

infestation stage.

(A) Shows a schematic representation of the biological activities of the aphid stylet inside the host plant and

its corresponding EPG waveform. The arrow points to the potential drop related to the stylet entry into the

sieve elements. C: Intercellular probing; pd: Cell puncture (potential drop); E1: Phloem salivation; E2:

Phloem ingestion; Ue: Upper epidermis; Vb: Vascular bundle; St: Stylet.

(B) Illustrates the EPG variables analyzed to define the timing of the early aphid infestation stage. Mean and

SE were calculated from n=20 (20 independent EPG recordings).

Figure 2. Early plant-aphid interaction increases global PME activity and decreases the degree of

HG methylesterification.

(A) Is a schematic representation of the HG demethylesterification process. Pectin methylesterases (PME)

catalysed by HG demethylesterification and their activity is regulated by their proteinaceous inhibitor

(PMEIs).

(B) Shows total PME activity that was measured after 6 hours of M. persicae- Arabidopsis interaction with

4-week-old WT Col-0 plants. Total protein extracts from rosette leaves of WT Col-0 plants were used to

measure global PME activity. Values are expressed as relative PME activity and normalized to the average

WT Col-0 activity (non-infested). Error bars represent SE from n=3 (Three individual plants).

(C) Is the degree of methylesterification after 6 hours of M. persicae- Arabidopsis interaction. Error bars

represent SE from n=3 (Three individual plants).

(D) Illustrates methanol emissions that was measured after 6 hours of M. persicae- Arabidopsis interaction

with 4-week-old WT Col-0 plants by full evaporation headspace gas chromatography (FE HS-GC). Values

correspond to part per million (ppm) of methanol in 1µl of collected transpiration vapour. Error bars

represent SE from n=4 (Four individual plants). Asterisks represent any significant differences determined

by the Student t-test: **(p < 0.005).

Figure 3. Early aphid infestation increases the abundance of de-methylesterified HG epitopes.

Representative transversal sections of 4-week-old WT Col-0 A. thaliana leaves immunolabeled with LM19

and LM20 monoclonal antibodies to target de-methylesterified HG (green) and highly methylesterified HG

(yellow) respectively. Calcofluor White was applied to stain all cell walls (magenta). The images show a

close up of the lower epidermis and parenchyma surrounding the main vascular bundle of leaves of non-

infested and infested plants. Le: Lower epidermis; Vb: Vascular bundle; Pa: Parenchyma. The scale bar is

equal to 50 µm.

The graphs show the relative fluorescence signal of each antibody. Values were normalized respect to the

non-infested condition. Error bars represent the SE obtained from 4 biological replicates (4 leaves from

different plants, from different culture batches). The asterisks represent significant differences determined

by the Student t-test: *(p < 0.05); **(p < 0.005).

Figure 4. Early stage of aphid infestation increases the calcium cross-linked HG and alters the total PL

activity.

(A) Is the schematic representation of the two possible fates of de-methylesterified HGs: (1) To form a stable ion cross-

linked structure with other de-methylesterified HG blocks via calcium, or (2), to be degraded by the hydrolytic action of

polygalacturonases (PGs) or pectate lyases (PLs).

(B) Shows the total PG activity that was measured after 6 hours of M. persicae- Arabidopsis interaction. Error bars

represent SE from n=3 (Three individual plants).

(C) Is the total PL activity that was measured after 6 hours of M. persicae- Arabidopsis interaction. Error bars represent

SE from n=4 (Four individual plants).

(D) Indicates the representative transversal sections of 4-week-old WT Col-0 A. thaliana leaves immunolabeled with

2F4 monoclonal antibody to target ion-cross linked HG (cyan). Calcofluor White was applied to stain all the cell walls

(magenta). Images show a close up of the lower epidermis and parenchyma surrounding the main vascular bundle of

leaves of non-infested and infested plants. Le: Lower epidermis; Vb: Vascular bundle; Pa: Parenchyma. Scale bar is

equal to 50 µm.

(E) Is the relative fluorescence signal of 2F4 antibody. Values are normalized with respect to the non-infested

condition. Error bars represent the SE obtained from 3 biological replicates (3 leaves from different plants, from

different culture batches). Asterisks represent any significant differences determined by the Student t-test: *(p < 0.05).

Figure 5. Exogenous modulation of total PME activity alters aphid host choice behavior.

(A) Is a schematic representation of PME activity and methanol release. A commercial PME cocktail and EGCG were

used to increase and to inhibit global PME activity, respectively. Infiltration of methanol was performed to mimic the

induction of HG demethylation during early aphid feeding.

(B) Shows the total PME activity that was exogenously modified in WT Col-0 plants by infiltrating leaves with

commercial orange peel PME, or the PME activity inhibitor EGCG. Total protein extracts from infiltrated leaves were

used to measure PME activity. Values are expressed as relative PME activity and normalized to the average value in

non-infiltrated WT Col-0 leaves (Mock). Error bars represent SE from n=3 (Three individual plants).

(C) Illustrates a choice assay that was performed to evaluate the aphid settling preference on plants with exogenously

altered PME activity. Thirty aphids were released equidistantly from orange peel PME- and EGCG-infiltrated plants

and allowed to freely choose their host. After 24 hours, the total number of aphids per plant was counted. Error bars

represent SE from 5 independent choice tests.

(D) Shows the methanol emissions measured on plants with exogenously modified PME activity by full evaporation

headspace gas chromatography (FE HS-GC). Values correspond to part per million (ppm) of methanol in 1µl of

collected transpiration vapour. Error bars represent SE from n=4 (Four individual plants).

(E) Is a choice assay which was performed to evaluate the aphid settling preference on plants infiltrated with methanol

(0.1% v/v). Thirty aphids were released equidistantly from mock and methanol treated plants and allowed to freely

choose their host. After 24 hours the total number of aphids per plant was counted. Error bars represent SE from 5

independent choice tests. Asterisks represent significant differences determined by the Student t-test: *(p<0.05); **(p <

0.005); **(p < 0.001).

Figure 6. pmei13 mutants possess increased abundance of de-methylesterified HG and are more

susceptible to M. persicae settling compared to WT genotypes.

(A) Are representative transversal sections of 4-week-old WT Col-0, WT WS4, pmei13-1 and pmei13-2 leaves

immunolabeled with LM19 monoclonal antibody to target de-methylesterified HG (green). Calcofluor White was

applied to stain all the cell walls (magenta). Le: Lower epidermis; Vb: Vascular bundle; Pa: Parenchyma. Scale bar

is equal to 50 µm.

(B) Illustrate the relative fluorescence signal of LM19 antibody. Values of pmei13 mutant lines were normalized

respect to its respective WT genotypes. Error bars represent the SE obtained from 3 biological replicates (3 leaves

from different plants, from different culture batches).

(C) Shows a choice assay performed to evaluate the aphid settling preference on pmei13 mutant lines. Thirty aphids

were released equidistantly from WT Col-0 and pmei13-1, or WT WS4 and pmei13-2. Aphids were allowed to freely

choose their host and the total number of aphids per plant was counted after 24 hours. Error bars represent SE from

5 independent choice tests. Asterisks represent significant differences as determined by the Student t-test:

*(p<0.05); *** (p<0.001).

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DOI 10.1105/tpc.19.00136; originally published online May 24, 2019;Plant Cell

Alberto Fereres, J. Paul Knox, Susana Saez-Aguayo and Francisca Blanco-HerreraBarbara Rojas, Patricio Olmedo, Miguel Angel Rubilar Romero, Ignacio Rios, Rodrigo A Chorbadjian, Christian Silva-Sanzana, Jonathan Celiz-Balboa, Elisa Garzo, Susan E Marcus, Juan Pablo Parra-Rojas,

Myzus persicaePectinmethyesterases Modulate Plant Homogalacturonan Status in Defenses Against the Aphid

 This information is current as of March 30, 2020

 

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