pectinmethyesterases modulate plant homogalacturonan ...mblanco@unab.cl) 22. susana saez-aguayo...
Post on 23-Mar-2020
3 Views
Preview:
TRANSCRIPT
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
REFERENCES 957
Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis 958
thaliana. Science 301: 653–657. 959
34
Baldwin, I. T., Halitschke, R., Paschold, A., Von Dahl, C. C., and Preston, C. A. 960
(2006). Volatile signaling in plant-plant interactions:" talking trees" in the genomics 961
era. science, 311(5762), 812-815. 962
Bethke, G., Grundman, R. E., Sreekanta, S., Truman, W., Katagiri, F., & 963
Glazebrook, J. (2014). Arabidopsis PECTIN METHYLESTERASEs contribute to 964
immunity against Pseudomonas syringae. Plant Physiology, 164(2), 1093-1107. 965
Braccini, I., Grasso, R. P., and Pérez, S. (1999). Conformational and 966
configurational features of acidic polysaccharides and their interactions with 967
calcium ions: a molecular modeling investigation. Carbohydrate Research, 317(1-968
4), 119-130. 969
Caffall, K. H., and Mohnen, D. (2009). The structure, function, and biosynthesis of 970
plant cell wall pectic polysaccharides. Carbohydrate research, 344(14), 1879-1900. 971
Cantu, D., Vicente, A. R., Labavitch, J. M., Bennett, A. B., and Powell, A. L. (2008). 972
Strangers in the matrix: plant cell walls and pathogen susceptibility. Trends in plant 973
science, 13(11), 610-617. 974
Dedryver, C. A., Le Ralec, A., and Fabre, F. (2010). The conflicting relationships 975
between aphids and men: a review of aphid damage and control 976
strategies. Comptes rendus biologies, 333(6-7), 539-553. 977
De Vos, M., et al. (2005). Signal signature and transcriptome changes of 978
Arabidopsis during pathogen and insect attack. Molecular plant-microbe 979
interactions, 18(9), 923-937. 980
Dixit, S., Upadhyay, S. K., Singh, H., Sidhu, O. P., Verma, P. C., and 981
Chandrashekar, K. (2013). Enhanced methanol production in plants provides broad 982
spectrum insect resistance. PLoS One, 8(11), e79664. 983
Dorokhov, Y. L., Komarova, T. V., Petrunia, I. V., Frolova, O. Y., Pozdyshev, D. V., 984
& Gleba, Y. Y. (2012). Airborne signals from a wounded leaf facilitate viral 985
35
spreading and induce antibacterial resistance in neighboring plants. PLoS 986
Pathogens, 8(4), e1002640. 987
Dreyer, D. L., and Campbell, B. C. (1987). Chemical basis of host‐plant resistance 988
to aphids. Plant, Cell & Environment, 10(5), 353-361. 989
Van Emden, H. F., and Harrington, R. (2017). Aphids as crop pests. Cabi. 990
Fraeye, I., et al. (2010). Influence of pectin structure on texture of pectin–calcium 991
gels.Innovative food science & emerging technologies, 11(2), 401-409. 992
Garzo, E., Soria, C., Gomez-Guillamon, M. L., and Fereres, A. (2002). Feeding 993
behavior ofAphis gossypii on resistant accessions of different melon genotypes 994
(Cucumis melo). Phytoparasitica, 30(2), 129-140. 995
Gramegna, G., Modesti, V., Savatin, D. V., Sicilia, F., Cervone, F., and De 996
Lorenzo, G. (2016). GRP-3 and KAPP, encoding interactors of WAK1, negatively 997
affect defense responses induced by oligogalacturonides and local response to 998
wounding. Journal of experimental botany, 67(6), 1715-1729. 999
Harrewijn, P., and Kayser, H. (1997). Pymetrozine, a fast‐acting and selective 1000
inhibitor of aphid feeding. In‐situ studies with electronic monitoring of feeding 1001
behaviour. Pesticide Science, 49(2), 130-140. 1002
Hothorn, M., Wolf, S., Aloy, P., Greiner, S., & Scheffzek, K. (2004). Structural 1003
insights into the target specificity of plant invertase and pectin methylesterase 1004
inhibitory proteins. The Plant Cell, 16(12), 3437-3447. 1005
Hooks, C. R., and Fereres, A. (2006). Protecting crops from non-persistently aphid-1006
transmitted viruses: a review on the use of barrier plants as a management 1007
tool. Virus research, 120(1-2), 1-16. 1008
Ibar, C., and Orellana, A. (2007). The import of S-adenosylmethionine into the 1009
Golgi apparatus is required for the methylation of homogalacturonan. Plant 1010
physiology, 145(2), 504-512. 1011
36
Jin, D. F., and West, C. A. (1984). Characteristics of galacturonic acid oligomers as 1012
elicitors of casbene synthetase activity in castor bean seedlings. Plant 1013
Physiology, 74(4), 989-992. 1014
Jolie, R. P., Duvetter, T., Van Loey, A. M., and Hendrickx, M. E. (2010). Pectin 1015
methylesterase and its proteinaceous inhibitor: a review. Carbohydrate 1016
Research, 345(18), 2583-2595. 1017
Kimmins, F. M. (1986). Ultrastructure of the stylet pathway of Brevicoryne 1018
brassicae in host plant tissue, Brassica oleracea. Entomologia experimentalis et 1019
applicata, 41(3), 283-290. 1020
Klingler, J., Powell, G., Thompson, G. A., and Isaacs, R. (1998). Phloem specific 1021
aphid resistance in Cucumis melo line AR 5: effects on feeding behaviour and 1022
performance of Aphis gossypii. Entomologia Experimentalis et Applicata, 86(1), 79-1023
88. 1024
Klingler, J., Creasy, R., Gao, L., Nair, R. M., Calix, A. S., Jacob, H. S., Edwards, 1025
O.R. and Singh, K. B. (2005). Aphid resistance in Medicago truncatula involves 1026
antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus 1027
flanked by NBS-LRR resistance gene analogs. Plant physiology, 137(4), 1445-1028
1455. 1029
Komarova, T. V., Sheshukova, E. V., and Dorokhov, Y. L. (2014). Cell wall 1030
methanol as a signal in plant immunity. Frontiers in plant science, 5, 101. 1031
Le Roux, V., Dugravot, S., Campan, E., Dubois, F., Vincent, C., and Giordanengo, 1032
P. (2008). Wild Solanum resistance to aphids: antixenosis or antibiosis?. Journal of1033
Economic Entomology, 101(2), 584-591 1034
Levesque-Tremblay, G., Pelloux, J., Braybrook, S. A., and Müller, K. (2015). 1035
Tuning of pectin methylesterification: consequences for cell wall biomechanics and 1036
development. Planta, 242(4), 791-811. 1037
37
Lewis, K. C., Selzer, T., Shahar, C., Udi, Y., Tworowski, D., and Sagi, I. (2008). 1038
Inhibition of pectin methyl esterase activity by green tea 1039
catechins. Phytochemistry, 69(14), 2586-2592. 1040
Liners, F., Letesson, J. J., Didembourg, C., and Van Cutsem, P. (1989). 1041
Monoclonal antibodies against pectin: recognition of a conformation induced by 1042
calcium. Plant physiology, 91(4), 1419-1424. 1043
Liners, F., and Van Cutsem, P. (1992). Distribution of pectic polysaccharides 1044
throughout walls of suspension-cultured carrot cells. Protoplasma, 170(1-2), 10-21. 1045
Lionetti, V., Fabri, E., De Caroli, M., Hansen, A. R., Willats, W. G., Piro, G., and 1046
Bellincampi, D. (2017). Three pectin methyl esterase inhibitors protect cell wall 1047
integrity for immunity to Botrytis. Plant physiology, pp-01185. 1048
Ma, R., Reese, J. C., Black IV, W. C., and Bramel-Cox, P. (1990). Detection of 1049
pectinesterase and polygalacturonase from salivary secretions of living greenbugs, 1050
Schizaphis graminum (Homoptera: Aphididae). Journal of Insect Physiology, 36(7), 1051
507-512.1052
Malinovsky, F. G., Fangel, J. U., and Willats, W. G. (2014). The role of the cell wall 1053
in plant immunity. Frontiers in plant science, 5, 178. 1054
McAllan, J. W., and Adams, J. B. (1961). The significance of pectinase in plant 1055
penetration by aphids. Canadian Journal of Zoology, 39(3), 305-310. 1056
Murray, D. A., Clarke, M. B., and Ronning, D. A. (2013). Estimating invertebrate 1057
pest losses in six major A ustralian grain crops. Australian Journal of 1058
Entomology, 52(3), 227-241. 1059
Navazio, L., et al., (2002). The role of calcium in oligogalacturonide-activated 1060
signalling in soybean cells. Planta, 215(4), 596-605. 1061
38
Ngouémazong, D. E., et al. (2012). Effect of de-methylesterification on network1062
development and nature of Ca2+ pectin gels: Towards understanding structure–1063
function relations of pectin. Food Hydrocolloids, 26(1), 89-98. 1064
Osorio, S., Castillejo, C., Quesada, M. A., Medina‐Escobar, N., Brownsey, G. J., 1065
Suau, R., Heredia, A., Botella, M., and Valpuesta, V. (2008). Partial demethylation 1066
of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence 1067
responses in wild strawberry (Fragaria vesca). The Plant Journal, 54(1), 43-55. 1068
Östman, Ö., Ekbom, B., and Bengtsson, J. (2003). Yield increase attributable to 1069
aphid predation by ground-living polyphagous natural enemies in spring barley in 1070
Sweden. Ecological economics, 45(1), 149-158. 1071
Peaucelle, A., Louvet, R., Johansen, J. N., Höfte, H., Laufs, P., Pelloux, J., and 1072
Mouille, G. (2008). Arabidopsis phyllotaxis is controlled by the methyl-esterification 1073
status of cell-wall pectins. Current Biology, 18(24), 1943-1948. 1074
Peaucelle, A., Braybrook, S. A., Le Guillou, L., Bron, E., Kuhlemeier, C., & Höfte, 1075
H. (2011). Pectin-induced changes in cell wall mechanics underlie organ initiation1076
in Arabidopsis. Current biology, 21(20), 1720-1726. 1077
Peng, H. C., and Walker, G. P. (2018). Sieve element occlusion provides 1078
resistance against Aphis gossypii in TGR‐1551 melons. Insect science. 1079
Poch, H. L. C., Ponz, F., and Fereres, A. (1998). Searching for resistance in 1080
Arabidopsis thaliana to the green peach aphid Myzus persicae. Plant 1081
science, 138(2), 209-216. 1082
Raiola, A., et al. (2011). Pectin methylesterase is induced in Arabidopsis upon 1083
infection and is necessary for a successful colonization by necrotrophic 1084
pathogens. Molecular Plant-Microbe Interactions, 24(4), 432-440. 1085
Ridley, B. L., O'Neill, M. A., and Mohnen, D. (2001). Pectins: structure, 1086
biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57(6), 929-1087
967.1088
39
Saez-Aguayo, S., Ralet, M. C., Berger, A., Botran, L., Ropartz, D., Marion-Poll, A., 1089
and North, H. M. (2013). PECTIN METHYLESTERASE INHIBITOR6 promotes 1090
Arabidopsis mucilage release by limiting methylesterification of homogalacturonan 1091
in seed coat epidermal cells. The Plant Cell, tpc-112. 1092
Saez-Aguayo, S., et al. (2017). UUAT1 Is a Golgi-Localized UDP-Uronic Acid 1093
Transporter that Modulates the Polysaccharide Composition of Arabidopsis Seed 1094
Mucilage. The Plant Cell, tpc-00465. 1095
Sarria, E., Cid, M., Garzo, E., and Fereres, A. (2009). Excel Workbook for 1096
automatic parameter calculation of EPG data. Computers and Electronics in 1097
Agriculture, 67(1-2), 35-42. 1098
Sénéchal, F., Mareck, A., Marcelo, P., Lerouge, P., & and Pelloux, J. (2015). 1099
Arabidopsis PME17 activity can be controlled by Pectin Methylesterase 1100
Inhibitor4. Plant signaling & behavior, 10(2), e983351. 1101
Schoonhoven, L. M., Van Loon, B., van Loon, J. J., and Dicke, M. (2005). Insect-1102
plant biology. Oxford University Press on Demand. 1103
Tiscione, N. B., Alford, I., Yeatman, D. T., & Shan, X. (2011). Ethanol analysis by 1104
headspace gas chromatography with simultaneous flame-ionization and mass 1105
spectrometry detection.Journal of analytical toxicology, 35(7), 501-511. 1106
Tjallingii, W. F. (1978). Electronic recording of penetration behaviour by aphids. 1107
Entomologia experimentalis et applicata, 24(3), 721-730. 1108
Tjallingii, W. F., and Mayoral, A. (1992). Criteria for host-plant acceptance by 1109
aphids. In Proceedings of the 8th International Symposium on Insect-Plant 1110
Relationships (pp. 280-282). Springer, Dordrecht. 1111
Tjallingii, W. F., and Esch, T. H. (1993). Fine structure of aphid stylet routes in 1112
plant tissues in correlation with EPG signals. Physiological entomology, 18(3), 317-1113
328.1114
40
Tjallingii, W. F. (2006). Salivary secretions by aphids interacting with proteins of 1115
phloem wound responses. Journal of experimental botany, 57(4), 739-745. 1116
Von Dahl, C. C., Hävecker, M., Schlögl, R., and Baldwin, I. T. (2006). Caterpillar‐1117
elicited methanol emission: a new signal in plant–herbivore interactions? The Plant 1118
Journal, 46(6), 948-960. 1119
Van Emden, H. F., and Harrington, R. (Eds.). (2017). Aphids as crop pests. Cabi. 1120
Verhertbruggen, Y., Marcus, S. E., Haeger, A., Ordaz-Ortiz, J. J., and Knox, J. P. 1121
(2009). An extended set of monoclonal antibodies to pectic 1122
homogalacturonan. Carbohydrate Research, 344(14), 1858-1862. 1123
Wang, D., Samsulrizal, N., Yan, C., Allcock, N.S., Craigon, J., Blanco-Ulate, B., 1124
Ortega-Salazar, I., Marcus, S.E., Bagheri, H.M., Perez-Fons, L., Fraser, P.D., 1125
Foster, T., Fray, R.G., Knox, J.P., Seymour, G.B., (2019). Characterisation of 1126
CRISPR mutants targeting genes modulating pectin degradation in ripening 1127
tomato. Plant Physiology 179, 544-557 1128
Willats, W. G., Orfila, C., Limberg, G., Buchholt, H. C., van Alebeek, G. J. W., 1129
Voragen, A. G., Marcus, S., Christensen, T., Mikkelsen, J., Murray, B. and Knox, J. 1130
P. (2001). Modulation of the degree and pattern of methyl esterification of pectic1131
homogalacturonan in plant cell walls: implications for pectin methyl esterase action, 1132
matrix properties and cell adhesion. Journal of Biological Chemistry. 1133
Wolf, S., Mouille, G., and Pelloux, J. (2009). Homogalacturonan methyl-1134
esterification and plant development. Molecular plant, 2(5), 851-860. 1135
Wolf, S., and Greiner, S. (2012). Growth control by cell wall 1136
pectins. Protoplasma, 249(2), 169-175. 1137
Zhang, C., et al. (2011). Harpin-induced expression and transgenic overexpression 1138
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).
Parsed CitationsAlonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Baldwin, I. T., Halitschke, R., Paschold, A., Von Dahl, C. C., and Preston, C. A. (2006). Volatile signaling in plant-plant interactions:"talking trees" in the genomics era. science, 311(5762), 812-815.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bethke, G., Grundman, R. E., Sreekanta, S., Truman, W., Katagiri, F., & Glazebrook, J. (2014). Arabidopsis PECTIN METHYLESTERASEscontribute to immunity against Pseudomonas syringae. Plant Physiology, 164(2), 1093-1107.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Braccini, I., Grasso, R. P., and Pérez, S. (1999). Conformational and configurational features of acidic polysaccharides and theirinteractions with calcium ions: a molecular modeling investigation. Carbohydrate Research, 317(1-4), 119-130.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Caffall, K. H., and Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydrateresearch, 344(14), 1879-1900.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cantu, D., Vicente, A. R., Labavitch, J. M., Bennett, A. B., and Powell, A. L. (2008). Strangers in the matrix: plant cell walls and pathogensusceptibility. Trends in plant science, 13(11), 610-617.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dedryver, C. A., Le Ralec, A., and Fabre, F. (2010). The conflicting relationships between aphids and men: a review of aphid damageand control strategies. Comptes rendus biologies, 333(6-7), 539-553.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
De Vos, M., et al. (2005). Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Molecularplant-microbe interactions, 18(9), 923-937.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dixit, S., Upadhyay, S. K., Singh, H., Sidhu, O. P., Verma, P. C., and Chandrashekar, K. (2013). Enhanced methanol production in plantsprovides broad spectrum insect resistance. PLoS One, 8(11), e79664.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dorokhov, Y. L., Komarova, T. V., Petrunia, I. V., Frolova, O. Y., Pozdyshev, D. V., & Gleba, Y. Y. (2012). Airborne signals from awounded leaf facilitate viral spreading and induce antibacterial resistance in neighboring plants. PLoS Pathogens, 8(4), e1002640.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dreyer, D. L., and Campbell, B. C. (1987). Chemical basis of host‐plant resistance to aphids. Plant, Cell & Environment, 10(5), 353-361.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Van Emden, H. F., and Harrington, R. (2017). Aphids as crop pests. Cabi.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fraeye, I., et al. (2010). Influence of pectin structure on texture of pectin–calcium gels.Innovative food science & emergingtechnologies, 11(2), 401-409.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Garzo, E., Soria, C., Gomez-Guillamon, M. L., and Fereres, A. (2002). Feeding behavior ofAphis gossypii on resistant accessions ofdifferent melon genotypes (Cucumis melo). Phytoparasitica, 30(2), 129-140.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Gramegna, G., Modesti, V., Savatin, D. V., Sicilia, F., Cervone, F., and De Lorenzo, G. (2016). GRP-3 and KAPP, encoding interactors ofWAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding. Journal of experimentalbotany, 67(6), 1715-1729.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Harrewijn, P., and Kayser, H. (1997). Pymetrozine, a fast‐acting and selective inhibitor of aphid feeding. In‐situ studies with electronicmonitoring of feeding behaviour. Pesticide Science, 49(2), 130-140.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hothorn, M., Wolf, S., Aloy, P., Greiner, S., & Scheffzek, K. (2004). Structural insights into the target specificity of plant invertase andpectin methylesterase inhibitory proteins. The Plant Cell, 16(12), 3437-3447.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hooks, C. R., and Fereres, A. (2006). Protecting crops from non-persistently aphid-transmitted viruses: a review on the use of barrierplants as a management tool. Virus research, 120(1-2), 1-16.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ibar, C., and Orellana, A. (2007). The import of S-adenosylmethionine into the Golgi apparatus is required for the methylation ofhomogalacturonan. Plant physiology, 145(2), 504-512.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jin, D. F., and West, C. A. (1984). Characteristics of galacturonic acid oligomers as elicitors of casbene synthetase activity in castorbean seedlings. Plant Physiology, 74(4), 989-992.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jolie, R. P., Duvetter, T., Van Loey, A. M., and Hendrickx, M. E. (2010). Pectin methylesterase and its proteinaceous inhibitor: a review.Carbohydrate Research, 345(18), 2583-2595.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kimmins, F. M. (1986). Ultrastructure of the stylet pathway of Brevicoryne brassicae in host plant tissue, Brassica oleracea.Entomologia experimentalis et applicata, 41(3), 283-290.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Klingler, J., Powell, G., Thompson, G. A., and Isaacs, R. (1998). Phloem specific aphid resistance in Cucumis melo line AR 5: effects onfeeding behaviour and performance of Aphis gossypii. Entomologia Experimentalis et Applicata, 86(1), 79-88.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Klingler, J., Creasy, R., Gao, L., Nair, R. M., Calix, A. S., Jacob, H. S., Edwards, O.R. and Singh, K. B. (2005). Aphid resistance inMedicago truncatula involves antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus flanked by NBS-LRRresistance gene analogs. Plant physiology, 137(4), 1445-1455.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Komarova, T. V., Sheshukova, E. V., and Dorokhov, Y. L. (2014). Cell wall methanol as a signal in plant immunity. Frontiers in plantscience, 5, 101.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Le Roux, V., Dugravot, S., Campan, E., Dubois, F., Vincent, C., and Giordanengo, P. (2008). Wild Solanum resistance to aphids:antixenosis or antibiosis?. Journal of Economic Entomology, 101(2), 584-591
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Levesque-Tremblay, G., Pelloux, J., Braybrook, S. A., and Müller, K. (2015). Tuning of pectin methylesterification: consequences forcell wall biomechanics and development. Planta, 242(4), 791-811.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lewis, K. C., Selzer, T., Shahar, C., Udi, Y., Tworowski, D., and Sagi, I. (2008). Inhibition of pectin methyl esterase activity by green teacatechins. Phytochemistry, 69(14), 2586-2592.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liners, F., Letesson, J. J., Didembourg, C., and Van Cutsem, P. (1989). Monoclonal antibodies against pectin: recognition of aconformation induced by calcium. Plant physiology, 91(4), 1419-1424.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liners, F., and Van Cutsem, P. (1992). Distribution of pectic polysaccharides throughout walls of suspension-cultured carrot cells.Protoplasma, 170(1-2), 10-21.
Pubmed: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lionetti, V., Fabri, E., De Caroli, M., Hansen, A. R., Willats, W. G., Piro, G., and Bellincampi, D. (2017). Three pectin methyl esteraseinhibitors protect cell wall integrity for immunity to Botrytis. Plant physiology, pp-01185.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ma, R., Reese, J. C., Black IV, W. C., and Bramel-Cox, P. (1990). Detection of pectinesterase and polygalacturonase from salivarysecretions of living greenbugs, Schizaphis graminum (Homoptera: Aphididae). Journal of Insect Physiology, 36(7), 507-512.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Malinovsky, F. G., Fangel, J. U., and Willats, W. G. (2014). The role of the cell wall in plant immunity. Frontiers in plant science, 5, 178.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
McAllan, J. W., and Adams, J. B. (1961). The significance of pectinase in plant penetration by aphids. Canadian Journal of Zoology, 39(3),305-310.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Murray, D. A., Clarke, M. B., and Ronning, D. A. (2013). Estimating invertebrate pest losses in six major A ustralian grain crops.Australian Journal of Entomology, 52(3), 227-241.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Navazio, L., et al., (2002). The role of calcium in oligogalacturonide-activated signalling in soybean cells. Planta, 215(4), 596-605.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ngouémazong, D. E., et al. (2012). Effect of de-methylesterification on network development and nature of Ca2+ pectin gels: Towardsunderstanding structure–function relations of pectin. Food Hydrocolloids, 26(1), 89-98.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Osorio, S., Castillejo, C., Quesada, M. A., Medina‐Escobar, N., Brownsey, G. J., Suau, R., Heredia, A., Botella, M., and Valpuesta, V.(2008). Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wildstrawberry (Fragaria vesca). The Plant Journal, 54(1), 43-55.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Östman, Ö., Ekbom, B., and Bengtsson, J. (2003). Yield increase attributable to aphid predation by ground-living polyphagous naturalenemies in spring barley in Sweden. Ecological economics, 45(1), 149-158.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peaucelle, A., Louvet, R., Johansen, J. N., Höfte, H., Laufs, P., Pelloux, J., and Mouille, G. (2008). Arabidopsis phyllotaxis is controlledby the methyl-esterification status of cell-wall pectins. Current Biology, 18(24), 1943-1948.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peaucelle, A., Braybrook, S. A., Le Guillou, L., Bron, E., Kuhlemeier, C., & Höfte, H. (2011). Pectin-induced changes in cell wallmechanics underlie organ initiation in Arabidopsis. Current biology, 21(20), 1720-1726.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peng, H. C., and Walker, G. P. (2018). Sieve element occlusion provides resistance against Aphis gossypii in TGR‐1551 melons. Insectscience.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Poch, H. L. C., Ponz, F., and Fereres, A. (1998). Searching for resistance in Arabidopsis thaliana to the green peach aphid Myzuspersicae. Plant science, 138(2), 209-216.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Raiola, A., et al. (2011). Pectin methylesterase is induced in Arabidopsis upon infection and is necessary for a successful colonizationby necrotrophic pathogens. Molecular Plant-Microbe Interactions, 24(4), 432-440.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ridley, B. L., O'Neill, M. A., and Mohnen, D. (2001). Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.Phytochemistry, 57(6), 929-967.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Saez-Aguayo, S., Ralet, M. C., Berger, A., Botran, L., Ropartz, D., Marion-Poll, A., and North, H. M. (2013). PECTIN METHYLESTERASEINHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells.The Plant Cell, tpc-112.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Saez-Aguayo, S., et al. (2017). UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter that Modulates the PolysaccharideComposition of Arabidopsis Seed Mucilage. The Plant Cell, tpc-00465.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sarria, E., Cid, M., Garzo, E., and Fereres, A. (2009). Excel Workbook for automatic parameter calculation of EPG data. Computers andElectronics in Agriculture, 67(1-2), 35-42.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sénéchal, F., Mareck, A., Marcelo, P., Lerouge, P., & and Pelloux, J. (2015). Arabidopsis PME17 activity can be controlled by PectinMethylesterase Inhibitor4. Plant signaling & behavior, 10(2), e983351.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schoonhoven, L. M., Van Loon, B., van Loon, J. J., and Dicke, M. (2005). Insect-plant biology. Oxford University Press on Demand.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tiscione, N. B., Alford, I., Yeatman, D. T., & Shan, X. (2011). Ethanol analysis by headspace gas chromatography with simultaneousflame-ionization and mass spectrometry detection.Journal of analytical toxicology, 35(7), 501-511.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tjallingii, W. F. (1978). Electronic recording of penetration behaviour by aphids. Entomologia experimentalis et applicata, 24(3), 721-730.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tjallingii, W. F., and Mayoral, A. (1992). Criteria for host-plant acceptance by aphids. In Proceedings of the 8th International Symposiumon Insect-Plant Relationships (pp. 280-282). Springer, Dordrecht.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tjallingii, W. F., and Esch, T. H. (1993). Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals.Physiological entomology, 18(3), 317-328.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tjallingii, W. F. (2006). Salivary secretions by aphids interacting with proteins of phloem wound responses. Journal of experimentalbotany, 57(4), 739-745.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Von Dahl, C. C., Hävecker, M., Schlögl, R., and Baldwin, I. T. (2006). Caterpillar‐elicited methanol emission: a new signal in plant–herbivore interactions? The Plant Journal, 46(6), 948-960.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Van Emden, H. F., and Harrington, R. (Eds.). (2017). Aphids as crop pests. Cabi.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Verhertbruggen, Y., Marcus, S. E., Haeger, A., Ordaz-Ortiz, J. J., and Knox, J. P. (2009). An extended set of monoclonal antibodies topectic homogalacturonan. Carbohydrate Research, 344(14), 1858-1862.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang, D., Samsulrizal, N., Yan, C., Allcock, N.S., Craigon, J., Blanco-Ulate, B., Ortega-Salazar, I., Marcus, S.E., Bagheri, H.M., Perez-Fons, L., Fraser, P.D., Foster, T., Fray, R.G., Knox, J.P., Seymour, G.B., (2019). Characterisation of CRISPR mutants targeting genesmodulating pectin degradation in ripening tomato. Plant Physiology 179, 544-557
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Willats, W. G., Orfila, C., Limberg, G., Buchholt, H. C., van Alebeek, G. J. W., Voragen, A. G., Marcus, S., Christensen, T., Mikkelsen, J.,Murray, B. and Knox, J. P. (2001). Modulation of the degree and pattern of methyl esterification of pectic homogalacturonan in plantcell walls: implications for pectin methyl esterase action, matrix properties and cell adhesion. Journal of Biological Chemistry.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wolf, S., Mouille, G., and Pelloux, J. (2009). Homogalacturonan methyl-esterification and plant development. Molecular plant, 2(5), 851-860.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wolf, S., and Greiner, S. (2012). Growth control by cell wall pectins. Protoplasma, 249(2), 169-175.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang, C., et al. (2011). Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsisrepress phloem feeding of the green peach aphid Myzus persicae. BMC plant biology, 11(1), 11.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Supplemental Data /content/suppl/2019/07/13/tpc.19.00136.DC2.html /content/suppl/2019/05/23/tpc.19.00136.DC1.html
Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs http://www.plantcell.org/cgi/alerts/ctmain
Sign up for eTOCs at:
CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain
Sign up for CiteTrack Alerts at:
Subscription Information http://www.aspb.org/publications/subscriptions.cfm
is available at:Plant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists
top related